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Sample records for thermostable l-tyrosine phenol-lyase

  1. Biochemical characterization of a novel tyrosine phenol-lyase from Fusobacterium nucleatum for highly efficient biosynthesis of l-DOPA.

    Science.gov (United States)

    Zheng, Ren-Chao; Tang, Xiao-Ling; Suo, Hui; Feng, Li-Lin; Liu, Xiao; Yang, Jian; Zheng, Yu-Guo

    2018-05-01

    Tyrosine phenol-lyase (TPL) catalyzes the reversible cleavage of l-tyrosine to phenol, pyruvate and ammonia. When pyrocatechol is substituted for phenol, l-dihydroxyphenylalanine (l-DOPA) is produced. The TPL-catalyzed route was regarded as the most economic process for l-DOPA production. In this study, a novel TPL from Fusobacterium nucleatum (Fn-TPL) was successfully overexpressed in Escherichia coli and screened for l-DOPA synthesis with a specific activity of 2.69Umg -1 . Fn-TPL was found to be a tetramer, and the optimal temperature and pH for α, β-elimination of l-tyrosine was 60°C and pH 8.5, respectively. The enzyme showed broad substrate specificity toward natural and synthetic l-amino acids. Kinetic analysis suggested that the k cat /K m value for l-tyrosine decomposition was much higher than that for l-DOPA decomposition, while Fn-TPL exhibited similar catalytic efficiency for synthesis of l-tyrosine and l-DOPA. With whole cells of recombinant E. coli as biocatalyst, l-DOPA yield reached 110gL -1 with a pyrocatechol conversion of 95%, which was comparable to the reported highest level. The results demonstrated the great potential of Fn-TPL for industrial production of l-DOPA. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Paraffin as oxygen vector modulates tyrosine phenol lyase production by Citrobacter freundii MTCC 2424.

    Science.gov (United States)

    Azmi, Wamik; Kumar, Ajay; Dev, Varun

    2013-06-01

    The efficiency of three oxygen-vectors liquid paraffin, silicone oil and n-dodecane in the production of tyrosine phenol lyase (TPL) by Citrobacter freundii MTCC 2424 was evaluated at 4% (v/v) concentration. The liquid paraffin as oxygenvectors was found to exhibit a stimulatory effect on TPL synthesis. The liquid paraffin at 6% (v/v) resulted in 34% increase in the TPL synthesis accompanied by a 13% increase in the production of cell mass at a 10 L scale. This improvement in TPL and cell mass production in the presence of liquid paraffin can be related to the fact that liquid paraffin was capable of maintaining dissolved O2 concentration above 28% throughout the course of the fermentation. Maintenance of the dissolved O2 concentration above 28% could be viewed in terms of an adequate oxygen supply to the rapidly dividing cells of the bacterium, which in turn resulted in enhanced synthesis of TPL and cell mass.

  3. Electrode Potentials of l-Tryptophan, l-Tyrosine, 3-Nitro-l-tyrosine, 2,3-Difluoro-l-tyrosine, and 2,3,5-Trifluoro-l-tyrosine.

    Science.gov (United States)

    Mahmoudi, Leila; Kissner, Reinhard; Nauser, Thomas; Koppenol, Willem H

    2016-05-24

    Electrode potentials for aromatic amino acid radical/amino acid couples were deduced from cyclic voltammograms and pulse radiolysis experiments. The amino acids investigated were l-tryptophan, l-tyrosine, N-acetyl-l-tyrosine methyl ester, N-acetyl-3-nitro-l-tyrosine ethyl ester, N-acetyl-2,3-difluoro-l-tyrosine methyl ester, and N-acetyl-2,3,5-trifluoro-l-tyrosine methyl ester. Conditional potentials were determined at pH 7.4 for all compounds listed; furthermore, Pourbaix diagrams for l-tryptophan, l-tyrosine, and N-acetyl-3-nitro-l-tyrosine ethyl ester were obtained. Electron transfer accompanied by proton transfer is reversible, as confirmed by detailed analysis of the current waves, and because the slopes of the Pourbaix diagrams obey Nernst's law. E°'(Trp(•),H(+)/TrpH) and E°'(TyrO(•),H(+)/TyrOH) at pH 7 are 0.99 ± 0.01 and 0.97 ± 0.01 V, respectively. Pulse radiolysis studies of two dipeptides that contain both amino acids indicate a difference in E°' of approximately 0.06 V. Thus, in small peptides, we recommend values of 1.00 and 0.96 V for E°'(Trp(•),H(+)/TrpH) and E°'(TyrO(•),H(+)/TyrOH), respectively. The electrode potential of N-acetyl-3-nitro-l-tyrosine ethyl ester is higher, while because of mesomeric stabilization of the radical, those of N-acetyl-2,3-difluoro-l-tyrosine methyl ester and N-acetyl-2,3,5-trifluoro-l-tyrosine methyl ester are lower than that of tyrosine. Given that the electrode potentials at pH 7 of E°'(Trp(•),H(+)/TrpH) and E°'(TyrO(•),H(+)/TyrOH) are nearly equal, they would be, in principle, interchangeable. Proton-coupled electron transfer pathways in proteins that use TrpH and TyrOH are thus nearly thermoneutral.

  4. Modular Engineering of l-Tyrosine Production in Escherichia coli

    Science.gov (United States)

    Juminaga, Darmawi; Baidoo, Edward E. K.; Redding-Johanson, Alyssa M.; Batth, Tanveer S.; Burd, Helcio; Mukhopadhyay, Aindrila; Petzold, Christopher J.

    2012-01-01

    Efficient biosynthesis of l-tyrosine from glucose is necessary to make biological production economically viable. To this end, we designed and constructed a modular biosynthetic pathway for l-tyrosine production in E. coli MG1655 by encoding the enzymes for converting erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to l-tyrosine on two plasmids. Rational engineering to improve l-tyrosine production and to identify pathway bottlenecks was directed by targeted proteomics and metabolite profiling. The bottlenecks in the pathway were relieved by modifications in plasmid copy numbers, promoter strength, gene codon usage, and the placement of genes in operons. One major bottleneck was due to the bifunctional activities of quinate/shikimate dehydrogenase (YdiB), which caused accumulation of the intermediates dehydroquinate (DHQ) and dehydroshikimate (DHS) and the side product quinate; this bottleneck was relieved by replacing YdiB with its paralog AroE, resulting in the production of over 700 mg/liter of shikimate. Another bottleneck in shikimate production, due to low expression of the dehydroquinate synthase (AroB), was alleviated by optimizing the first 15 codons of the gene. Shikimate conversion to l-tyrosine was improved by replacing the shikimate kinase AroK with its isozyme, AroL, which effectively consumed all intermediates formed in the first half of the pathway. Guided by the protein and metabolite measurements, the best producer, consisting of two medium-copy-number, dual-operon plasmids, was optimized to produce >2 g/liter l-tyrosine at 80% of the theoretical yield. This work demonstrates the utility of targeted proteomics and metabolite profiling in pathway construction and optimization, which should be applicable to other metabolic pathways. PMID:22020510

  5. Evaluation of Brachypodium distachyon L-Tyrosine Decarboxylase Using L-Tyrosine Over-Producing Saccharomyces cerevisiae.

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    Shuhei Noda

    Full Text Available To demonstrate that herbaceous biomass is a versatile gene resource, we focused on the model plant Brachypodium distachyon, and screened the B. distachyon for homologs of tyrosine decarboxylase (TDC, which is involved in the modification of aromatic compounds. A total of 5 candidate genes were identified in cDNA libraries of B. distachyon and were introduced into Saccharomyces cerevisiae to evaluate TDC expression and tyramine production. It is suggested that two TDCs encoded in the transcripts Bradi2g51120.1 and Bradi2g51170.1 have L-tyrosine decarboxylation activity. Bradi2g51170.1 was introduced into the L-tyrosine over-producing strain of S. cerevisiae that was constructed by the introduction of mutant genes that promote deregulated feedback inhibition. The amount of tyramine produced by the resulting transformant was 6.6-fold higher (approximately 200 mg/L than the control strain, indicating that B. distachyon TDC effectively converts L-tyrosine to tyramine. Our results suggest that B. distachyon possesses enzymes that are capable of modifying aromatic residues, and that S. cerevisiae is a suitable host for the production of L-tyrosine derivatives.

  6. Radiolytic dimerization of tyrosine in alkaline solutions of poly-L-tyrosine, glycyl-L-tyrosine and tyrosine

    International Nuclear Information System (INIS)

    Boguta, G.; Dancewicz, A.M.

    1982-01-01

    Blue fluorescence characteristic of dityrosine appeared in γ-irradiated solutions of tyrosine, glycyl-L-tyrosine or polytyrosine (MW 110,000). The intensity of fluorescence was used for the determination of the dityrosine concentration in hydrolysed samples. The radiation-induced formation of dityrosine depended on pH and on the presence of oxygen during radiolysis carried out with a total dose of the order of 1000 Gy. The presence of oxygen in the system suppressed the formation of dityrosine in solution at low or neutral pH but had no effect on this process in alkaline solutions. Except for the radiolysis of air-saturated poly-L-tyrosine solutions, where G(Dityrosine) = 0.35, the yields of dityrosine at high pH were lower than the yields obtained during radiolysis at low pH and in the absence of oxygen. (author)

  7. Mechanistic deductions from kinetic isotope effects and pH studies of pyridoxal phosphate dependent carbon-carbon lyases: Erwinia herbicola and Citrobacter freundii tyrosine phenol-lyase

    International Nuclear Information System (INIS)

    Kiick, D.M.; Phillips, R.S.

    1988-01-01

    The pH dependence of the kinetic parameters and primary deuterium isotope effects have been determined for tyrosine phenol-lyase from both Erwinia herbicola and Citrobacter freundii. The primary deuterium isotope effects indicate that proton abstraction from the 2-position of the substrate is partially rate-limiting for both enzymes. The C. freundii enzyme primary deuterium isotope effects [DV = 3.5 and D(V/Ktyr) = 2.5] are pH independent, indicating that tyrosine is not sticky (i.e., does not dissociate slower than it reacts to give products). Since Vmax for both tyrosine and the alternate substrate S-methyl-L-cysteine is also pH independent, substrate binds only to the correctly protonated form of the enzyme. For the E. herbicola enzyme, both Vmax and V/K for tyrosine or S-methyl-L-cysteine are pH dependent, as well as both DV and D(V/Ktyr). Thus, while both the protonated and unprotonated enzyme can bind substrate, and may be interconverted directly, only the unprotonated Michaelis complex is catalytically competent. At pH 9.5, DV = 2.5 and D(V/Ktyr) = 1.5. However, at pH 6.4 the isotope effect on both parameters is equal to 4.1. From these data, the forward commitment factor (cf = 5.2) and catalytic ratio (cvf = 1.1) for tyrosine and S-methyl-L-cysteine (cf = 2.2, cvf = 24) are calculated. Also, the Michaelis complex partition ratio (cf/cvf) for substrate and products is calculated to be 4.7 for tyrosine and 0.1 for S-methyl-L-cysteine

  8. Subcutaneous L-tyrosine elicits cutaneous analgesia in response to local skin pinprick in rats.

    Science.gov (United States)

    Hung, Ching-Hsia; Chiu, Chong-Chi; Liu, Kuo-Sheng; Chen, Yu-Wen; Wang, Jhi-Joung

    2015-10-15

    The purpose of the study was to estimate the ability of L-tyrosine to induce cutaneous analgesia and to investigate the interaction between L-tyrosine and the local anesthetic lidocaine. After subcutaneously injecting the rats with L-tyrosine and lidocaine in a dose-dependent manner, cutaneous analgesia (by blocking the cutaneous trunci muscle reflex-CTMR) was evaluated in response to the local pinprick. The drug-drug interaction was analyzed by using an isobolographic method. We showed that both L-tyrosine and lidocaine produced dose-dependent cutaneous analgesia. On the 50% effective dose (ED50) basis, the rank of drug potency was lidocaine (5.09 [4.88-5.38] μmol)>L-tyrosine (39.1 [36.5-41.8] μmol) (Ptyrosine lasted longer than that caused by lidocaine (Ptyrosine exhibited an additive effect on infiltrative cutaneous analgesia. Our pre-clinical study demonstrated that L-tyrosine elicits the local/cutaneous analgesia, and the interaction between L-tyrosine and lidocaine is additive. L-tyrosine has a lower potency but much greater duration of cutaneous analgesia than lidocaine. Adding L-tyrosine to lidocaine preparations showed greater duration of cutaneous analgesia compared with lidocaine alone. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Evaluation of o-[11C]methyl-L-tyrosine and o-[18F]fluoromethyl-L-tyrosine as tumor imaging tracers by PET

    International Nuclear Information System (INIS)

    Ishiwata, Kiichi; Kawamura, Kazunori; Wang Weifang; Furumoto, Shozo; Kubota, Kazuo; Pascali, Claudio; Bogni, Anna; Iwata, Ren

    2004-01-01

    We investigated the potential of O-[ 11 C]methyl-L-tyrosine and O-[ 18 F]fluoromethyl-L-tyrosine as positron-emitting tracers for tumor imaging. The two tracers had similar distribution patterns in rats bearing AH109A hepatoma, with pancreas and, on a lesser extent, AH109A showing the highest uptake. Uptake of both tracers in the AH109A and uptake ratios of AH109A-to-tissues (with the exception of AH109A-to-bone) gradually increased for 60 min. O-[ 11 C]methyl-L-tyrosine was metabolically stable, whereas a negligible low amount of metabolites was observed for O-[ 18 F]fluoromethyl-L-tyrosine. Both tracers showed the potential for tumor imaging

  10. Effective L-Tyrosine Hydroxylation by Native and Immobilized Tyrosinase.

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    Małgorzata Cieńska

    Full Text Available Hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA by immobilized tyrosinase in the presence of ascorbic acid (AH2, which reduces DOPA-quinone to L-DOPA, is characterized by low reaction yields that are mainly caused by the suicide inactivation of tyrosinase by L-DOPA and AH2. The main aim of this work was to compare processes with native and immobilized tyrosinase to identify the conditions that limit suicide inactivation and produce substrate conversions to L-DOPA of above 50% using HPLC analysis. It was shown that immobilized tyrosinase does not suffer from partitioning and diffusion effects, allowing a direct comparison of the reactions performed with both forms of the enzyme. In typical processes, additional aeration was applied and boron ions to produce the L-DOPA and AH2 complex and hydroxylamine to close the cycle of enzyme active center transformations. It was shown that the commonly used pH 9 buffer increased enzyme stability, with concomitant reduced reactivity of 76%, and that under these conditions, the maximal substrate conversion was approximately 25 (native to 30% (immobilized enzyme. To increase reaction yield, the pH of the reaction mixture was reduced to 8 and 7, producing L-DOPA yields of approximately 95% (native enzyme and 70% (immobilized. A three-fold increase in the bound enzyme load achieved 95% conversion in two successive runs, but in the third one, tyrosinase lost its activity due to strong suicide inactivation caused by L-DOPA processing. In this case, the cost of the immobilized enzyme preparation is not overcome by its reuse over time, and native tyrosinase may be more economically feasible for a single use in L-DOPA production. The practical importance of the obtained results is that highly efficient hydroxylation of monophenols by tyrosinase can be obtained by selecting the proper reaction pH and is a compromise between complexation and enzyme reactivity.

  11. Effective L-Tyrosine Hydroxylation by Native and Immobilized Tyrosinase

    Science.gov (United States)

    Lewańczuk, Marcin; Koźlecki, Tomasz; Liesiene, Jolanta; Bryjak, Jolanta

    2016-01-01

    Hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA) by immobilized tyrosinase in the presence of ascorbic acid (AH2), which reduces DOPA-quinone to L-DOPA, is characterized by low reaction yields that are mainly caused by the suicide inactivation of tyrosinase by L-DOPA and AH2. The main aim of this work was to compare processes with native and immobilized tyrosinase to identify the conditions that limit suicide inactivation and produce substrate conversions to L-DOPA of above 50% using HPLC analysis. It was shown that immobilized tyrosinase does not suffer from partitioning and diffusion effects, allowing a direct comparison of the reactions performed with both forms of the enzyme. In typical processes, additional aeration was applied and boron ions to produce the L-DOPA and AH2 complex and hydroxylamine to close the cycle of enzyme active center transformations. It was shown that the commonly used pH 9 buffer increased enzyme stability, with concomitant reduced reactivity of 76%, and that under these conditions, the maximal substrate conversion was approximately 25 (native) to 30% (immobilized enzyme). To increase reaction yield, the pH of the reaction mixture was reduced to 8 and 7, producing L-DOPA yields of approximately 95% (native enzyme) and 70% (immobilized). A three-fold increase in the bound enzyme load achieved 95% conversion in two successive runs, but in the third one, tyrosinase lost its activity due to strong suicide inactivation caused by L-DOPA processing. In this case, the cost of the immobilized enzyme preparation is not overcome by its reuse over time, and native tyrosinase may be more economically feasible for a single use in L-DOPA production. The practical importance of the obtained results is that highly efficient hydroxylation of monophenols by tyrosinase can be obtained by selecting the proper reaction pH and is a compromise between complexation and enzyme reactivity. PMID:27711193

  12. Effective L-Tyrosine Hydroxylation by Native and Immobilized Tyrosinase.

    Science.gov (United States)

    Cieńska, Małgorzata; Labus, Karolina; Lewańczuk, Marcin; Koźlecki, Tomasz; Liesiene, Jolanta; Bryjak, Jolanta

    2016-01-01

    Hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA) by immobilized tyrosinase in the presence of ascorbic acid (AH2), which reduces DOPA-quinone to L-DOPA, is characterized by low reaction yields that are mainly caused by the suicide inactivation of tyrosinase by L-DOPA and AH2. The main aim of this work was to compare processes with native and immobilized tyrosinase to identify the conditions that limit suicide inactivation and produce substrate conversions to L-DOPA of above 50% using HPLC analysis. It was shown that immobilized tyrosinase does not suffer from partitioning and diffusion effects, allowing a direct comparison of the reactions performed with both forms of the enzyme. In typical processes, additional aeration was applied and boron ions to produce the L-DOPA and AH2 complex and hydroxylamine to close the cycle of enzyme active center transformations. It was shown that the commonly used pH 9 buffer increased enzyme stability, with concomitant reduced reactivity of 76%, and that under these conditions, the maximal substrate conversion was approximately 25 (native) to 30% (immobilized enzyme). To increase reaction yield, the pH of the reaction mixture was reduced to 8 and 7, producing L-DOPA yields of approximately 95% (native enzyme) and 70% (immobilized). A three-fold increase in the bound enzyme load achieved 95% conversion in two successive runs, but in the third one, tyrosinase lost its activity due to strong suicide inactivation caused by L-DOPA processing. In this case, the cost of the immobilized enzyme preparation is not overcome by its reuse over time, and native tyrosinase may be more economically feasible for a single use in L-DOPA production. The practical importance of the obtained results is that highly efficient hydroxylation of monophenols by tyrosinase can be obtained by selecting the proper reaction pH and is a compromise between complexation and enzyme reactivity.

  13. l-Tyrosine Contained in Dietary Supplement by Chemiluminescence Reaction of an Iron-Phthalocyanine Complex

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    Takao Ohtomo

    2012-01-01

    Full Text Available The chemiluminescence (CL signal immediately appeared when a hydrogen peroxide solution was injected into an iron-phthalocyanine tetrasulfonic acid (Fe-PTS aqueous solution. Moreover, the CL intensity of Fe-PTS decreased by adding L-tyrosine. Based on these results, the determination of trace amounts of L-tyrosine was developed using the quenching-chemiluminescence. The calibration curve of L-tyrosine was obtained in the concentration range of 2.0×10−7 M to 2.0×10−5 M. Moreover, the relative standard deviation (RSD was 1.63 % (=5 for 2.0×10−6 M L-tyrosine, and its detection limits (3σ were 1.81×10−7 M. The spike and recovery experiments for L-tyrosine were performed using a soft drink. Furthermore, the determination of L-tyrosine was applied to supplements containing various kinds of amino acids. Each satisfactory relative recovery was obtained at 98 to 102%.

  14. Development of amperometric L-tyrosine sensor based on Fe-doped hydroxyapatite nanoparticles

    International Nuclear Information System (INIS)

    Kanchana, P.; Lavanya, N.; Sekar, C.

    2014-01-01

    A novel biosensor based on Fe-doped hydroxyapatite (Fe-HA) nanoparticles and tyrosinase has been developed for the detection of L-tyrosine. Nanostructured Fe-HA was synthesized by a simple microwave irradiation method, and its phase formation, morphology and magnetic property were examined by powder X-ray diffraction (XRD), transmission electron microscopy (TEM) and vibrating sample magnetometer (VSM). Electrochemical performance of the nano Fe-HA/tyrosinase modified glassy carbon electrode (GCE) for detection of L-tyrosine was investigated by cyclic voltammetry (CV) and amperometric methods. The fabricated biosensor exhibited a linear response to L-tyrosine over a wide concentration range of 1.0 × 10 −7 to 1.0 × 10 −5 M with a detection limit of 245 nM at pH 7.0. In addition, the fabricated sensor showed an excellent selectivity, good reproducibility, long-term stability and anti-interference towards the determination of L-tyrosine. - Highlights: • A novel amperometric L-tyrosine biosensor has been fabricated using nanostructured Fe-HA. • The fabricated sensor exhibits a wide linear range, good stability and high reproducibility. • Fe-HA assists microenvironment and direct electron transfer between enzyme and electrode surface. • The nano Fe-HA and electrode fabrication procedure are simple and less expensive

  15. Synthesis of 2-[18F]fluoro-L-tyrosine via regiospecific fluoro-de-stannylation

    International Nuclear Information System (INIS)

    Hess, E.; Sichler, S.; Kluge, A.; Coenen, H.H.

    2002-01-01

    2-[ 18 F]Fluoro-L-tyrosine is a fluorine labelled amino acid, known to be incorporated into newly synthesised proteins, rendering it a potentially suitable tracer to image protein metabolism in vivo using positron emission tomography. For the electrophilic preparation of 2-[ 18 F]fluoro-L-tyrosine three protected 2-trialkylstannyl tyrosine derivatives have been synthesised for the first time as precursors. While O,N-di-Boc-2-triethylstannyl-L-tyrosine ethylester has proved to be suitable as precursor for radiosynthesis, imidazolidinon-derivatives of 2-triaklylstannyl tyrosine have not because of difficult fast hydrolysis of a phenolic O-methyl protective group. The di-Boc-tin derivative of tyrosine ethylester readily reacted with [ 18 F]F 2 , which was prepared via the 18 O(p,n) 18 F nuclear reaction. 2-[ 18 F]Fluoro-L-tyrosine was isolated after full deprotection with aqueous hydrobromic acid and HPLC purification with activities of 1.41±0.32 GBq. The isomeric and enantiomeric purity is high (both >99%). The preparation procedure is facile and easy to automate. The chemical yields of this fluoro-de-stannylation reaction as well as of the synthesis of 6-[ 18 F]fluoro-L-dopa, determined with an analogous precursor and non-radioactive fluorine under identical conditions, amounted to 42.7±1.6% and 60.2±2.8%, respectively

  16. Preclinical and clinical evaluation of O-[11C]methyl-L-tyrosine for tumor imaging by positron emission tomography

    International Nuclear Information System (INIS)

    Ishiwata, Kiichi; Tsukada, Hideo; Kubota, Kazuo; Nariai, Tadashi; Harada, Norihiro; Kawamura, Kazunori; Kimura, Yuichi; Oda, Keiichi; Iwata, Ren; Ishii, Kenji

    2005-01-01

    We performed preclinical and clinical studies of O-[ 11 C]methyl-L-tyrosine, a potential tracer for imaging amino acid transport of tumors by positron emission tomography (PET). Examinations of the radiation-absorbed dose by O-[ 11 C]methyl-L-tyrosine and the acute toxicity and mutagenicity of O-methyl-L-tyrosine showed suitability of the tracer for clinical use. The whole-body imaging of monkeys and healthy humans by PET showed low uptake of O-[ 11 C]methyl-L-tyrosine in all normal organs except for the urinary track and bladder, suggesting that the O-[ 11 C]methyl-L-tyrosine PET has the potential for tumor imaging in the whole-body. Finally, the brain tumor imaging was preliminarily demonstrated

  17. Facile, one-step controlled synthesis of Se nanocrystals in the presence of L-tyrosine

    International Nuclear Information System (INIS)

    Wang Xiufang; Zhang Weiqiang; Shen Yuhua; Xie Anjian; Huang Lachun

    2011-01-01

    Highlights: → Se was synthesized via a novel and facile pathway. → The morphologies and the crystalline phases of Se can be easily controlled. → Tyrosine is excellent reducing agent and soft template for the synthesis of Se. → It is possible to provide an excellent route to obtain nanomaterials. - Abstract: Se with different morphologies was synthesized using L-tyrosine as reducing agent and soft template by means of hydrothermal method. The method was simple and convenient to handle. The reaction process was monitored using ultraviolet-visible spectroscopy (UV-vis) and Fourier-transform infrared spectroscopy (FTIR). The morphology and crystalline phase were determined by transmission electron microscopy (TEM), scanning electronic microscopy (SEM) and X-ray diffraction (XRD) pattern. The results show that the morphologies and the crystalline phases of Se can be easily controlled. By varying the concentration ratio of L-tyrosine to selenious acid, the morphologies and crystalline phases of Se were not changed, but the diameter of Se was different. Se nano-rods are obtained in the lower temperature, and there is a transformation of microspheres into nanorods of Se with the increase of reaction time or reaction temperature. In addition, we discuss the possible mechanism of the reduction of SeO 3 2- ions by L-tyrosine. The eco-friendly, biogenic synthesis strategy could be widely used for preparing inorganic/organic biocomposites.

  18. Thermostating highly confined fluids.

    Science.gov (United States)

    Bernardi, Stefano; Todd, B D; Searles, Debra J

    2010-06-28

    In this work we show how different use of thermostating devices and modeling of walls influence the mechanical and dynamical properties of confined nanofluids. We consider a two dimensional fluid undergoing Couette flow using nonequilibrium molecular dynamics simulations. Because the system is highly inhomogeneous, the density shows strong fluctuations across the channel. We compare the dynamics produced by applying a thermostating device directly to the fluid with that obtained when the wall is thermostated, considering also the effects of using rigid walls. This comparison involves an analysis of the chaoticity of the fluid and evaluation of mechanical properties across the channel. We look at two thermostating devices with either rigid or vibrating atomic walls and compare them with a system only thermostated by conduction through vibrating atomic walls. Sensitive changes are observed in the xy component of the pressure tensor, streaming velocity, and density across the pore and the Lyapunov localization of the fluid. We also find that the fluid slip can be significantly reduced by rigid walls. Our results suggest caution in interpreting the results of systems in which fluid atoms are thermostated and/or wall atoms are constrained to be rigid, such as, for example, water inside carbon nanotubes.

  19. Identification and Partial Characterization of an L-Tyrosine Aminotransferase (TAT from Arabidopsis thaliana

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    Pranav R. Prabhu

    2010-01-01

    Full Text Available The aminotransferase gene family in the model plant Arabidopsis thaliana consists of 44 genes. Twenty six of these enzymes are classified as characterized meaning that the reaction(s that the enzyme catalyzes are documented using experimental means. The remaining 18 enzymes are uncharacterized and are therefore deemed putative. Our laboratory is interested in elucidating the function(s of the remaining putative aminotransferase enzymes. To this end, we have identified and partially characterized an aminotransferase (TAT enzyme from Arabidopsis annotated by the locus tag At5g36160. The full-length cDNA was cloned and the purified recombinant enzyme was characterized using in vitro and in vivo experiments. In vitro analysis showed that the enzyme is capable of interconverting L-Tyrosine and 4-hydroxyphenylpyruvate, and L-Phenylalanine and phenylpyruvate. In vivo analysis by functional complementation showed that the gene was able to complement an E. coli with a background of aminotransferase mutations that confers auxotrophy for L-Tyrosine and L-Phenylalanine.

  20. High-pressure Raman spectra and DFT calculations of L-tyrosine hydrochloride crystal

    Science.gov (United States)

    dos Santos, C. A. A. S. S.; Carvalho, J. O.; da Silva Filho, J. G.; Rodrigues, J. L.; Lima, R. J. C.; Pinheiro, G. S.; Freire, P. T. C.; Façanha Filho, P. F.

    2018-02-01

    High-pressure Raman spectra of L-tyrosine hydrochloride crystal were obtained from 1.0 atm to 7.0 GPa in the 90-1800 cm-1 spectral region. At atmospheric pressure, the Raman spectrum was obtained in the 50-3200 cm-1 spectral range and the assignment of the normal modes based on density functional theory calculations was provided. We found good correspondence between the calculated and the observed intramolecular geometry parameters. This confirms the correct assignment of the normal modes, since it was crucial to understand the meaning of the changes observed in particular Raman active modes. Here we show that bands associated with internal modes undergo slight modifications during compression. However, an inversion of the relative intensity of bands around 125 cm-1 as well as a change of slope dω/dP from 1.0 to 1.5 GPa was understood as a conformational change involving a torsion of the L-tyrosine molecule. As a consequence, it is possible to conclude that the crystal remained in the same monoclinic structure in the 1 atm-7.0 GPa interval, although conformational change of the molecule was verified. A comparison of our results with other selected studies provided insights about the role of the amino acid side chain on the arrangement of hydrogen bonds. Finally, when the pressure was released back to 1 atm, the Raman spectrum was recovered and no hysteresis was observed.

  1. L-tyrosine and L-DOPA as hormone-like regulators of melanocytes functions

    Science.gov (United States)

    Slominski, Andrzej; Zmijewski, Michal; Pawelek, John

    2011-01-01

    Summary Evidence reveals that L-tyrosine and L-DOPA, besides serving as substrates and intermediates of melanogenesis, are also bioregulatory agents acting not only as inducers and positive regulators of melanogenesis but also as regulators of other cellular functions. These can be mediated through action on specific receptors or through non-receptor mediated mechanisms. The substrate induced (L-tyrosine and/or L-DOPA) melanogenic pathway would autoregulate itself as well as it would regulate the melanocyte functions through activity of its structural or regulatory proteins and through intermediates of melanogenesis and melanin itself. Dissection of regulatory and autoregulatory elements of this process may elucidate how substrate induced autoregulatory pathways have evolved from prokaryotic or simple eukaryotic organisms to complex systems in vertebrates. This could substantiate older theory proposing that receptors for amino-acid derived hormones arose from the receptors for those amino acids, and that nuclear receptors evolved from primitive intracellular receptors binding nutritional factors or metabolic intermediates. PMID:21834848

  2. Oral l-tyrosine supplementation augments the vasoconstriction response to whole-body cooling in older adults.

    Science.gov (United States)

    Lang, James A; Smaller, Kevin A

    2017-07-01

    What is the central question of this study? Ageing is associated with altered sympathetic responses to stress, which are explained in part by reduced noradrenergic function. The impact of supplementation with oral l-tyrosine, the amino acid precursor for catecholamine synthesis, on the effector responses to cold and exercise stress has yet to be examined. What is the main finding and its importance? Oral l-tyrosine ingestion augmented the sympathetically mediated vasoconstriction response to cold exposure in aged skin. This suggests that l-tyrosine supplementation might improve thermoregulatory function in older adults. l-Tyrosine is the primary substrate for noradrenaline biosynthesis within sympathetic axon terminals. In stressful conditions requiring increased catecholamine production, the axonal l-tyrosine concentration may limit the full expression of the sympathetic effector response and this may be particularly evident in older adults. We hypothesize that oral l-tyrosine supplementation will increase the sympathetic response to whole-body cooling and muscle metaboreflex activation. In a randomized, double-blind design, 11 young (Y = 24 ± 1 years) and 11 older participants (O = 68 ± 4 years) ingested either 150 mg kg -1 of l-tyrosine or placebo before commencing 30 min of whole-body cooling to induce a gradual decline in skin temperature from 34 to 30.5°C. Laser Doppler flux (LDF) was measured at the ventral forearm, and cutaneous vascular conductance (CVC) was calculated as CVC = LDF/mean arterial pressure and expressed as a percentage change from baseline (%ΔCVC). Two minutes of static hand-grip exercise (35% maximal voluntary contraction) followed by 3 min of postexercise ischaemia were implemented before and toward the end of the cooling bout. l-Tyrosine supplementation did not affect blood pressure or heart rate responses to exercise or postexercise ischaemia. However, the blunted vasoconstriction response to whole-body cooling in

  3. Excess amounts of 3-iodo-l-tyrosine induce Parkinson-like features in experimental approaches of Parkinsonism.

    Science.gov (United States)

    Fernández-Espejo, Emilio; Bis-Humbert, Cristian

    2018-06-06

    3-iodo-l-tyrosine might play a role in Parkinson's disease since this molecule is able, at high concentration, to inhibit tyrosine-hydroxylase activity, the rate-limiting enzyme in dopamine biosynthesis. The possible Parkinson-like effects of 3-iodo-l-tyrosine were tested on three experimental approaches in mice: cultured substantia nigra neurons, the enteric nervous system of the jejunum after intra-peritoneal infusions, and the nigrostriatal system following unilateral intrabrain injections. 3-iodo-l-tyrosine, a physiological molecule, was used at concentrations higher than its serum levels in humans. Parkinson-like signs were evaluated through abnormal aggregation of α-synuclein and tyrosine-hydroxylase, loss of tyrosine-hydroxylase-expressing and striatum-projecting neurons and fibers, reduced tyrosine-hydroxylase density, and Parkinson-like motor and non-motor deficits. The retrograde tracer FluoroGold was used in the brain model. The findings revealed that excess amounts of 3-iodo-l-tyrosine induce Parkinson-like effects in the three experimental approaches. Thus, culture neurons of substantia nigra show, after 3-iodo-l-tyrosine exposure, intracytoplasmic inclusions that express α-synuclein and tyrosine-hydroxylase. Intra-peritoneal infusions of 3-iodo-l-tyrosine cause, in the long-term, α-synuclein aggregation, thicker α-synuclein-positive fibers, and loss of tyrosine-hydroxylase-positive cells and fibers in intramural plexuses and ganglia of the jejunum. Infusion of 3-iodo-l-tyrosine into the left dorsal striata of mice damages the nigrostriatal system, as revealed through lower striatal tyrosine-hydroxylase density, reduced number of tyrosine-hydroxylase-expressing and striatum-projecting neurons in the left substantia nigra, as well as the emergence of Parkinson-like behavioral deficits such as akinesia, bradykinesia, motor disbalance, and locomotion directional bias. In conclusion, excess amounts of 3-iodo-l-tyrosine induce Parkinson-like features in

  4. Overeating Behavior and Striatal Dopamine with 6-[18F]-Fluoro-L--Tyrosine PET

    Directory of Open Access Journals (Sweden)

    Claire E. Wilcox

    2010-01-01

    Full Text Available Eating behavior may be affected by dopamine synthesis capacity. In this study, 6-[18F]-fluoro-L--tyrosine (FMT positron emission tomography (PET uptake in striatal subregions was correlated with BMI (kg/m2 and an estimate of the frequency of prior weight loss attempts in 15 healthy subjects. BMI was negatively correlated with FMT uptake in the dorsal caudate. Although the association between BMI and FMT uptake in the dorsal caudate was not significant upon correction for age and sex, the association fell within the range of a statistical trend. Weight loss attempts divided by years trying was also negatively correlated with FMT uptake in the dorsal putamen (=.05. These results suggest an association between low dorsal striatal presynaptic dopamine synthesis capacity and overeating behavior.

  5. L-tyrosine hydrochloride crystals under high pressures via Raman Spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Santos, C.A.A.S.; Facanha Filho, P.F.; Santos, A.O. dos; Ribeiro, L.H.L.; Victor, F.M.S.; Abreu, D.C.; Carvalho, J.O.; Soares, R.A.; Sousa, J.C.F.; Lima, R.C.; Cavaignac, A.O. [Universidade Federal do Maranhao (UFMA), MA (Brazil)

    2016-07-01

    Full text: Amino acid single crystals have been attracted researchers in recent years due to their potential applications as second harmonic generator. The goal of this work is to produce semi organic single crystals of L-tyrosine hydrochloride (LTHCl) and verify the behavior of their vibrational normal modes under high pressures and the stability of material in these conditions extremes. The LTHCl single crystals were produced for solubilization of amino acid L-tyrosine in hydrochloric acid by slow evaporation technique of the solvent in room temperature. The technique of X-ray diffraction (XRD) and the refinement of structure by the Rietveld method were used to confirm the crystal structure. The LTHCl crystal belongs to the monoclinic crystal system having two molecules per unit cell. The refinement by the Rietveld method showed good results with Rwp = 8.49% and Rp = 6.29% with S = 1.13. Raman scattering measurements as a function of pressure was performed in a piece of crystal from the ambient pressure to 7.2 GPa and Nujol was used as pressure medium. It was observed the appearance of a weak band around 163 cm-1 between pressures of 0.5 and 1.0 GPa, which characterize an phase transition undergone by the crystal. Moreover, this band gains intensity as pressure increases while gradual decreasing relative intensity of the very strong band at 123 cm-1 for all range of pressure also was observed. In fact, almost all bands of the spectra have undergone strong decreasing up to 7.2 GPa. However, on release of pressure the crystal has reached the original phase again. Therefore, the results showed this material cannot be suitable for the application (NLO) in this range of pressure. (author)

  6. Synthesis and characterization of L-tyrosine hydrochloride crystals submitted to high and low temperatures

    Energy Technology Data Exchange (ETDEWEB)

    Santos, C.A.A.S.; Facanha Filho, P.F.; Ribeiro, L.H.L.; Victor, F.M.S.; Abreu, D.C.; Santos, A.O. dos; Carvalho, J.O.; Soares, R.A.; Sousa, J.C.F.; Lima, R.C.; Cavaignac, A.O. [Universidade Federal do Maranhao (UFMA), MA (Brazil)

    2016-07-01

    Full text: New materials are emerging and generate advances in nonlinear optics that studies the phenomena related to changes in optical properties when occurs interaction of light with the matter. Semi organic crystals present such properties. The goal is this work is to produce semi organic single crystal of L-tyrosine hydrochloride (LTHCl) and verify their thermal stability when subjected to high and low temperatures. The single crystals of LTHCl were produced for solubilization of amino acid L-tyrosine in hydrochloric acid using slow solvent evaporation technique at a constant temperature of 25 deg C. The X-ray diffraction (XRD) and refining by the Rietveld method were used to confirm the structure of the material. The thermal stability was investigated using DSC, TGA-DTA. The LTHCl crystal belongs to the monoclinic system, with two molecules per unit cell. The refinement by the Rietveld method showed good results with Rwp= 8.49% and Rp= 6.29% with S=1.13. Thermal analysis shown an endothermic event at about 160°C, which can be associated with phase transition occurred in LTHCl crystal. It was also observed that the crystal melting point occurs at a temperature of 230°C. No water of crystallization was found in the crystal structure, which was confirmed by Raman spectroscopy and thermal analysis. From the Raman spectroscopy experiments in function of temperature, no significant changes was observe in the behavior of vibrational normal modes between temperatures of -253 and 170 deg C. Finally, a monoclinic crystal system LTHCl is stable up to 160°C at high temperatures and -253°C at low temperatures. Therefore, our investigation has proved that LTHCl crystals can be used in this range of temperature without the lost of their nonlinear optical properties. (author)

  7. Preclinical and clinical evaluation of O-[{sup 11}C]methyl-L-tyrosine for tumor imaging by positron emission tomography

    Energy Technology Data Exchange (ETDEWEB)

    Ishiwata, Kiichi [Positron Medical Center, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173-0022 (Japan)]. E-mail: ishiwata@pet.tmig.or.jp; Tsukada, Hideo [Central Research Laboratory, Hamamatsu Photonics K.K., Hamakita 434-8601 (Japan); Kubota, Kazuo [Department of Radiology, Division of Nuclear Medicine, International Medical Center of Japan, Shinjuku-ku, Tokyo 162-8655 (Japan); Nariai, Tadashi [Department of Neurosurgery, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8519 (Japan); Harada, Norihiro [Department of Radiology, Division of Nuclear Medicine, International Medical Center of Japan, Shinjuku-ku, Tokyo 162-8655 (Japan); Kawamura, Kazunori [Positron Medical Center, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173-0022 (Japan); SHI Accelerator Service Ltd., Shinagawa-ku, Tokyo 141-8686 (Japan); Kimura, Yuichi [Positron Medical Center, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173-0022 (Japan); Oda, Keiichi [Positron Medical Center, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173-0022 (Japan); Iwata, Ren [CYRIC, Tohoku University, Aoba-ku, Sendai 980-8578 (Japan); Ishii, Kenji [Positron Medical Center, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173-0022 (Japan)

    2005-04-01

    We performed preclinical and clinical studies of O-[{sup 11}C]methyl-L-tyrosine, a potential tracer for imaging amino acid transport of tumors by positron emission tomography (PET). Examinations of the radiation-absorbed dose by O-[{sup 11}C]methyl-L-tyrosine and the acute toxicity and mutagenicity of O-methyl-L-tyrosine showed suitability of the tracer for clinical use. The whole-body imaging of monkeys and healthy humans by PET showed low uptake of O-[{sup 11}C]methyl-L-tyrosine in all normal organs except for the urinary track and bladder, suggesting that the O-[{sup 11}C]methyl-L-tyrosine PET has the potential for tumor imaging in the whole-body. Finally, the brain tumor imaging was preliminarily demonstrated.

  8. Pharmacologic inhibition of L-tyrosine degradation ameliorates cerebral dopamine deficiency in murine phenylketonuria (PKU)

    Science.gov (United States)

    Harding, Cary O.; Winn, Shelley R.; Gibson, K. Michael; Arning, Erland; Bottiglieri, Teodoro; Grompe, Markus

    2014-01-01

    Summary Monoamine neurotransmitter deficiency has been implicated in the etiology of neuropsychiatric symptoms associated with chronic hyperphenylalaninemia in phenylketonuria (PKU). Two proposed explanations for neurotransmitter deficiency in PKU include first, that chronically elevated blood L-phenylalanine (Phe) inhibits the transport of L-tyrosine (Tyr) and L-tryptophan (Trp), the substrates for dopamine and serotonin synthesis respectively, into brain. In the second hypothesis, elevated Phe competitively inhibits brain tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) activities, the rate limiting steps in dopamine and serotonin synthesis. Dietary supplementation with large neutral amino acids (LNAA) including Tyr and Trp has been recommended for individuals with chronically elevated blood Phe in an attempt to restore amino acid and monoamine homeostasis in brain. As a potential alternative treatment approach, we demonstrate that pharmacologic inhibition of Tyr degradation through oral administration of nitisinone (NTBC) yielded sustained increases in blood and brain Tyr, decreased blood and brain Phe, and consequently increased dopamine synthesis in a murine model of PKU. Our results suggest that Phe-mediated inhibition of TH activity is the likely mechanism of impaired dopamine synthesis in PKU. Pharmacologic inhibition of Tyr degradation may be a promising adjunct therapy for CNS monoamine neurotransmitter deficiency in hyperphenylalaninemic individuals with PKU. PMID:24487571

  9. Whole-body distribution and dosimetry of O-(2-[18F]fluoroethyl)-l-tyrosine

    International Nuclear Information System (INIS)

    Pauleit, Dirk; Floeth, Frank; Herzog, Hans; Tellmann, Lutz; Langen, Karl-J.; Hamacher, Kurt; Coenen, Heinz H.; Mueller, Hans-W.

    2003-01-01

    The whole-body distribution of O-(2-[ 18 F]fluoroethyl)-l-tyrosine (FET) was studied in seven patients with brain tumours by positron emission tomography (PET). Based on the IMEDOSE and MIRDOSE procedures, radiation absorbed doses were estimated from whole-body PET scans acquired approximately 70 and 200 min after i.v. injection of 400 MBq FET. After injection of FET, the peak of radioactivity in the blood was observed after 1.5 min, and a plateau of nearly constant radioactivity was reached at 20 min. The whole-body distribution of FET showed the highest activities in the urinary tract. All other organs exhibited only moderate FET uptake (SUV ≤1.6) which remained constant between early and late PET scans. No increased uptake was seen in the bone, the biliary tract or the pancreas. Twenty-two percent of the injected activity was excreted 5 h p.i. (approx. 5.3% ID/h). The highest absorbed dose was found for the urinary bladder wall. The effective dose according to ICRP 60 was 16.5 μSv/MBq for adults, which would lead to an effective dose of 6.1 mSv in a PET study using 370 MBq FET. (orig.)

  10. Whole-body distribution and dosimetry of O-(2-[{sup 18}F]fluoroethyl)-l-tyrosine

    Energy Technology Data Exchange (ETDEWEB)

    Pauleit, Dirk [Clinic of Nuclear Medicine, Heinrich-Heine-University Duesseldorf, Duesseldorf (Germany); Clinic of Nuclear Medicine, Research Center Juelich, P.O. Box 1913, 52425, Juelich (Germany); Floeth, Frank [Department of Neurosurgery, Heinrich-Heine-University Duesseldorf, Duesseldorf (Germany); Herzog, Hans; Tellmann, Lutz; Langen, Karl-J. [Institute of Medicine, Research Center Juelich, Juelich (Germany); Hamacher, Kurt; Coenen, Heinz H. [Institute of Nuclear Chemistry, Research Center Juelich, Juelich (Germany); Mueller, Hans-W. [Clinic of Nuclear Medicine, Heinrich-Heine-University Duesseldorf, Duesseldorf (Germany)

    2003-04-01

    The whole-body distribution of O-(2-[{sup 18}F]fluoroethyl)-l-tyrosine (FET) was studied in seven patients with brain tumours by positron emission tomography (PET). Based on the IMEDOSE and MIRDOSE procedures, radiation absorbed doses were estimated from whole-body PET scans acquired approximately 70 and 200 min after i.v. injection of 400 MBq FET. After injection of FET, the peak of radioactivity in the blood was observed after 1.5 min, and a plateau of nearly constant radioactivity was reached at 20 min. The whole-body distribution of FET showed the highest activities in the urinary tract. All other organs exhibited only moderate FET uptake (SUV {<=}1.6) which remained constant between early and late PET scans. No increased uptake was seen in the bone, the biliary tract or the pancreas. Twenty-two percent of the injected activity was excreted 5 h p.i. (approx. 5.3% ID/h). The highest absorbed dose was found for the urinary bladder wall. The effective dose according to ICRP 60 was 16.5 {mu}Sv/MBq for adults, which would lead to an effective dose of 6.1 mSv in a PET study using 370 MBq FET. (orig.)

  11. Whole-body distribution and dosimetry of O-(2-[18F]fluoroethyl)-L-tyrosine.

    Science.gov (United States)

    Pauleit, Dirk; Floeth, Frank; Herzog, Hans; Hamacher, Kurt; Tellmann, Lutz; Müller, Hans-W; Coenen, Heinz H; Langen, Karl-J

    2003-04-01

    The whole-body distribution of O-(2-[(18)F]fluoroethyl)- l-tyrosine (FET) was studied in seven patients with brain tumours by positron emission tomography (PET). Based on the IMEDOSE and MIRDOSE procedures, radiation absorbed doses were estimated from whole-body PET scans acquired approximately 70 and 200 min after i.v. injection of 400 MBq FET. After injection of FET, the peak of radioactivity in the blood was observed after 1.5 min, and a plateau of nearly constant radioactivity was reached at 20 min. The whole-body distribution of FET showed the highest activities in the urinary tract. All other organs exhibited only moderate FET uptake (SUV

  12. Simple automated preparation of O-[{sup 11}C]methyl-L-tyrosine for routine clinical use

    Energy Technology Data Exchange (ETDEWEB)

    Ishikawa, Yoichi [CYRIC Tohoku University, Aramaki, Aoba-ku, Sendai 980-8578 (Japan); Iwata, Ren [CYRIC Tohoku University, Aramaki, Aoba-ku, Sendai 980-8578 (Japan)]. E-mail: rencyric@cyric.tohoku.ac.jp; Furumoto, Shozo [TUBERO, Tohoku University, Sendai 980-8575 (Japan); Pascali, Claudio [National Cancer Institute, 20133 Milan (Italy); Bogni, Anna [National Cancer Institute, 20133 Milan (Italy); Kubota, Kazuo [International Medical Center, Tokyo 162-8655 (Japan); Ishiwata, Kiichi [Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0022 (Japan)

    2005-07-01

    The previously reported preparation of O-[{sup 11}C]methyl-L-tyrosine ([{sup 11}C]MT), a promising tumor imaging agent, has been now considerably simplified and automated. Main changes were the use of [{sup 11}C]methyl iodide ([{sup 11}C]MeI) in the reaction with L-tyrosine disodium and the use of solid phase extraction on commercially available cartridges instead of HPLC for the final purification. An injectable saline solution of [{sup 11}C]MT was obtained within 30 min after EOB with radiochemical yield of ca. 60% (decay-corrected, based on [{sup 11}C]MeI). Radiochemical purity was over 97%. The automated preparation was carried out using a miniature module employing manifold valves.

  13. Synthesis of O-(2-[18F]fluoroethyl)-L-tyrosine based on a cartridge purification method

    International Nuclear Information System (INIS)

    Mueller, Dirk; Klette, Ingo; Kalb, Fabrizia; Baum, Richard P.

    2011-01-01

    Introduction: O-(2-[ 18 F]fluoroethyl)-L-tyrosine (FET) is widely used as a positron emission tomography tracer for brain tumors. Usually, a high-performance liquid chromatography (HPLC) purification at the end of the two-step synthesis is applied. In this work, we report an automatic radiosynthesis of FET with a purification procedure based on standard cartridges. Methods: O-(2-[ 18 F]fluoroethyl)-L-tyrosine was prepared by [ 18 F]fluoroethylation of L-tyrosine by a two-step synthesis using a modified [ 11 C]methionine module (Nuclear Interface). In the first reaction step, we synthesized [ 18 F]fluoroethyltosylate starting from [ 18 F]fluoride. After a purification step, L-tyrosine was [ 18 F]fluoroethylated with [ 18 F]fluoroethyltosylate. The final reaction mixture was purified by means of solid phase extraction. The FET was trapped on an SCX cartridge, eluted with saline solution and trapped again on an HRX cartridge. For a second purification step, the FET was eluted from the HRX cartridge with ammonium acetate buffer and collected on two SCX cartridges followed by a washing step with water. The final product was eluted with saline solution and neutralised with 450 μl NaHCO 3 solution (8.4%). Results: The synthesis was finished after 50 min and delivered the FET in a range of 3-16 GBq. The synthesis typically yielded 41% (21 experiments) of FET (d.c.) without an HPLC purification step. The radiochemical purity ranged between 97% and 100%. Conclusion: We present a radiosynthesis of FET where the usually used HPLC purification procedure has been substituted by a purification step based on standard cartridges. This method is useful for automatic modules without an expensive HPLC purification unit and for the routine production of FET.

  14. High performance microbiological transformation of L-tyrosine to L-dopa by Yarrowia lipolytica NRRL-143

    Directory of Open Access Journals (Sweden)

    Shultz Jeffry L

    2007-08-01

    Full Text Available Abstract Background The 3,4-dihydroxy phenyl L-alanine (L-dopa is a drug of choice for Parkinson's disease, controlling changes in energy metabolism enzymes of the myocardium following neurogenic injury. Aspergillus oryzae is commonly used for L-dopa production; however, potential improvements in ease of handling, growth rate and environmental impact have led to an interest in exploiting alternative yeasts. The two important elements required for L-dopa production are intracellular tyrosinases (thus pre-grown yeast cells are required for the transformation of L-tyrosine to L-dopa and L-ascorbate, which acts as a reducing agent. Results Pre-grown cells of Yarrowia lipolytica NRRL-143 were used for the microbiological transformation of L-tyrosine to L-dopa. Different diatomite concentrations (0.5–3.0 mg/ml were added to the acidic (pH 3.5 reaction mixture. Maximum L-dopa biosynthesis (2.96 mg/ml L-dopa from 2.68 mg/ml L-tyrosine was obtained when 2.0 mg/ml diatomite was added 15 min after the start of the reaction. After optimizing reaction time (30 min, and yeast cell concentration (2.5 mg/ml, an overall 12.5 fold higher L-dopa production rate was observed when compared to the control. Significant enhancements in Yp/s, Qs and qs over the control were observed. Conclusion Diatomite (2.0 mg/ml addition 15 min after reaction commencement improved microbiological transformation of L-tyrosine to L-dopa (3.48 mg/ml; p ≤ 0.05 by Y. lipolytica NRRL-143. A 35% higher substrate conversion rate was achieved when compared to the control.

  15. Combinatorial analysis of enzymatic bottlenecks of L-tyrosine pathway by p-coumaric acid production in Saccharomyces cerevisiae.

    Science.gov (United States)

    Mao, Jiwei; Liu, Quanli; Song, Xiaofei; Wang, Hesuiyuan; Feng, Hui; Xu, Haijin; Qiao, Mingqiang

    2017-07-01

    To identify new enzymatic bottlenecks of L-tyrosine pathway for further improving the production of L-tyrosine and its derivatives. When ARO4 and ARO7 were deregulated by their feedback resistant derivatives in the host strains, the ARO2 and TYR1 genes, coding for chorismate synthase and prephenate dehydrogenase were further identified as new important rate-limiting steps. The yield of p-coumaric acid in the feedback-resistant strain overexpressing ARO2 or TYR1, was significantly increased from 6.4 to 16.2 and 15.3 mg l -1 , respectively. Subsequently, we improved the strain by combinatorial engineering of pathway genes increasing the yield of p-coumaric acid by 12.5-fold (from 1.7 to 21.3 mg l -1 ) compared with the wild-type strain. Batch cultivations revealed that p-coumaric acid production was correlated with cell growth, and the formation of by-product acetate of the best producer NK-M6 increased to 31.1 mM whereas only 19.1 mM acetate was accumulated by the wild-type strain. Combinatorial metabolic engineering provides a new strategy for further improvement of L-tyrosine or other metabolic biosynthesis pathways in S. cerevisiae.

  16. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  17. Enhancement of L-phenylalanine production by engineered Escherichia coli using phased exponential L-tyrosine feeding combined with nitrogen source optimization.

    Science.gov (United States)

    Yuan, Peipei; Cao, Weijia; Wang, Zhen; Chen, Kequan; Li, Yan; Ouyang, Pingkai

    2015-07-01

    Nitrogen source optimization combined with phased exponential L-tyrosine feeding was employed to enhance L-phenylalanine production by a tyrosine-auxotroph strain, Escherichia coli YP1617. The absence of (NH4)2SO4, the use of corn steep powder and yeast extract as composite organic nitrogen source were more suitable for cell growth and L-phenylalanine production. Moreover, the optimal initial L-tyrosine level was 0.3 g L(-1) and exponential L-tyrosine feeding slightly improved L-phenylalanine production. Nerveless, L-phenylalanine production was greatly enhanced by a strategy of phased exponential L-tyrosine feeding, where exponential feeding was started at the set specific growth rate of 0.08, 0.05, and 0.02 h(-1) after 12, 32, and 52 h, respectively. Compared with exponential L-tyrosine feeding at the set specific growth rate of 0.08 h(-1), the developed strategy obtained a 15.33% increase in L-phenylalanine production (L-phenylalanine of 56.20 g L(-1)) and a 45.28% decrease in L-tyrosine supplementation. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  18. Solvent 1H/2H isotopic effects in the reaction of the L-Tyrosine oxidation catalyzed by Tyrosinase

    International Nuclear Information System (INIS)

    Kozlowska, M.; Kanska, M.

    2006-01-01

    Tyrosinase is well known catalyst in the oxidation of L-Tyrosine to L-DOPA and following oxidation of L-DOPA to dopachinone. The aim of communication is to present the results of studies on the solvent isotopic effects (SIE) in the above reactions for the 1 H/ 2 H in the 3',5' and 2',6' substituted tyrosine. Obtained dependence of the reaction rate on the substrate concentration were applied for optimization of the kinetic parameters, k cat and k cat /K m , in the Michaelis-Menten equation. As a result - better understanding of the L-DOPA creation can be achieved

  19. Solvation thermodynamics of L-cystine, L-tyrosine, and L-leucine in aqueous-electrolyte media

    Science.gov (United States)

    Roy, Sanjay; Guin, Partha Sarathi; Mahali, Kalachand; Dolui, Bijoy Krishna

    2017-12-01

    Solubilities of L-cystine, L-tyrosine, and L-leucine in aqueous NaCl media at 298.15 K have been studied. Indispensable and related solvent parameters such as molar mass, molar volume, etc., were also determined. The results are used to evaluate the standard transfer Gibbs free energy, cavity forming enthalpy of transfer, cavity forming transfer Gibbs free energy and dipole-dipole interaction effects during the course of solvation. Various weak interactions involving solute-solvent or solvent-solvent molecules were characterized in order to find their role on the solvation of these amino acids.

  20. Aspergillus niger PA2: a novel strain for extracellular biotransformation of L-tyrosine into L-DOPA.

    Science.gov (United States)

    Agarwal, Pragati; Pareek, Nidhi; Dubey, Swati; Singh, Jyoti; Singh, R P

    2016-05-01

    L-DOPA (3,4-dihydroxyphenyl-L-alanine), an amino acid derivative is the most widely used drug of choice for the treatment of Parkinson's disease and other neurologic injuries. The present study deals with the elevated biochemical transformation of L-tyrosine to L-DOPA by Aspergillus niger PA2, a potent tyrosinase producer, isolated from decomposed food wastes. This appears to be the first report on A. niger as a notable extracellular tyrosinase producer. The extracellular tyrosinase activity produced remarkably higher levels of L-DOPA, i.e. 2.44 mg mL(-1) when the media was supplemented with 5 mg mL(-1) L-tyrosine. The optimum pH for tyrosinase production was 6.0, with the maximal L-DOPA production at the same pH. The product thus produced was analyzed by thin-layer chromatography, UV spectroscopy, high-performance liquid chromatography and Fourier transform infrared spectroscopy, that had denoted this to be L-DOPA. Kinetic parameters viz. Y p/s, Q s and Q p had further indicated the notable levels of production. Thus, Aspergillus niger PA2 could be a promising resource and may be further exploited for large-scale production of L-DOPA.

  1. A simple and sensitive fluorescent sensor for methyl parathion based on L-tyrosine methyl ester functionalized carbon dots.

    Science.gov (United States)

    Hou, Juying; Dong, Jing; Zhu, Haishuang; Teng, Xue; Ai, Shiyun; Mang, Minglin

    2015-06-15

    In this paper, a simple and sensitive fluorescent sensor for methyl parathion is developed based on L-tyrosine methyl ester functionalized carbon dots (Tyr-CDs) and tyrosinase system. The carbon dots are obtained by simple hydrothermal reaction using citric acid as carbon resource and L-tyrosine methyl ester as modification reagent. The carbon dots are characterized by transmission electron microscope, high resolution transmission electron microscopy, X-ray diffraction spectrum, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy. The carbon dots show strong and stable photoluminescence with a quantum yield of 3.8%. Tyrosinase can catalyze the oxidation of tyrosine methyl ester on the surface of carbon dots to corresponding quinone products, which can quench the fluorescence of carbon dots. When organophosphorus pesticides (OPs) are introduced in system, they can decrease the enzyme activity, thus decrease the fluorescence quenching rate. Methyl parathion, as a model of OPs, was detected. Experimental results show that the enzyme inhibition rate is proportional to the logarithm of the methyl parathion concentration in the range 1.0×10(-10)-1.0×10(-4) M with the detection limit (S/N=3) of 4.8×10(-11) M. This determination method shows a low detection limit, wide linear range, good selectivity and high reproducibility. This sensing system has been successfully used for the analysis of cabbage, milk and fruit juice samples. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Dosimetric estimation of O-(3-18F-fluoropropyl)-L-tyrosine in human based on mice biodistribution data

    International Nuclear Information System (INIS)

    Tang Ganghua; Wang Mingfang; Luo Lei; Gan Manquan; Tang Xiaolan

    2002-01-01

    Objective: To estimate the radiation absorbed doses in humans due to intravenous administration of O-(3- 18 F-fluoropropyl)-L-tyrosine (FPT) based on mice biodistribution data and appraise the security of FPT in humans. Methods: FPT was injected into mice through a tail vein. At 10, 30, 60, 120 and 180 min after injection, the mice were killed by cervical fracture and biodistribution in mice were determined. Human dosimetric estimation was performed from the biodistribution of FPT in mice and the standard MIRD method using fractional radioactivity-time curves for humans. Results: The bone in human was the organ receiving highest dose of 4.29 x 10 -3 mGy/MBq, the brain received lowest dose of 1.57 x 10 -3 mGy/MBq, and other organs received doses between 1.8 x 10 -3 and 2.4 x 10 -3 mGy/MBq. The effective dose was estimated to be 9.15 x 10 -3 mSv/MBq. These results were comparable to values reported by foreign authors on the radiation dosimetry of O-(2- 18 F-fluoroethyl)-L-tyrosine. Conclusion: Human dosimetric estimation can be performed based on mice biodistribution data. The study provides an important data for clinical safety of FPT

  3. Properties of the humic-like material arising from the photo-transformation of L-tyrosine

    Energy Technology Data Exchange (ETDEWEB)

    Berto, Silvia, E-mail: silvia.berto@unito.it [Università di Torino, Dipartimento Chimica, via P. Giuria, 7, 10125 Torino (Italy); De Laurentiis, Elisa; Tota, Tiziana; Chiavazza, Enrico; Daniele, Pier Giuseppe; Minella, Marco [Università di Torino, Dipartimento Chimica, via P. Giuria, 7, 10125 Torino (Italy); Isaia, Marco [Università di Torino, Dipartimento di Scienze della Vita e Biologia dei Sistemi, Via Accademia Albertina 13, Torino 10123 (Italy); Brigante, Marcello [Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448, F-63000 Clermont-Ferrand (France); CNRS, UMR 6296, ICCF, BP 80026, F-63177 Aubière (France); Vione, Davide, E-mail: davide.vione@unito.it [Università di Torino, Dipartimento Chimica, via P. Giuria, 7, 10125 Torino (Italy)

    2016-03-01

    The UVB photolysis of L-tyrosine yields species with fluorescence and absorption spectra that are very similar to those of humic substances. By potentiometric measurements, chemical modeling and the application of NMR, mass spectrometry and laser flash photolysis, it was possible to get insights into the structural and chemical properties of the compounds derived by the L-tyrosine phototransformation. The photolytic process follows aromatic-ring hydroxylation and dimerization. The latter is presumably linked with the photoinduced generation of tyrosyl (phenoxy-type) radicals, which have a marked tendency to dimerize and possibly oligomerize. Interestingly, photoinduced transformation gives compounds with protogenic and complexation capabilities similar to those of the humic substances that occur naturally in surface waters. This finding substantiates a new and potentially important abiotic (photolytic) pathway for the formation of humic compounds in surface-water environments. - Highlights: • Tyrosine photolysis proceeds through deamination, hydroxylation and dimerization. • Dimerization could be linked to the photoinduced formation of tyrosyl radicals. • New protogenic sites are formed by irradiation, compared to the parent amino acid. • The irradiated material has higher copper complexation capacity than tyrosine. • Humic-like substances derived from tyrosine could complex Cu in surface waters.

  4. Thermostable Cellulases: Why & How?

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Manoj [Royal DSM, San Francisco, CA (United States)

    2010-03-24

    These are a set of slides from the conference. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.

  5. Thermostable Cellulases: Why & How?

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Manoj [DSM Innovation, Incorporated, San Francisco, CA (United States)

    2010-04-19

    Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.

  6. Towards the conception of an amperometric sensor of L-tyrosine based on Hemin/PAMAM/MWCNT modified glassy carbon electrode

    International Nuclear Information System (INIS)

    Ma Qiang; Ai Shiyun; Yin Huanshun; Chen Quanpeng; Tang Tiantian

    2010-01-01

    A novel amperometric sensor was fabricated based on the immobilization of hemin onto the poly (amidoamine)/multi-walled carbon nanotube (PAMAM/MWCNT) nanocomposite film modified glassy carbon electrode (GCE). Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and ultraviolet visible (UV-vis) adsorption spectroscopy were used to investigate the possible state and electrochemical activity of the immobilized hemin. In the Hemin/PAMAM/MWCNT nanocomposite film, MWCNT layer possessed excellent inherent conductivity to enhance the electron transfer rate, while the layer of PAMAM greatly enlarged the surface average concentration of hemin (Γ) on the modified electrode. Therefore, the nanocomposite film showed enhanced electrocatalytical activity towards the oxidation of L-tyrosine. The kinetic parameters of the modified electrode were investigated. In pH 7.0 phosphate buffer solution (PBS), the sensor exhibits a wide linear range from 0.1 μM to 28.8 μM L-tyrosine with a detection limit of 0.01 μM and a high sensitivity of 0.31 μA μM -1 cm -2 . In addition, the response time of the L-tyrosine sensor is less than 5 s. The excellent performance of the sensor is largely attributed to the electro-generated high reactive oxoiron (IV) porphyrin (O = Fe IV -P) which effectively catalyzed the oxidation of L-tyrosine. A mechanism was herein proposed for the catalytic oxidation of L-tyrosine by oxoiron (IV) porphyrin complexes.

  7. O-(2-[{sup 18}F]Fluorethyl)-L-tyrosine in the diagnostics of brain tumors; O-(2-[{sup 18}F]Fluorethyl)-L-Tyrosin (FET) in der Diagnostik von Hirntumoren

    Energy Technology Data Exchange (ETDEWEB)

    Langen, K.J.; Stoffels, G. [Forschungszentrum Juelich (Germany). Inst. fuer Neurowissenschaften und Biophysik - Medizin

    2009-06-15

    Positron emission tomography (PET) using radiolabeled amino acids has shown great potential for a more accurate diagnosis of cerebral gliomas. Magnetic resonance imaging (MRI) is the investigation of choice for diagnosing cerebral glioma, but its capacity to differentiate tumor tissue from non-specific tissue changes is limited. ([{sup 18}F] Fluorethyl)-L-tyrosine (FET) is a new tracer for PET that can be produced with high efficiency and distributed on a wide clinical scale like [{sup 18}F]-Fluorodeoxyglucose (FDG). The use of FET PET allows better delineation of tumor margins and improves targeting of biopsy and radiotherapy, and planning surgery. In addition, amino acid imaging appears useful in distinguishing tumor recurrence from non-specific post-therapeutic scar tissue, predicting prognosis in low grade gliomas, and monitoring metabolic response during treatment. (orig.)

  8. Remote-controlled module-assisted synthesis of O-(2-[18F]fluoroethyl)-L-tyrosine as tumor PET tracer using two different radiochemical routes

    International Nuclear Information System (INIS)

    Wang Mingwei; Yin Duanzhi; Zhang Lan; Zhou Wei; Wang Yongxian

    2006-01-01

    The positron-emitter fluorine-18 labeled amino acid O-(2-[ 18 F]fluoroethyl)-L-tyrosine ([ 18 F]FET) has shown very promising perspectives for brain tumor diagnosis with positron emission tomography (PET). There have been two existing preparation routes of [ 18 F]FET named direct nucleophilic radiofiuorination of protected L-tyrosine and radiofiuoroallcylation of unprotected L-tyrosine, respectively. A general module was designed specifically for the routine synthesis of [ 18 F]FET, which could be suitable for the present two chemical methods with simple modifications. The fluorinated intermediates and the final product were separated and purified using solid phase extraction (SPE) on the Sep-Pak silica plus cartridge instead of the time-consuming high performance liquid chromatography (HPLC) procedures. The total synthesis time was about 50-60 rain with good radiochemical yield (about 20-40%, no-decay-corrected) and good radiochemical purity (more than 97%) for both the synthetic methods. (authors)

  9. Effects of a common worldwide drink (Beer) on L-Phenylalanine and L-Tyrosine fibrillar assemblies

    Science.gov (United States)

    Banik, Debasis; Banerjee, Pavel; Sabeehuddin, Ghazi; Sarkar, Nilmoni

    2017-11-01

    In this letter, small amount of beer [0.42-2.08% (v/v)] is employed to investigate the fibril inhibition kinetics of 1 mM L-Phenylalanine and L-Tyrosine (relevant to disease condition) using Fluorescence Lifetime imaging Microscopy (FLIM), Field Emission Scanning Electron Microscopy (FESEM) and High Resolution Transmission Electron Microscopic (HR-TEM) techniques. Our results indicate that 1.67 and 0.42% of beer is sufficient for effective breakdown of L-Phe and L-Tyr assemblies, respectively. Quantitative information about fibril inhibition is obtained from Fluorescence Correlation Spectroscopic (FCS) measurements. We have shown that the morphology of L-Phe changes to L-Tyr in presence of 2,2‧-Bipyridine-3,3‧-diol (BP(OH)2).

  10. Urinary excretion of deuterated metabolites in patients with tyrosinemia type I after oral loading with deuterated L-tyrosine

    International Nuclear Information System (INIS)

    Wadman, S.K.; Duran, M.; Ketting, D.; Bruinvis, L.; Sprang, F.J. van; Berger, R.; Smit, G.P.A.; Divry, P.; Farriaux, J.P.; Cartigny, B.

    1983-01-01

    The metabolic fate of orally given deuterated L-tyrosine, 50 mg/kg body weight, was investigated in seven patients with tyrosinemia type I in order to obtain evidence that the primary defect is at the level of fumarylacetoacetase. The absence of fumarylacetoacetase could be proved in liver biopsy specimens obtained from four patients. All patients excreted deuterated succinylacetoacetate and deuterated succinylacetone was detected in six out of seven. The total amount of these compounds was rather low; maximal 8.3% of the dose. The peak of the excretion occurred 3-6 h after loading, indicating an endogenous formation of the metabolites. All patients excreted deuterated 4-hydroxyphenyl acids, probably reflecting secondary 4-hydroxyphenylpyruvate dioxygenase deficiency connected with liver damage. 5. No evidence for other secondary routes of tyrosine metabolism was found. (Auth.)

  11. The diagnostic accuracy of detecting malignant transformation of low-grade glioma using O-(2-[F]fluoroethyl)-l-tyrosine positron emission tomography

    DEFF Research Database (Denmark)

    Bashir, Asma; Brennum, Jannick; Broholm, Helle

    2018-01-01

    OBJECTIVE The diagnostic accuracy of O-(2-[18F]fluoroethyl)-l-tyrosine (FET) PET scanning in detecting the malignant transformation of low-grade gliomas (LGGs) is controversial. In this study, the authors retrospectively assessed the diagnostic potential of FET PET in patients with MRI-suspected ...

  12. Attainment of unstable β nucleation of glycine in presence of L-tyrosine and its analytical interpretation-A combined approach

    Science.gov (United States)

    Renuka Devi, K.; Srinivasan, K.

    2015-05-01

    The ability of L-tyrosine molecules to act as a template and to facilitate the nucleation of unstable β polymorph in the solution has been revealed through in-situ nucleation study. This nucleation of β occurs along with the existing α nucleation at the critical concentration of additive in the solution. The presence of L-tyrosine molecules lowers the inherent barrier that exists for β nucleation in the solution. No nucleation of γ was observed over the entire range of concentrations studied. The molecular recognition capability and stereo selective inhibitory action of the added L-tyrosine molecules towards glycine molecule have been successfully revealed in terms of habit modification observed in the nucleated polymorphs. In the case of α polymorph, L-tyrosine induces a change in the morphology along the enantiopolar -b direction while in the case of β polymorph, habit modification from needle to plate like structure is observed. With the increase in time span, solution mediated phase transformation from β to α polymorph has been observed in the solution. Analytically the nucleation parameters of α and β polymorphs were estimated based on Classical Nucleation Theory. Form of crystallization of the nucleated polymorphs of glycine was confirmed by a powder x-ray diffraction analysis.

  13. Human dosimetric estimation of O-(2-18F-fluoroethyl)-L-tyrosine based on mice biodistribution data

    International Nuclear Information System (INIS)

    Tang Ganghua; Wang Mingfang; Luo Lei; Gan Manquan; Tang Xiaolan

    2004-01-01

    To estimate the human radiation absorbed doses of O-(2- 18 F-fluoroethyl)-L-tyrosine (FET), mice are considered as model. FET is injected into mice through a tail vein. At 10, 30, 60, 120 and 180 min after injection, the mice are killed by cervical fracture and the biodistribution in mice are determined. Human dosimetric estimation is performed from the biodistribution of FET in mice and the standard Medical Internal Radiation Dose (MIRD) method using time-fractional radioactivity curves for humans. The bone in human is the organ receiving highest dose of 4.78 pGy/Bq, the brain and the whole body receive the lowest dose of 1.6 pGy/Bq, and other organs receive doses between 1.6 and 3.5 pGy/Bq. The effective dose is estimated to be 9.0 pSv/Bq. The data show that a 370 MBq injection of FET leads to an estimated effective dose of 3.3 mSv, which is in the range of routine nuclear medicine investigations. The potential radiation risks associated with this study are well within accepted limits

  14. Biotransformation of L-tyrosine to Dopamine by a Calcium Alginate Immobilized Mutant Strain of Aspergillus oryzae.

    Science.gov (United States)

    Ali, Sikander; Nawaz, Wajeeha

    2016-08-01

    The present research work is concerned with the biotransformation of L-tyrosine to dopamine (DA) by calcium alginate entrapped conidiospores of a mutant strain of Aspergillus oryzae. Different strains of A. oryzae were isolated from soil. Out of 13 isolated strains, isolate-2 (I-2) was found to be a better DA producer. The wild-type I-2 was chemically improved by treating it with different concentrations of ethyl methyl sulfonate (EMS). Among seven mutant variants, EMS-6 exhibiting maximal DA activity of 43 μg/ml was selected. The strain was further exposed with L-cysteine HCl to make it resistant against diversion and environmental stress. The conidiospores of selected mutant variant A. oryzae EMS-6 strain were entrapped in calcium alginate beads. Different parameters for immobilization were investigated. The activity was further improved from 44 to 62 μg/ml under optimized conditions (1.5 % sodium alginate, 2 ml inoculum, and 2 mm bead size). The best resistant mutant variable exhibited over threefold increase in DA activity (62 μg/ml) than did wild-type I-2 (21 μg/ml) in the reaction mixture. From the results presented in the study, it was observed that high titers of DA activity in vitro could effectively be achieved by the EMS-induced mutagenesis of filamentous fungus culture used.

  15. Fully automated one-pot radiosynthesis of O-(2-[{sup 18}F]fluoroethyl)-L-tyrosine on the TracerLab FX{sub FN} module

    Energy Technology Data Exchange (ETDEWEB)

    Bourdier, Thomas, E-mail: bts@ansto.gov.au [LifeSciences, Australian Nuclear Science and Technology Organisation, Locked Bag 2001, Kirrawee DC NSW 2232, Sydney (Australia); Greguric, Ivan [LifeSciences, Australian Nuclear Science and Technology Organisation, Locked Bag 2001, Kirrawee DC NSW 2232, Sydney (Australia); Roselt, Peter [Centre for Molecular Imaging, Peter MacCallum Cancer Centre, 12 St Andrew' s Place, East Melbourne, VIC, 3002 (Australia); Jackson, Tim; Faragalla, Jane; Katsifis, Andrew [LifeSciences, Australian Nuclear Science and Technology Organisation, Locked Bag 2001, Kirrawee DC NSW 2232, Sydney (Australia)

    2011-07-15

    Introduction: An efficient fully automated method for the radiosynthesis of enantiomerically pure O-(2-[{sup 18}F]fluoroethyl)-L-tyrosine ([{sup 18}F]FET) using the GE TracerLab FX{sub FN} synthesis module via the O-(2-tosyloxyethyl)-N-trityl-L-tyrosine tert-butylester precursor has been developed. Methods: The radiolabelling of [{sup 18}F]FET involved a classical [{sup 18}F]fluoride nucleophilic substitution performed in acetonitrile using potassium carbonate and Kryptofix 222, followed by acid hydrolysis using 2N hydrochloric acid. Results: [{sup 18}F]FET was produced in 35{+-}5% (n=22) yield non-decay-corrected (55{+-}5% decay-corrected) and with radiochemical and enantiomeric purity of >99% with a specific activity of >90 GBq/{mu}mol after 63 min of radiosynthesis including HPLC purification and formulation. Conclusion: The automated radiosynthesis provides high and reproducible yields suitable for routine clinical use.

  16. L-tyrosine induces the production of a pyomelanin-like pigment by the parasitic yeast-form of Histoplasma capsulatum.

    Science.gov (United States)

    Almeida-Paes, Rodrigo; Almeida-Silva, Fernando; Pinto, Gabriela Costa Maia; Almeida, Marcos de Abreu; Muniz, Mauro de Medeiros; Pizzini, Claudia Vera; Gerfen, Gary J; Nosanchuk, Joshua Daniel; Zancopé-Oliveira, Rosely Maria

    2018-06-01

    Melanization of Histoplasma capsulatum remains poorly described, particularly in regards to the forms of melanin produced. In the present study, 30 clinical and environmental H. capsulatum strains were grown in culture media with or without L-tyrosine under conditions that produced either mycelial or yeast forms. Mycelial cultures were not melanized under the studied conditions. However, all strains cultivated under yeast conditions produced a brownish to black soluble pigment compatible with pyomelanin when grew in presence of L-tyrosine. Sulcotrione inhibited pigment production in yeast cultures, strengthening the hyphothesis that H. capsulatum yeast forms produce pyomelanin. Since pyomelanin is produced by the fungal parasitic form, this pigment may be involved in H. capsulatum virulence.

  17. Dual-time-point O-(2-[18F]fluoroethyl)-L-tyrosine PET for grading of cerebral gliomas

    International Nuclear Information System (INIS)

    Lohmann, Philipp; Herzog, Hans; Rota Kops, Elena; Stoffels, Gabriele; Judov, Natalie; Filss, Christian; Tellmann, Lutz; Galldiks, Norbert; Weiss, Carolin; Sabel, Michael; Coenen, Heinz Hubert; Shah, Nadim Jon; Langen, Karl-Josef

    2015-01-01

    We aimed to evaluate the diagnostic potential of dual-time-point imaging with positron emission tomography (PET) using O-(2-[ 18 F]fluoroethyl)-L-tyrosine ( 18 F-FET) for non-invasive grading of cerebral gliomas compared with a dynamic approach. Thirty-six patients with histologically confirmed cerebral gliomas (21 primary, 15 recurrent; 24 high-grade, 12 low-grade) underwent dynamic PET from 0 to 50 min post-injection (p.i.) of 18 F-FET, and additionally from 70 to 90 min p.i. Mean tumour-to-brain ratios (TBR mean ) of 18 F-FET uptake were determined in early (20-40 min p.i.) and late (70-90 min p.i.) examinations. Time-activity curves (TAC) of the tumours from 0 to 50 min after injection were assigned to different patterns. The diagnostic accuracy of changes of 18 F-FET uptake between early and late examinations for tumour grading was compared to that of curve pattern analysis from 0 to 50 min p.i. of 18 F-FET. The diagnostic accuracy of changes of the TBR mean of 18 F-FET PET uptake between early and late examinations for the identification of HGG was 81 % (sensitivity 83 %; specificity 75 %; cutoff - 8 %; p < 0.001), and 83 % for curve pattern analysis (sensitivity 88 %; specificity 75 %; p < 0.001). Dual-time-point imaging of 18 F-FET uptake in gliomas achieves diagnostic accuracy for tumour grading that is similar to the more time-consuming dynamic data acquisition protocol. (orig.)

  18. Dual-time-point O-(2-[{sup 18}F]fluoroethyl)-L-tyrosine PET for grading of cerebral gliomas

    Energy Technology Data Exchange (ETDEWEB)

    Lohmann, Philipp; Herzog, Hans; Rota Kops, Elena; Stoffels, Gabriele; Judov, Natalie; Filss, Christian; Tellmann, Lutz [Forschungszentrum Juelich, Institute of Neuroscience and Medicine, Juelich (Germany); Galldiks, Norbert [Forschungszentrum Juelich, Institute of Neuroscience and Medicine, Juelich (Germany); University of Cologne, Department of Neurology, Cologne (Germany); Weiss, Carolin [University of Cologne, Department of Neurosurgery, Cologne (Germany); Sabel, Michael [Heinrich-Heine University, Department of Neurosurgery, Duesseldorf (Germany); Coenen, Heinz Hubert [Forschungszentrum Juelich, Institute of Neuroscience and Medicine, Juelich (Germany); Juelich-Aachen Research Alliance (JARA) - Section JARA-Brain, Juelich (Germany); Shah, Nadim Jon [Forschungszentrum Juelich, Institute of Neuroscience and Medicine, Juelich (Germany); Juelich-Aachen Research Alliance (JARA) - Section JARA-Brain, Juelich (Germany); RWTH Aachen University Hospital, Department of Neurology, Aachen (Germany); Langen, Karl-Josef [Forschungszentrum Juelich, Institute of Neuroscience and Medicine, Juelich (Germany); RWTH Aachen University Hospital, Department of Nuclear Medicine, Aachen (Germany); Juelich-Aachen Research Alliance (JARA) - Section JARA-Brain, Juelich (Germany)

    2015-10-15

    We aimed to evaluate the diagnostic potential of dual-time-point imaging with positron emission tomography (PET) using O-(2-[{sup 18}F]fluoroethyl)-L-tyrosine ({sup 18}F-FET) for non-invasive grading of cerebral gliomas compared with a dynamic approach. Thirty-six patients with histologically confirmed cerebral gliomas (21 primary, 15 recurrent; 24 high-grade, 12 low-grade) underwent dynamic PET from 0 to 50 min post-injection (p.i.) of {sup 18}F-FET, and additionally from 70 to 90 min p.i. Mean tumour-to-brain ratios (TBR{sub mean}) of {sup 18}F-FET uptake were determined in early (20-40 min p.i.) and late (70-90 min p.i.) examinations. Time-activity curves (TAC) of the tumours from 0 to 50 min after injection were assigned to different patterns. The diagnostic accuracy of changes of {sup 18}F-FET uptake between early and late examinations for tumour grading was compared to that of curve pattern analysis from 0 to 50 min p.i. of {sup 18}F-FET. The diagnostic accuracy of changes of the TBR{sub mean} of {sup 18}F-FET PET uptake between early and late examinations for the identification of HGG was 81 % (sensitivity 83 %; specificity 75 %; cutoff - 8 %; p < 0.001), and 83 % for curve pattern analysis (sensitivity 88 %; specificity 75 %; p < 0.001). Dual-time-point imaging of {sup 18}F-FET uptake in gliomas achieves diagnostic accuracy for tumour grading that is similar to the more time-consuming dynamic data acquisition protocol. (orig.)

  19. Diagnosis of pseudoprogression in patients with glioblastoma using O-(2-[{sup 18}F]fluoroethyl)-l-tyrosine PET

    Energy Technology Data Exchange (ETDEWEB)

    Galldiks, Norbert [University of Cologne, Department of Neurology, Cologne (Germany); Forschungszentrum Juelich, Institute of Neuroscience and Medicine, Juelich (Germany); University of Cologne, Center of Integrated Oncology (CIO), Cologne (Germany); Dunkl, Veronika; Fink, Gereon R. [University of Cologne, Department of Neurology, Cologne (Germany); Forschungszentrum Juelich, Institute of Neuroscience and Medicine, Juelich (Germany); Stoffels, Gabriele [Forschungszentrum Juelich, Institute of Neuroscience and Medicine, Juelich (Germany); Hutterer, Markus; Hau, Peter [University of Regensburg, Department of Neurology and Wilhelm Sander-NeuroOncology Unit, Regensburg (Germany); Rapp, Marion; Sabel, Michael [Heinrich Heine University Duesseldorf, Department of Neurosurgery, Duesseldorf (Germany); Reifenberger, Guido [Heinrich Heine University Duesseldorf, Department of Neuropathology, Duesseldorf (Germany); Kebir, Sied [University of Bonn, Department of Neurology, Bonn (Germany); Dorn, Franziska [University of Cologne, Department of Neuroradiology, Cologne (Germany); Blau, Tobias [University of Cologne, Department of Neuropathology, Cologne (Germany); Herrlinger, Ulrich [University of Cologne, Center of Integrated Oncology (CIO), Cologne (Germany); University of Bonn, Department of Neurology, Bonn (Germany); Ruge, Maximilian I. [University of Cologne, Center of Integrated Oncology (CIO), Cologne (Germany); University of Cologne, Department of Stereotaxy and Functional Neurosurgery, Cologne (Germany); Kocher, Martin [University of Cologne, Center of Integrated Oncology (CIO), Cologne (Germany); University of Cologne, Department of Radiation Oncology, Cologne (Germany); Goldbrunner, Roland [University of Cologne, Center of Integrated Oncology (CIO), Cologne (Germany); University of Cologne, Department of Neurosurgery, Cologne (Germany); Drzezga, Alexander; Schmidt, Matthias [University of Cologne, Department of Nuclear Medicine, Cologne (Germany); Langen, Karl-Josef [Forschungszentrum Juelich, Institute of Neuroscience and Medicine, Juelich (Germany); University of Aachen, Department of Nuclear Medicine, Aachen (Germany)

    2015-04-01

    The follow-up of glioblastoma patients after radiochemotherapy with conventional MRI can be difficult since reactive alterations to the blood-brain barrier with contrast enhancement may mimic tumour progression (i.e. pseudoprogression, PsP). The aim of this study was to assess the clinical value of O-(2-{sup 18}F-fluoroethyl)-l-tyrosine ({sup 18}F-FET) PET in the differentiation of PsP and early tumour progression (EP) after radiochemotherapy of glioblastoma. A group of 22 glioblastoma patients with new contrast-enhancing lesions or lesions showing increased enhancement (>25 %) on standard MRI within the first 12 weeks after completion of radiochemotherapy with concomitant temozolomide (median 7 weeks) were additionally examined using amino acid PET with {sup 18}F-FET. Maximum and mean tumour-to-brain ratios (TBR{sub max}, TBR{sub mean}) were determined. {sup 18}F-FET uptake kinetic parameters (i.e. patterns of time-activity curves, TAC) were also evaluated. Classification as PsP or EP was based on the clinical course (no treatment change at least for 6 months), follow-up MR imaging and/or histopathological findings. Imaging results were also related to overall survival (OS). PsP was confirmed in 11 of the 22 patients. In patients with PsP, {sup 18}F-FET uptake was significantly lower than in patients with EP (TBR{sub max} 1.9 ± 0.4 vs. 2.8 ± 0.5, TBR{sub mean} 1.8 ± 0.2 vs. 2.3 ± 0.3; both P < 0.001) and presence of MGMT promoter methylation was significantly more frequent (P = 0.05). Furthermore, a TAC type II or III was more frequently present in patients with EP (P = 0.04). Receiver operating characteristic analysis showed that the optimal {sup 18}F-FET TBR{sub max} cut-off value for identifying PsP was 2.3 (sensitivity 100 %, specificity 91 %, accuracy 96 %, AUC 0.94 ± 0.06; P < 0.001). Univariate survival analysis showed that a TBR{sub max} <2.3 predicted a significantly longer OS (median OS 23 vs. 12 months; P = 0.046). {sup 18}F-FET PET may facilitate

  20. L-Tyrosine availability affects basal and stimulated catecholamine indices in prefrontal cortex and striatum of the rat.

    Science.gov (United States)

    Brodnik, Zachary D; Double, Manda; España, Rodrigo A; Jaskiw, George E

    2017-09-01

    We previously found that L-tyrosine (L-TYR) but not D-TYR administered by reverse dialysis elevated catecholamine synthesis in vivo in medial prefrontal cortex (MPFC) and striatum of the rat (Brodnik et al., 2012). We now report L-TYR effects on extracellular levels of catecholamines and their metabolites. In MPFC, reverse dialysis of L-TYR elevated in vivo levels of dihydroxyphenylacetic acid (DOPAC) (L-TYR 250-1000 μM), homovanillic acid (HVA) (L-TYR 1000 μM) and 3-methoxy-4-hydroxyphenylglycol (MHPG) (L-TYR 500-1000 μM). In striatum L-TYR 250 μM elevated DOPAC. We also examined L-TYR effects on extracellular dopamine (DA) and norepinephrine (NE) levels during two 30 min pulses (P2 and P1) of K+ (37.5 mM) separated by t = 2.0 h. L-TYR significantly elevated the ratio P2/P1 for DA (L-TYR 125 μM) and NE (L-TYR 125-250 μM) in MPFC but lowered P2/P1 for DA (L-TYR 250 μM) in striatum. Finally, we measured DA levels in brain slices using ex-vivo voltammetry. Perfusion with L-TYR (12.5-50 μM) dose-dependently elevated stimulated DA levels in striatum. In all the above studies, D-TYR had no effect. We conclude that acute increases within the physiological range of L-TYR levels can increase catecholamine metabolism and efflux in MPFC and striatum. Chronically, such repeated increases in L-TYR availability could induce adaptive changes in catecholamine transmission while amplifying the metabolic cost of catecholamine synthesis and degradation. This has implications for neuropsychiatric conditions in which neurotoxicity and/or disordered L-TYR transport have been implicated. Published by Elsevier Ltd.

  1. Optimization of medium composition for thermostable protease ...

    African Journals Online (AJOL)

    SERVER

    2008-04-17

    Apr 17, 2008 ... Optimization of the fermentation medium for maximization of thermostable neutral protease production by Bacillus sp. ..... Each contour curve represented an infinite number of combinations of two ..... Production in sea-water of.

  2. Purification and Characterization of Thermostable Cellulase from ...

    African Journals Online (AJOL)

    Available online at http://www.tjpr.org ... Methods: Molecular community structure of the newly selected thermophilic bacterial ... Keywords: Thermostable cellulase, Sugarcane bagasse, Purification, Characterization, Hot spring ... Currently, one.

  3. Conformation of L-Tyrosine Studied by Fluorescence-Detected UV-UV and IR-UV Double-Resonance Spectroscopy

    OpenAIRE

    Inokuchi, Yoshiya; Kobayashi, Yusuke; Ito, Takafumi; Ebata, Takayuki

    2007-01-01

    The laser-induced fluorescence spectrum of jet-cooled L-tyrosine exhibits more than 20 vibronic bands in the 35450-35750 cm-1 region. We attribute these bands to eight conformers by using results of UV-UV hole-burning spectroscopy. These isomers are classified into four groups; each group consists of two rotational isomers that have a similar side-chain conformation but different orientations of the phenolic OH. The splitting of band origins of rotational isomers is 31, 21, 5, and 0 cm-1 for ...

  4. Human phenylalanine hydroxylase is activated by H2O2: a novel mechanism for increasing the L-tyrosine supply for melanogenesis in melanocytes

    International Nuclear Information System (INIS)

    Schallreuter, Karin U.; Wazir, Umar; Kothari, Sonal; Gibbons, Nicholas C.J.; Moore, Jeremy; Wood, John M.

    2004-01-01

    Epidermal phenylalanine hydroxylase (PAH) produces L-tyrosine from the essential amino acid L-phenylalanine supporting melanogenesis in human melanocytes. Those PAH activities increase linearly in the different skin phototypes I-VI (Fitzpatrick classification) and also increase up to 24 h after UVB light with only one minimal erythemal dose. Since UVB generates also H 2 O 2 , we here asked the question whether this reactive oxygen species could influence the activity of pure recombinant human PAH. Under saturating conditions with the substrate L-phenylalanine (1 x 10 -3 M), the V max for enzyme activity increased 4-fold by H 2 O 2 (>2.0 x 10 -3 M). Lineweaver-Burk analysis identified a mixed activation mechanism involving both the regulatory and catalytic domains of PAH. Hyperchem molecular modelling and Deep View analysis support oxidation of the single Trp 120 residue to 5-OH-Trp 120 by H 2 O 2 causing a conformational change in the regulatory domain. PAH was still activated by H 2 O 2 in the presence of the electron donor/cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin despite slow oxidation of this cofactor. In vivo FT-Raman spectroscopy confirmed decreased epidermal phenylalanine in association with increased tyrosine after UVB exposure. Hence, generation of H 2 O 2 by UVB can activate epidermal PAH leading to an increased L-tyrosine pool for melanogenesis

  5. Unsupervised consensus cluster analysis of [18F]-fluoroethyl-L-tyrosine positron emission tomography identified textural features for the diagnosis of pseudoprogression in high-grade glioma.

    Science.gov (United States)

    Kebir, Sied; Khurshid, Zain; Gaertner, Florian C; Essler, Markus; Hattingen, Elke; Fimmers, Rolf; Scheffler, Björn; Herrlinger, Ulrich; Bundschuh, Ralph A; Glas, Martin

    2017-01-31

    Timely detection of pseudoprogression (PSP) is crucial for the management of patients with high-grade glioma (HGG) but remains difficult. Textural features of O-(2-[18F]fluoroethyl)-L-tyrosine positron emission tomography (FET-PET) mirror tumor uptake heterogeneity; some of them may be associated with tumor progression. Fourteen patients with HGG and suspected of PSP underwent FET-PET imaging. A set of 19 conventional and textural FET-PET features were evaluated and subjected to unsupervised consensus clustering. The final diagnosis of true progression vs. PSP was based on follow-up MRI using RANO criteria. Three robust clusters have been identified based on 10 predominantly textural FET-PET features. None of the patients with PSP fell into cluster 2, which was associated with high values for textural FET-PET markers of uptake heterogeneity. Three out of 4 patients with PSP were assigned to cluster 3 that was largely associated with low values of textural FET-PET features. By comparison, tumor-to-normal brain ratio (TNRmax) at the optimal cutoff 2.1 was less predictive of PSP (negative predictive value 57% for detecting true progression, p=0.07 vs. 75% with cluster 3, p=0.04). Clustering based on textural O-(2-[18F]fluoroethyl)-L-tyrosine PET features may provide valuable information in assessing the elusive phenomenon of pseudoprogression.

  6. Functional analysis of thermostable proteins involved in carbohydrate metabolism

    NARCIS (Netherlands)

    Akerboom, A.P.

    2007-01-01

    Thermostable proteins can resist temperature stress whilst keeping their integrity and functionality. In many cases, thermostable proteins originate from hyperthermophilic microorganisms that thrive in extreme environments. These systems are generally located close to geothermal (volcanic) activity,

  7. Development of a new paper based nano-biosensor using the co-catalytic effect of tyrosinase from banana peel tissue (Musa Cavendish) and functionalized silica nanoparticles for voltammetric determination of l-tyrosine.

    Science.gov (United States)

    Rahimi-Mohseni, Mohadeseh; Raoof, Jahan Bakhsh; Ojani, Reza; Aghajanzadeh, Tahereh A; Bagheri Hashkavayi, Ayemeh

    2018-07-01

    In this paper, a new and facile method for the electrochemical determination of l-tyrosine was designed. First, 3-mercaptopropyl trimethoxysilane-functionalized silica nanoparticles were added to a paper disc. Then, the banana peel tissue and the mediator potassium hexacyanoferrate were dropped onto the paper, respectively. The modified paper disc was placed on the top of the graphite screen printed electrode and electrochemical characterization of this biosensor was studied by cyclic voltammetry and electrochemical impedance spectroscopy methods. The effective parameters like pH, banana peel tissue percentage, and the amount of mediator loading were optimized. l-tyrosine measurements were done by differential pulse voltammetry with a little sample (3 μL) for analysis. The biosensor showed a linear response for l-tyrosine in the wide concentration range of 0.05-600 μM and a low detection limit about 0.02 μM because of the co-catalytic effect of enzyme and nanoparticles. The stability of the biosensor and its selectivity were evaluated. This biosensor was applied for the voltammetric determination of l-tyrosine in the blood plasma sample. The results of the practical application study were comparable with the standard method (HPLC). In conclusion, a simple, inexpensive, rapid, sensitive and selective technique was successfully applied to the l-tyrosine analysis of the little samples. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. Schiff bases derived from L-Tyrosine L-Tryptophan and their Cu(II) chelates as effective means for preventive-treatment of radiation injuries

    International Nuclear Information System (INIS)

    Malakyan, M.H.; Bajinyan, S.A.; Matosyan, V.H.; Tonoyan, V.J.; Babayan, K.N.; Boyajyan, A.S.; Yeghiazaryan, D.E.; Vardevanyan, L.A.; Sorenson, J.R.J.

    2008-01-01

    Full text: Study on essential metallo element chelates as radioprotectors presents a promising direction in a search for and development of novel anti-radiation agents and offers a new approach to overcome the pathological effects of ionizing radiation. The key idea elucidating the radioprotective effects of metallo element-containing chelates of amino acid derivatives is their role in stimulation of de novo synthesis of metallo element-dependent enzymes required for recovery of hemopoietic activity and immuno competency lost as a consequence of radiation damage. Aimed to develop novel anti-radiation remedies of less toxicity and high efficacy, Schiff bases derived from L-Tyrosine and L-Tryptophan and their Cu(II) chelates were synthesized. In experiments in vitro and in vivo biological and pharmacological properties of the mentioned Schiff Bases and their copper complexes are under study. According to the results obtained, L-Tyrosinate and L-Tryptophanate Schiff bases are low toxic compounds with a weak antioxidant activity and exert radioprotective effects in case of animal X-ray irradiation at a dose level equal or less than LD 50/30 . Unlike Schiff Bases, their appropriate Cu(II) chelates possess high anti radical/antioxidant activity and manifest expressed radio-protective action at LD 100/30 dose of ionizing radiation. Anti-radiation effects of amino acid Schiff bases and their metallo chelates are manifested in case of both subcutaneous and oral single administration to the animal organism at 10, 20, or 40 mg/kg 1, 3, 6, or 24 hours prior to radiation exposure. Conclusions are drawn basing on determinations of survival and average life-span indices of irradiated animals, as well as on studies for their hematological, biochemical, immunological, biophysical indices. It is revealed that on the background of preliminary administration of the compounds studied to the animal organism the characteristics of DNA are significantly improved, the immune status

  9. Thermostable Carbonic Anhydrases in Biotechnological Applications

    Directory of Open Access Journals (Sweden)

    Anna Di Fiore

    2015-07-01

    Full Text Available Carbonic anhydrases are ubiquitous metallo-enzymes which catalyze the reversible hydration of carbon dioxide in bicarbonate ions and protons. Recent years have seen an increasing interest in the utilization of these enzymes in CO2 capture and storage processes. However, since this use is greatly limited by the harsh conditions required in these processes, the employment of thermostable enzymes, both those isolated by thermophilic organisms and those obtained by protein engineering techniques, represents an interesting possibility. In this review we will provide an extensive description of the thermostable carbonic anhydrases so far reported and the main processes in which these enzymes have found an application.

  10. Structure-activity relationships of N-beta-phenylpropionyl-L-tyrosine and its derivatives on the inhibition of an identifiable giant neurone of an African giant snail (Achatina fulica Férussac).

    Science.gov (United States)

    Ariyoshi, Y.; Takeuchi, H.

    1982-01-01

    1 Inhibitory effects of N-beta-phenylpropionyl-L-tyrosine, N-beta-phenylpropionyl-L-tryptophan and their derivatives on an identifiable giant neurone, TAN (tonically autoactive neurone) of an African giant snail (Achatina fulica Férussac) were examined in an attempt to elucidate which structural features are necessary to produce the effect. 2 Of the compounds examined, N-beta-cyclohexylpropionyl-L-tyrosine showed the strongest effect. Its critical concentration (c.c.) was 3 X 10(-8)-10(-7)M, about ten times lower than that of N-beta-phenylpropionyl-L-tyrosine (c.c., 3 X 10(-7)-10(-6)M). N-beta-cyclohexylpropionyl-L-tryptophan (c.c., 10(-6)M) had an effect almost similar to that of N-beta-phenylpropionyl-L-tryptophan (c.c., 10(-6)M). 3 N-beta-Phenylpropionyl-N-methyl-L-tyrosine had no effect at a high concentration. 4 Effects of N-beta-phenylpropionyl-L-tyrosine amide (c.c., 3 X 10(-7)-10(-6)M) and N-beta-phenylpropionyl-L-tryptophan amide (c.c., 10(-6)M) were very similar to those of N-beta-phenylpropionyl-L-tyrosine and N-beta-phenylpropionyl-L-tryptophan respectively. 5 N-beta-Phenylpropionyl-p-amino-L-phenylalanine (c.c., 3 X 10(-5)-10(-4)M) and N-beta-phenylpropionyl-p-chloro-L-phenylalanine (c.c., 10(-4)M) had only a weak effect. 6 It is proposed that the structural features producing the effect are as follows: the active compound has a phenyl or a cyclohexyl group (hydrophobic binding group), after a suitable distance a peptide bond (proton donor and proton acceptor), adjacently a carbonyl group (proton acceptor), and a phenolic hydroxyl or an indolyl imino group (proton donor) in the molecule. PMID:7150871

  11. The antimicrobial efficacy of sustained release silver–carbene complex-loaded l-tyrosine polyphosphate nanoparticles: Characterization, in vitro and in vivo studies

    Science.gov (United States)

    Hindi, Khadijah M.; Ditto, Andrew J.; Panzner, Matthew J.; Medvetz, Douglas A.; Han, Daniel S.; Hovis, Christine E.; Hilliard, Julia K.; Taylor, Jane B.; Yun, Yang H.; Cannon, Carolyn L.; Youngs, Wiley J.

    2009-01-01

    The pressing need to treat multi-drug resistant bacteria in the chronically infected lungs of cystic fibrosis (CF) patients has given rise to novel nebulized antimicrobials. We have synthesized a silver–carbene complex (SCC10) active against a variety of bacterial strains associated with CF and chronic lung infections. Our studies have demonstrated that SCC10-loaded into l-tyrosine polyphosphate nanoparticles (LTP NPs) exhibits excellent antimicrobial activity in vitro and in vivo against the CF relevant bacteria Pseudomonas aeruginosa. Encapsulation of SCC10 in LTP NPs provides sustained release of the antimicrobial over the course of several days translating into efficacious results in vivo with only two administered doses over a 72 h period. PMID:19395021

  12. L-tyrosine immobilized on multiwalled carbon nanotubes: a new substrate for thallium separation and speciation using stabilized temperature platform furnace-electrothermal atomic absorption spectrometry.

    Science.gov (United States)

    Pacheco, Pablo H; Gil, Raúl A; Smichowski, Patricia; Polla, Griselda; Martinez, Luis D

    2009-12-10

    An approach for the separation and determination of inorganic thallium species is described. A new sorbent, L-tyrosine-carbon nanotubes (L-tyr-CNTs), was used and applied to the analysis of tap water samples. At pH 5.0, L-tyr was selective only towards Tl(III), while total thallium was determined directly by stabilized temperature platform furnace-electrothermal atomic absorption spectrometry (STPF-ETAAS). The Tl(III) specie, which was retained by L-tyrosine, was quantitatively eluted from the column with 10% of nitric acid. An on-line breakthrough curve was used to determine the column capacity, which resulted to be 9.00 micromol of Tl(III) g(-1) of L-tyr-CNTs with a molar ratio of 0.14 (moles of Tl bound to moles of L-tyr at pH 5). Transient peak areas revealed that Tl stripping from the column occurred instantaneously. Effects of sample flow rate, concentration and flow rate of the eluent, and interfering ions on the recovery of the analyte were systematically investigated. The detection limit for the determination of total thallium (3sigma) by STPF-ETAAS was 150 ng L(-1). The detection limit (3sigma) for Tl(III) employing the separation system was 3 ng L(-1), with an enrichment factor of 40. The precision of the method expressed as the relative standard deviation (RSD) resulted to be 3.4%. The proposed method was applied to the speciation and determination of inorganic thallium in tap water samples. The found concentrations were in the range of 0.88-0.91 microg L(-1) of Tl(III), and 3.69-3.91 microg L(-1) of total thallium.

  13. L-Tyrosine immobilized on multiwalled carbon nanotubes: A new substrate for thallium separation and speciation using stabilized temperature platform furnace-electrothermal atomic absorption spectrometry

    International Nuclear Information System (INIS)

    Pacheco, Pablo H.; Gil, Raul A.; Smichowski, Patricia; Polla, Griselda; Martinez, Luis D.

    2009-01-01

    An approach for the separation and determination of inorganic thallium species is described. A new sorbent, L-tyrosine-carbon nanotubes (L-tyr-CNTs), was used and applied to the analysis of tap water samples. At pH 5.0, L-tyr was selective only towards Tl(III), while total thallium was determined directly by stabilized temperature platform furnace-electrothermal atomic absorption spectrometry (STPF-ETAAS). The Tl(III) specie, which was retained by L-tyrosine, was quantitatively eluted from the column with 10% of nitric acid. An on-line breakthrough curve was used to determine the column capacity, which resulted to be 9.00 μmol of Tl(III) g -1 of L-tyr-CNTs with a molar ratio of 0.14 (moles of Tl bound to moles of L-tyr at pH 5). Transient peak areas revealed that Tl stripping from the column occurred instantaneously. Effects of sample flow rate, concentration and flow rate of the eluent, and interfering ions on the recovery of the analyte were systematically investigated. The detection limit for the determination of total thallium (3σ) by STPF-ETAAS was 150 ng L -1 . The detection limit (3σ) for Tl(III) employing the separation system was 3 ng L -1 , with an enrichment factor of 40. The precision of the method expressed as the relative standard deviation (RSD) resulted to be 3.4%. The proposed method was applied to the speciation and determination of inorganic thallium in tap water samples. The found concentrations were in the range of 0.88-0.91 μg L -1 of Tl(III), and 3.69-3.91 μg L -1 of total thallium.

  14. Thermostable crude endoglucanase produced by Aspergillus ...

    African Journals Online (AJOL)

    Cellulases are used in many industries worldwide and there is an ever increasing need to isolate, produce or develop thermostable cellulases. Manipulation of fermentation techniques in order to obtain desirable product(s) can be one line of action. In this study Aspergillus fumigatus was grown on chopped wheat straw in a ...

  15. Purification and Characterization of Thermostable Cellulase from ...

    African Journals Online (AJOL)

    The thermostable CMCase was purified with ion-exchange and gel filtration chromatography. Results: ... Conclusion: Due to its high temperature stability, the purified XM70-CMCase may be useful for industrial application such as biofuel, animal feed industry, paper industry and clarification of fruit juices. Keywords: ...

  16. Evaluation of thermostable enzymes for bioethanol processing

    DEFF Research Database (Denmark)

    Skovgaard, Pernille Anastasia

    of fermentable sugars (glucose) as cellulose is tightly linked to hemicellulose and lignin. Lignocellulose is disrupted during pretreatment, but to degrade cellulose to single sugars, lignocellulolytic enzymes such as cellulases and hemicellulases are needed. Lignocellulolytic enzymes are costly...... for the ioethanol production, but the expenses can be reduced by using thermostable enzymes, which are known for their increased stability and inhibitor olerance. However, the advantage of using thermostable enzymes has not been studied thoroughly and more knowledge is needed for development of bioethanol processes....... Enzymes are added to the bioethanol process after pretreatment. For an efficient sugar and ethanol yield, the solids content of biomass is normally increased, which results in highly viscous slurries that are difficult to mix. Therefore, the first enzymatic challenge is to ensure rapid reduction...

  17. Stimulation of {sup 125}I-3-iodo-{alpha}-methyl-L-tyrosine uptake in Chinese hamster ovary (CHO-K1) cells by tyrosine esters

    Energy Technology Data Exchange (ETDEWEB)

    Shikano, Naoto [Department of Radiological Sciences, Center for Medical Sciences and Center for Humanities and Sciences, Ibaraki Prefectural University of Health Sciences, Inashiki-gun, Ibaraki (Japan)], E-mail: sikano@ipu.ac.jp; Ogura, Masato; Sagara, Jun-ichi; Nakajima, Syuichi [Department of Radiological Sciences, Center for Medical Sciences and Center for Humanities and Sciences, Ibaraki Prefectural University of Health Sciences, Inashiki-gun, Ibaraki (Japan); Kobayashi, Masato [Division of Health Science, Graduate School of Health Sciences, Kanazawa University, Kanazawa, Ishikawa (Japan); Baba, Takeshi; Yamaguchi, Naoto; Iwamura, Yukio; Kubota, Nobuo [Department of Radiological Sciences, Center for Medical Sciences and Center for Humanities and Sciences, Ibaraki Prefectural University of Health Sciences, Inashiki-gun, Ibaraki (Japan); Kawai, Keiichi [Division of Health Science, Graduate School of Health Sciences, Kanazawa University, Kanazawa, Ishikawa (Japan)

    2010-02-15

    Introduction: Transport of the amino acid analog {sup 123}I-3-iodo-{alpha}-methyl-L-tyrosine, which is used in clinical SPECT imaging, occurs mainly via L-type amino acid transporter type 1 (LAT1; an amino acid exchanger). As LAT1 is highly expressed in actively proliferating tumors, we made a preliminary investigation of the effects of amino acid esters on enhancement of {sup 125}I-3-iodo-{alpha}-methyl-L-tyrosine (IMT) uptake via LAT1 in Chinese hamster ovary (CHO-K1) cells. Methods: Because the sequence of the CHO-K1 LAT1 gene is not available, we confirmed LAT1 expression through IMT (18.5 kBq) uptake mechanisms using specific inhibitors. L-Gly, L-Ser, L-Leu, L-Phe, L-Met, L-Tyr, D-Tyr, L-Val and L-Lys ethyl/methyl esters were tested in combination with IMT. Time-course studies over a 3-h period were conducted, and the concentration dependence of L-Tyr ethyl and methyl esters (0.001 to 10 mM) in combination with IMT was also examined. For a proof of de-esterification of L- and D-Tyr ethyl and methyl esters in the cells (by enzymatic attack or other cause), the concentration of L- and D-Tyr was analyzed by high-performance liquid chromatography of the esters in phosphate buffer (pH 7.4) and cell homogenates at 37 deg. C or under ice-cold conditions. Results: Inhibition tests suggested that LAT1 is involved in IMT uptake by CHO-K1 cells. Co-administration of 1 mM of L-Tyr ethyl or methyl ester with IMT produced the greatest enhancement. The de-esterification reaction was stereo selective and temperature dependent in the homogenate. De-esterification kinetics were very fast in the homogenate and very slow in the phosphate buffer. Conclusions: The L-Tyr ethyl or methyl esters were the most effective enhancers of IMT uptake into CHO-K1 cells and acted by trans-stimulation of the amino acid exchange function of LAT1. This result suggests that de-esterification in the cells may be caused by enzymatic attack. We will use IMT and L-Tyr ethyl or methyl esters to examine

  18. Demonstration of tyrosinase in the vitiligo skin of human beings by a sensitive fluorometric method as well as by 14C(U)-L-tyrosine incorporation into melanin

    International Nuclear Information System (INIS)

    Husain, I.; Vijayan, E.; Ramaiah, A.; Pasricha, J.S.; Madan, N.C.

    1982-01-01

    Tyrosinase activity (Monophenol, dihydroxyphenylalanine: oxygen oxidoreductase EC 1.14.18.1) in vitiligo and normal epidermal homogenates of skin from human beings was measured by estimating beta 3,4-dihydroxyphenylalanine (dopa) by a highly sensitive fluorometric method described in this paper. The tyrosine activity in the vitiligo skin was about 4 to 37% of corresponding normal skin. The activity of tyrosinase in normal human skin from different individuals and from different regions of the body was in the range of 4 to 140 picomoles of beta 3,4-dihydroxyphenylalanine formed per min/mg protein of epidermal homogenate. The enzyme from vitiligo and normal skin was severely inhibited by substance(s) of low molecular weight. The enzyme exhibits a lag of about 4 hr in the absence of added beta 3,4-dihydroxyphenylalanine and 1 hr in presence of 5 microM dopa. Tyrosinase from the normal and vitiligo skin was inhibited by excess concentration of tyrosine. The homogenates from vitiligo skin could synthesize melanin from C14(U)-L-Tyrosine. The rate of tyrosine incorporation into melanin by the epidermal homogenates is increased by 3,4-dihydroxyphenylalanine (dopa) disproportionate to its effect on tyrosinase activity. Based on the data presented in this paper it is concluded that melanocytes are present in the vitiligo skin. A tentative hypothesis is put forward to explain the lack of melanin synthesis by the vitiligo skin under in vivo conditions, although melanocytes are present

  19. Tracer experiment administering L-phenylalanine-U-14C and L-tyrosine-U-14C to the tissue slices of bamboo shoots

    International Nuclear Information System (INIS)

    Kozukue, E.; Mizuno, S.

    1987-01-01

    Uniformly 14 C-labeled L-phenylalanine and L-tyrosine were administered to tissue slices of both top and base sections of bamboo shoots. Alcohol soluble substances were extracted and then separated into organic acid, sugar and amino acid fractions by ion exchange chromatography. The homogentisic acid fraction among the organic acids was collected by high-performance liquid chromatography (HPLC) and its radioactivity was measured, while the alcohol insoluble residue was used for the analysis of lignin aldehyde by the method of alkaline nitrobenzene oxidation. 1. The two labeled amino acids were steadily incorporated into the tissues during incubation and rapidly converted to organic acid, sugar and alcohol insoluble residue, especially the latter. 2. On determining the amount of phenylalanine converted to tyrosine, it was found that this was extremely small. 3. The incorporation of phenylalanine-U- 14 C into alcohol insoluble residue was higher than that of tyrosine in both sections. 4. Although the conversion into lignin aldehyde from phenylalanine-U- 14 C was higher than that from tyrosine-U- 14 C, it was found that tyrosine incorporated into the shoots was converted to a remarkable extent for formation of lignin aldehyde. 5. The incorporation of phenylalanine and tyrosine into homogentisic acid was very low. From these results, we assume that the conversion of phenylalanine to tyrosine or of tyrosine to homogentisic acid is very small, and that a part of the high amount of tyrosine in the shoots may be used for formation of lignin

  20. The usefulness of dynamic O-(2-18F-fluoroethyl)-L-tyrosine PET in the clinical evaluation of brain tumors in children and adolescents

    DEFF Research Database (Denmark)

    Dunkl, Veronika; Cleff, Corvin; Stoffels, Gabriele

    2015-01-01

    UNLABELLED: Experience regarding O-(2-(18)F-fluoroethyl)-L-tyrosine ((18)F-FET) PET in children and adolescents with brain tumors is limited. METHODS: Sixty-nine (18)F-FET PET scans of 48 children and adolescents (median age, 13 y; range, 1-18 y) were analyzed retrospectively. Twenty-six scans...... to assess newly diagnosed cerebral lesions, 24 scans for diagnosing tumor progression or recurrence, 8 scans for monitoring of chemotherapy effects, and 11 scans for the detection of residual tumor after resection were obtained. Maximum and mean tumor-to-brain ratios (TBRs) were determined at 20-40 min...... after injection, and time-activity curves of (18)F-FET uptake were assigned to 3 different patterns: constant increase; peak at greater than 20-40 min after injection, followed by a plateau; and early peak (≤ 20 min), followed by a constant descent. The diagnostic accuracy of (18)F-FET PET was assessed...

  1. Monitoring of Radiochemotherapy in Patients with Glioblastoma Using O-(2-[18F]Fluoroethyl-L-Tyrosine Positron Emission Tomography: Is Dynamic Imaging Helpful?

    Directory of Open Access Journals (Sweden)

    Marc D. Piroth

    2013-09-01

    Full Text Available Monitoring of radiochemotherapy (RCX in patients with glioblastoma is difficult because unspecific alterations in magnetic resonance imaging with contrast enhancement can mimic tumor progression. Changes in tumor to brain ratios (TBRs in positron emission tomography (PET using O-(2-[18F]fluoroethyl-L-tyrosine (18F-FET after RCX with temozolomide of patients with glioblastoma have been shown to be valuable parameters to predict survival. The kinetic behavior of 18F-FET in the tumors is another promising parameter to analyze tumor metabolism. In this study, we investigated the predictive value of dynamic 18F-FET PET during RCX of glioblastoma. Time-activity curves (TACs of 18F-FET uptake of 25 patients with glioblastoma were evaluated after surgery (FET-1, early (7–10 days after completion of RCX (FET-2, and 6 to 8 weeks later (FET-3. Changes in the time to peak (TTP and the slope of the TAC (10–50 minutes postinjection were analyzed and related to survival. Changes in kinetic parameters of 18F-FET uptake after RCX showed no relationship with survival time. In contrast, the high predictive value of changes of TBR to predict survival was confirmed. We conclude that dynamic 18F-FET PET does not provide additional prognostic information during RCX. Static 18F-FET PET imaging (20–40 minutes postinjection appears to be sufficient for this purpose and reduces costs.

  2. [F-18]fluoro-meta-L-tyrosine is a better PET tracer than [F-18]fluoro-L-dopa for the delineation of dopaminergic structures in the human brain

    International Nuclear Information System (INIS)

    Firnau, G.; Chirakal, R.; Nahmias, C.; Garnett, E.S.

    1990-01-01

    Fluorine-18 labelled fluoro-m-L-tyrosine (FmLtyr) and fluoro-L-Dopa (F-Dopa) have been synthesized, and the utility of FmLtyr for PET investigations of dopaminergic brain regions has been compared to that of F-dopa. Experimental results from both monkey and human studies indicate that FmLtyr gives better delineation of striatum, and is a better PET tracer than F-dopa

  3. Thermostable cellulase from a thermomonospora gene

    Science.gov (United States)

    Wilson, D.B.; Walker, L.P.; Zhang, S.

    1997-10-14

    The invention relates to a gene isolated from Thermomonospora fusca, wherein the gene encodes a thermostable cellulase. Disclosed is the nucleotide sequence of the T. fusca gene; and nucleic acid molecules comprising the gene, or a fragment of the gene, that can be used to recombinantly express the cellulase or a catalytically active polypeptide thereof, respectively. The isolated and purified recombinant cellulase or catalytically active polypeptide may be used to hydrolyze substrate either by itself; or in combination with other cellulases, with the resultant combination having unexpected hydrolytic activity. 3 figs.

  4. O-(2-18F-fluoroethyl)-L-tyrosine PET for evaluation of brain metastasis recurrence after radiotherapy: an effectiveness and cost-effectiveness analysis.

    Science.gov (United States)

    Heinzel, Alexander; Müller, Dirk; Yekta-Michael, Sareh Said; Ceccon, Garry; Langen, Karl-Josef; Mottaghy, Felix M; Wiesmann, Martin; Kocher, Martin; Hattingen, Elke; Galldiks, Norbert

    2017-09-01

    Conventional MRI is the standard method to diagnose recurrence of brain metastases after radiation. However, following radiation therapy, reactive transient blood-brain barrier alterations with consecutive contrast enhancement can mimic brain metastasis recurrence. Recent studies have suggested that O-(2-18F-fluoroethyl)-L-tyrosine (FET) PET improves the correct differentiation of brain metastasis recurrence from radiation injury. Based on published evidence and clinical expert opinion, we analyzed effectiveness and cost-effectiveness of the use of FET PET in addition to MRI compared with MRI alone for the diagnosis of recurrent brain metastases. A decision-tree model was designed to compare the 2 diagnostic strategies from the perspective of the German Statutory Health Insurance (SHI) system. Effectiveness was defined as correct diagnosis of recurrent brain metastasis and was compared between FET PET with MRI and MRI alone. Costs were calculated for a baseline scenario and for a more expensive scenario. Robustness of the results was tested using sensitivity analyses. Compared with MRI alone, FET PET in combination with MRI increases the rate of correct diagnoses by 42% (number needed to diagnose of 3) with an incremental cost-effectiveness ratio of €2821 (baseline scenario) and €4014 (more expensive scenario) per correct diagnosis. The sensitivity analyses confirmed the robustness of the results. The model suggests that the additional use of FET PET with conventional MRI for the diagnosis of recurrent brain metastases may be cost-effective. Integration of FET PET has the potential to avoid overtreatment with corresponding costs as well as unnecessary side effects. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

  5. Tracer experiment administering L-phenylalanine-U-{sup 14}C and L-tyrosine-U-{sup 14}C to the tissue slices of bamboo shoots

    Energy Technology Data Exchange (ETDEWEB)

    Kozukue, E. [Kenmei Women' s Junior Coll., Himeji, Hyogo (Japan); Mizuno, S.

    1987-09-15

    Uniformly {sup 14}C-labeled L-phenylalanine and L-tyrosine were administered to tissue slices of both top and base sections of bamboo shoots. Alcohol soluble substances were extracted and then separated into organic acid, sugar and amino acid fractions by ion exchange chromatography. The homogentisic acid fraction among the organic acids was collected by high-performance liquid chromatography (HPLC) and its radioactivity was measured, while the alcohol insoluble residue was used for the analysis of lignin aldehyde by the method of alkaline nitrobenzene oxidation. 1. The two labeled amino acids were steadily incorporated into the tissues during incubation and rapidly converted to organic acid, sugar and alcohol insoluble residue, especially the latter. 2. On determining the amount of phenylalanine converted to tyrosine, it was found that this was extremely small. 3. The incorporation of phenylalanine-U-{sup 14}C into alcohol insoluble residue was higher than that of tyrosine in both sections. 4. Although the conversion into lignin aldehyde from phenylalanine-U-{sup 14}C was higher than that from tyrosine-U-{sup 14}C, it was found that tyrosine incorporated into the shoots was converted to a remarkable extent for formation of lignin aldehyde. 5. The incorporation of phenylalanine and tyrosine into homogentisic acid was very low. From these results, we assume that the conversion of phenylalanine to tyrosine or of tyrosine to homogentisic acid is very small, and that a part of the high amount of tyrosine in the shoots may be used for formation of lignin.

  6. Reproducibility of O-(2-{sup 18}F-fluoroethyl)-L-tyrosine uptake kinetics in brain tumors and influence of corticoid therapy: an experimental study in rat gliomas

    Energy Technology Data Exchange (ETDEWEB)

    Stegmayr, Carina; Schoeneck, Michael; Oliveira, Dennis; Willuweit, Antje [Institute of Neuroscience and Medicine, Research Center Juelich, Juelich (Germany); Filss, Christian; Coenen, Heinz H.; Langen, Karl-Josef [Institute of Neuroscience and Medicine, Research Center Juelich, Juelich (Germany); University of Aachen, Department of Nuclear Medicine and Neurology, Aachen (Germany); Galldiks, Norbert [Institute of Neuroscience and Medicine, Research Center Juelich, Juelich (Germany); University of Cologne, Department of Neurology, Cologne (Germany); Shah, N. Jon [Institute of Neuroscience and Medicine, Research Center Juelich, Juelich (Germany); University of Aachen, Department of Nuclear Medicine and Neurology, Aachen (Germany); Juelich-Aachen Research Alliance (JARA) - Section JARA-Brain, Juelich (Germany)

    2016-06-15

    Positron emission tomography (PET) using O-(2-{sup 18}F-fluoroethyl)-L-tyrosine ({sup 18}F-FET) is a well-established method for the diagnostics of brain tumors. This study investigates reproducibility of {sup 18}F-FET uptake kinetics in rat gliomas and the influence of the frequently used dexamethasone (Dex) therapy. F98 glioma or 9L gliosarcoma cells were implanted into the striatum of 31 Fischer rats. After 10-11 days of tumor growth, the animals underwent dynamic PET after injection of {sup 18}F-FET (baseline). Thereafter, animals were divided into a control group and a group receiving Dex injections, and all animals were reinvestigated 2 days later. Tumor-to-brain ratios (TBR) of {sup 18}F-FET uptake (18-61 min p.i.) and the slope of the time-activity-curves (TAC) (18-61 min p.i.) were evaluated using a Volume-of-Interest (VOI) analysis. Data were analyzed by two-way repeated measures ANOVA and reproducibility by the intraclass correlation coefficient (ICC). The slope of the tumor TACs showed high reproducibility with an ICC of 0.93. A systematic increase of the TBR in the repeated scans was noted (3.7 ± 2.8 %; p < 0.01), and appeared to be related to tumor growth as indicated by a significant correlation of TBR and tumor volume (r = 0.77; p < 0.0001). After correction for tumor growth TBR showed high longitudinal stability with an ICC of 0.84. Dex treatment induced a significant decrease of the TBR (-8.2 ± 6.1 %; p < 0.03), but did not influence the slope of the tumor TAC. TBR of {sup 18}F-FET uptake and tracer kinetics in brain tumors showed high longitudinal stability. Dex therapy may induce a minor decrease of the TBR; this needs further investigation. (orig.)

  7. The use of dynamic O-(2-18F-fluoroethyl)-l-tyrosine PET in the diagnosis of patients with progressive and recurrent glioma.

    Science.gov (United States)

    Galldiks, Norbert; Stoffels, Gabriele; Filss, Christian; Rapp, Marion; Blau, Tobias; Tscherpel, Caroline; Ceccon, Garry; Dunkl, Veronika; Weinzierl, Martin; Stoffel, Michael; Sabel, Michael; Fink, Gereon R; Shah, Nadim J; Langen, Karl-Josef

    2015-09-01

    We evaluated the diagnostic value of static and dynamic O-(2-[(18)F]fluoroethyl)-L-tyrosine ((18)F-FET) PET parameters in patients with progressive or recurrent glioma. We retrospectively analyzed 132 dynamic (18)F-FET PET and conventional MRI scans of 124 glioma patients (primary World Health Organization grade II, n = 55; grade III, n = 19; grade IV, n = 50; mean age, 52 ± 14 y). Patients had been referred for PET assessment with clinical signs and/or MRI findings suggestive of tumor progression or recurrence based on Response Assessment in Neuro-Oncology criteria. Maximum and mean tumor/brain ratios of (18)F-FET uptake were determined (20-40 min post-injection) as well as tracer uptake kinetics (ie, time to peak and patterns of the time-activity curves). Diagnoses were confirmed histologically (95%) or by clinical follow-up (5%). Diagnostic accuracies of PET and MR parameters for the detection of tumor progression or recurrence were evaluated by receiver operating characteristic analyses/chi-square test. Tumor progression or recurrence could be diagnosed in 121 of 132 cases (92%). MRI and (18)F-FET PET findings were concordant in 84% and discordant in 16%. Compared with the diagnostic accuracy of conventional MRI to diagnose tumor progression or recurrence (85%), a higher accuracy (93%) was achieved by (18)F-FET PET when a mean tumor/brain ratio ≥2.0 or time to peak dynamic (18)F-FET PET parameters differentiate progressive or recurrent glioma from treatment-related nonneoplastic changes with higher accuracy than conventional MRI. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Thermostability in endoglucanases is fold-specific

    Science.gov (United States)

    2011-01-01

    Background Endoglucanases are usually considered to be synergistically involved in the initial stages of cellulose breakdown-an essential step in the bioprocessing of lignocellulosic plant materials into bioethanol. Despite their economic importance, we currently lack a basic understanding of how some endoglucanases can sustain their ability to function at elevated temperatures required for bioprocessing, while others cannot. In this study, we present a detailed comparative analysis of both thermophilic and mesophilic endoglucanases in order to gain insights into origins of thermostability. We analyzed the sequences and structures for sets of endoglucanase proteins drawn from the Carbohydrate-Active enZymes (CAZy) database. Results Our results demonstrate that thermophilic endoglucanases and their mesophilic counterparts differ significantly in their amino acid compositions. Strikingly, these compositional differences are specific to protein folds and enzyme families, and lead to differences in intramolecular interactions in a fold-dependent fashion. Conclusions Here, we provide fold-specific guidelines to control thermostability in endoglucanases that will aid in making production of biofuels from plant biomass more efficient. PMID:21291533

  9. Thermostability in endoglucanases is fold-specific

    Directory of Open Access Journals (Sweden)

    Wolt Jeffrey D

    2011-02-01

    Full Text Available Abstract Background Endoglucanases are usually considered to be synergistically involved in the initial stages of cellulose breakdown-an essential step in the bioprocessing of lignocellulosic plant materials into bioethanol. Despite their economic importance, we currently lack a basic understanding of how some endoglucanases can sustain their ability to function at elevated temperatures required for bioprocessing, while others cannot. In this study, we present a detailed comparative analysis of both thermophilic and mesophilic endoglucanases in order to gain insights into origins of thermostability. We analyzed the sequences and structures for sets of endoglucanase proteins drawn from the Carbohydrate-Active enZymes (CAZy database. Results Our results demonstrate that thermophilic endoglucanases and their mesophilic counterparts differ significantly in their amino acid compositions. Strikingly, these compositional differences are specific to protein folds and enzyme families, and lead to differences in intramolecular interactions in a fold-dependent fashion. Conclusions Here, we provide fold-specific guidelines to control thermostability in endoglucanases that will aid in making production of biofuels from plant biomass more efficient.

  10. Approaches for improving thermostability characteristics in cellulases.

    Science.gov (United States)

    Anbar, Michael; Bayer, Edward A

    2012-01-01

    Many efforts have been invested to reduce the cost of biofuel production to substitute renewable sources of energy for fossil-based fuels. At the forefront of these efforts are the initiatives to convert plant-derived cellulosic material to biofuels. Although significant improvements have been achieved recently in cellulase engineering in both efficiency and cost reduction, complete degradation of lignocellulosic material still requires very long periods of time and high enzyme loads. Thermostable cellulases offer many advantages in the bioconversion process, which include increase in specific activity, higher levels of stability, inhibition of microbial growth, increase in mass transfer rate due to lower fluid viscosity, and greater flexibility in the bioprocess. Besides rational design methods, which require deep understanding of protein structure-function relationship, two of the major methods for improvement in specific cellulase properties are directed evolution and knowledge-based library design based on multiple sequence alignments. In this chapter, we provide protocols for constructing and screening of improved thermostable cellulases. Modifications of these protocols may also be used for screening for other improved properties of cellulases such as pH tolerance, high salt, and more. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Thermostable enzymes as biocatalysts in the biofuel industry.

    Science.gov (United States)

    Yeoman, Carl J; Han, Yejun; Dodd, Dylan; Schroeder, Charles M; Mackie, Roderick I; Cann, Isaac K O

    2010-01-01

    Lignocellulose is the most abundant carbohydrate source in nature and represents an ideal renewable energy source. Thermostable enzymes that hydrolyze lignocellulose to its component sugars have significant advantages for improving the conversion rate of biomass over their mesophilic counterparts. We review here the recent literature on the development and use of thermostable enzymes for the depolymerization of lignocellulosic feedstocks for biofuel production. Furthermore, we discuss the protein structure, mechanisms of thermostability, and specific strategies that can be used to improve the thermal stability of lignocellulosic biocatalysts. Copyright 2010 Elsevier Inc. All rights reserved.

  12. Influence of blood-brain barrier permeability on O-(2-{sup 18}F-fluoroethyl)-L-tyrosine uptake in rat gliomas

    Energy Technology Data Exchange (ETDEWEB)

    Stegmayr, Carina; Bandelow, Ulrike; Oliveira, Dennis; Lohmann, Philipp; Willuweit, Antje; Galldiks, Norbert; Luebke, Joachim H.R. [Institute of Neuroscience and Medicine, Forschungszentrum Juelich, Juelich (Germany); Filss, Christian; Ermert, Johannes; Langen, Karl-Josef [Institute of Neuroscience and Medicine, Forschungszentrum Juelich, Juelich (Germany); RWTH/University Hospital Aachen, Department of Nuclear Medicine and Neurology, Aachen (Germany); Shah, N. Jon [Institute of Neuroscience and Medicine, Forschungszentrum Juelich, Juelich (Germany); RWTH/University Hospital Aachen, Department of Nuclear Medicine and Neurology, Aachen (Germany); Juelich-Aachen Research Alliance (JARA) - Section JARA-Brain, Aachen (Germany)

    2017-03-15

    O-(2-{sup 18}F-fluoroethyl)-L-tyrosine ({sup 18}F-FET) is an established tracer for the diagnosis of brain tumors with PET. This study investigates the influence of blood-brain barrier (BBB) permeability on {sup 18}F-FET uptake in two rat glioma models and one human xenograft model. F98 glioma, 9L gliosarcoma or human U87 glioblastoma cells were implanted into the striatum of 56 Fischer or RNU rats. Thereafter, animals were divided into a control group and a group receiving injections of the glucocorticoid dexamethasone (Dex). After 12-13 days of tumor growth animals received injection of Evans blue dye (EBD) to visualize BBB disturbance and underwent {sup 18}F-FET PET followed by autoradiography. Time activity curves, standardized uptake values (SUV) and Tumor-to-brain ratios (TBR) of {sup 18}F-FET uptake [18-61 min post injection (p.i.)] were evaluated using a volume-of-Interest (VOI) analysis. BBB disturbance was quantitatively evaluated by EBD fluorescence. The membrane gaps of blood vessel endothelial tight junctions were measured using electron microscopy to visualize ultrastructural BBB alterations in one untreated and one Dex treated F98 glioma. Data were analyzed by two-way ANOVAs. In Dex treated animals EBD extravasation was significantly reduced in 9L (P < 0.001) and U87 (P = 0.008) models and showed a trend in F98 models (P = 0.053). In contrast, no significant differences of {sup 18}F-FET uptake were observed between Dex treated animals and control group except a decrease of the TBR in the 9L tumor model in PET (P < 0.01). Ultrastructural evaluation of tumor blood vessel endothelia revealed significant reduction of the cleft diameter between endothelial cells after Dex treatment in F98 model (P = 0.010). Despite a considerable reduction of BBB permeability in rat gliomas after Dex treatment, no relevant changes of {sup 18}F-FET uptake were noted in this experimental study. Thus, {sup 18}F-FET uptake in gliomas appears to be widely independent of the

  13. O-(2-[18F]fluoroethyl)-L-tyrosine uptake is an independent prognostic determinant in patients with glioma referred for radiation therapy

    International Nuclear Information System (INIS)

    Sweeney, Reinhart; Polat, Bülent; Flentje, Michael; Samnick, Samuel; Reiners, Christoph; Verburg, Frederik A.

    2014-01-01

    To evaluate the prognostic value of O-(2-[ 18 F]fluoroethyl)-L-tyrosine positron emission tomography (FET-PET) uptake intensity in World Health Organization (WHO) tumor grade II-IV gliomas. We studied 28 patients with WHO tumor grade II-IV gliomas who were referred to our department for radiation therapy. We acquired a FET-PET in all patients, as well as magnetic resonance imaging (MRI) of the brain consisting of at least T2-weighted imaging, flair and pre- and post-contrast T1-weighted imaging. SUVmax was measured and the tumor-to-brain uptake ratio (TBR) of all lesions was calculated based on the SUVmax (TBRmax) or SUVmean (TBRmean) of the contralateral healthy tissue. For this study, volumes were calculated using MRI alone, MRI + the volume with a SUVmax on FET-PET ≥ 2.2 as well as MRI + the volume with an uptake of at least 40% of the SUVmax. Tumor volumes were a median (range) of 88.6 (2.6-467.4) ml (MRI alone), 84.2 (2.8-474.4) ml (MRI + SUVmax on FET-PET ≥ 2.2) and 101.5 (4.0-512.1) ml (MRI + FET-PET uptake ≥ 40% SUVmax), respectively. TBR-SUVmean was 2.36 (1.46-4.08); TBR-SUVmax was 1.71 (0.97-2.85). During a follow-up of 18.7 (2.5-36.1) months after FET-PET, 12 patients died of malignant glioma. Patients with a SUVmax ≥ 2.6 had a significantly worse tumor-related mortality (p=0.005) and progression-free survival (p=0.038) than those with a lower SUVmax. Multivariate analysis showed that WHO tumor grade (p=0.001) and SUVmax ≥ 2.6 (p < 0.001) were independent predictors for tumor-related mortality, but not tumor volume or TBRmax or TBRmean. SUVmax ≥ 2.6 (p=0.007) and being treated for a recurrence rather than for a primary tumor manifestation (p=0.014) were predictors for progression-free survival, but not TBRmax or TBRmean. In this heterogeneous patient population, higher tracer uptake in FET-PET appears to be associated with a worse tumor-related mortality and a shorter duration of the disease-free interval. (author)

  14. Transcellular transport of radioiodinated 3-iodo-α-methyl-L-tyrosine across monolayers of kidney epithelial cell line LLC-PK1

    International Nuclear Information System (INIS)

    Shikano, Naoto; Nakajima, Syuichi; Kubota, Nobuo; Ishikawa, Nobuyoshi; Kawai, Keiichi; Kubodera, Akiko; Saji, Hideo

    2004-01-01

    3-[ 123 I]iodo-α-methyl-L-tyrosine ([ 123 I]IMT) is an imaging agent for amino acid transport. In order to obtain fundamental data related to tumor imaging with [ 123 I]IMT and renal physiological accumulation of [ 123 I]IMT, we investigated the transport characteristics of [ 125 I]IMT in porcine kidney epithelial cell line LLC-PK 1 using cell monolayers grown on microporous membrane filters. LLC-PK 1 monolayers were created on a collagen-coated microporous (3 μm) membrane (4.7 cm 2 ). To examine transcellular transport (secretion and reabsorption) and accumulation, the monolayers were incubated for up to 90 min at 37 deg C with 18.5 kBq [ 125 I]IMT in Dulbecco's phosphate-buffered saline (pH 7.4) as an uptake solution. After incubation, transcellular transport was assessed by quantifying the radioactivity of the solutions on each side of the monolayer. For the accumulation experiment, the cells were solubilized in NaOH solution, and the radioactivity was quantified. For the inhibition experiment, the inhibitor was added at a final concentration of 1 mM. For the pH dependence experiment, the pH of the apical-side uptake solution was varied from pH 5 to pH 8. Transport of [ 14 C]Tyr was examined for comparison. Bi-directional transcellular transport of [ 125 I]IMT was observed, corresponding to secretion and reabsorption in proximal tubule. Accumulation of [ 125 I]IMT from the basolateral side (1.62±0.15%) and the apical side (2.62±0.35%) was observed at 90 min. 2-Amino-bicyclo[2,2,1]heptane-2-carboxylic acid (a specific inhibitor of system L), L -Tyr (mother compound of [ 125 I]IMT) and 2-aminoisobutyric acid (an inhibitor of system L and A) inhibited both directional transport (p 125 I]IMT from both sides (p 125 I]IMT transport is system L, rather than Na + -dependent transport, in both apical and basolateral membrane. [ 125 I]IMT was transported by the system that transported L-Tyr, but the observed pH dependence of transport suggests that different

  15. Engineering of pectinolytic enzymes for enhanced thermostability

    DEFF Research Database (Denmark)

    Larsen, Dorte Møller

    Conversion of waste materials into valuable compounds is promising concerning transformation of byproduct streams such as sugar beet and potato pulp. In order to obtain those compounds with reduced energy consumption, carbohydrate active enzymes can be used as catalysts. Sugar beet and potato pulp...... consist of pectin that can be converted into beneficial polymeric and oligomeric carbohydrates requiring enzymes such as pectin lyases, rhamnogalacturonan I (RGI) lyases, polygalacturonases and galactanases. Enzymatic conversion of such pectinaceous biomasses at high temperatures is advantageous...... as it gives rise to lower substrate viscosity, easier mixing, higher substrate solubility and lowers the risk of contamination. The overall objective of this thesis was to discover enzymes for degradation of RGI structures in pectin and further engineer for enhanced thermostability. The hypotheses were...

  16. Thermostable Alginate degrading enzymes and their methods of use

    NARCIS (Netherlands)

    Hreggvidsson, Gudmundur Oli; Jonsson, Oskar W.J.; Bjornsdottir, Bryndis; Fridjonsson, Hedinn O; Altenbuchner, Josef; Watzlawick, Hildegard; Dobruchowska, Justyna; Kamerling, Johannis

    2015-01-01

    The present invention relates to the identification, production and use of thermostable alginate lyase enzymes that can be used to partially degrade alginate to yield oligosaccharides or to give complete degradation of alginate to yield (unsaturated) mono-uronates.

  17. Do the metabolites of 6-[F-18]fluoro-L-dopa and of [F-18]fluoro-meta-L-tyrosine contribute to the F-18 accumulation in the human brain?

    International Nuclear Information System (INIS)

    Firnau, G.; Chirakal, R.; Nahmias, C.; Garnett, E.S.

    1990-01-01

    The purpose of this study was to determine if the metabolites of 6-[F-18]fluoro-L-dopa (F-dopa) and of [F-18]fluoro-meta-L-tyrosine (FmLtyr) contribute to the accumulation of fluorine-18 in the brain through unspecific retention. PET studies were conducted on a healthy human subject who was treated with both of the radiopharmaceuticals and their labelled metabolites. Results indicated that in contrast to F-dopa, the metabolite of FmLtyr does not 'contaminate' the brain with extraneous fluorine-18

  18. RESOLUTE PET/MRI Attenuation Correction for O-(2-18F-fluoroethyl-L-tyrosine (FET in Brain Tumor Patients with Metal Implants

    Directory of Open Access Journals (Sweden)

    Claes N. Ladefoged

    2017-08-01

    Full Text Available Aim: Positron emission tomography (PET imaging is a useful tool for assisting in correct differentiation of tumor progression from reactive changes, and the radiolabeled amino acid analog tracer O-(2-18F-fluoroethyl-L-tyrosine (FET-PET is amongst the most frequently used. The FET-PET images need to be quantitatively correct in order to be used clinically, which require accurate attenuation correction (AC in PET/MRI. The aim of this study was to evaluate the use of the subject-specific MR-derived AC method RESOLUTE in post-operative brain tumor patients.Methods: We analyzed 51 post-operative brain tumor patients (68 examinations, 200 MBq [18F]-FET investigated in a PET/MRI scanner. MR-AC maps were acquired using: (1 the Dixon water fat separation sequence, (2 the ultra short echo time (UTE sequences, (3 calculated using our new RESOLUTE methodology, and (4 a same day low-dose CT used as reference “gold standard.” For each subject and each AC method the tumor was delineated by isocontouring tracer uptake above a tumor(T-to-brain background (B activity ratio of 1.6. We measured B, tumor mean and maximal activity (TMEAN, TMAX, biological tumor volume (BTV, and calculated the clinical metrics TMEAN/B and TMAX/B.Results: When using RESOLUTE 5/68 studies did not meet our predefined acceptance criteria of TMAX/B difference to CT-AC < ±0.1 or 5%, TMEAN/B < ±0.05 or 5%, and BTV < ±2 mL or 10%. In total, 46/68 studies failed our acceptance criteria using Dixon, and 26/68 using UTE. The 95% limits of agreement for TMAX/B was for RESOLUTE (−3%; 4%, Dixon (−9%; 16%, and UTE (−7%; 10%. The absolute error when measuring BTV was 0.7 ± 1.9 mL (N.S with RESOLUTE, 5.3 ± 10 mL using Dixon, and 1.7 ± 3.7 mL using UTE. RESOLUTE performed best in the identification of the location of peak activity and in brain tumor follow-up monitoring using clinical FET PET metrics.Conclusions: Overall, we found RESOLUTE to be the AC method that most robustly

  19. Response assessment of bevacizumab in patients with recurrent malignant glioma using [{sup 18}F]Fluoroethyl-l-tyrosine PET in comparison to MRI

    Energy Technology Data Exchange (ETDEWEB)

    Galldiks, Norbert; Fink, Gereon R. [Institute of Neuroscience and Medicine (INM-3,-4,-5), Forschungszentrum Juelich, Juelich (Germany); University of Cologne, Department of Neurology, Cologne (Germany); Rapp, Marion; Sabel, Michael [University of Duesseldorf, Department of Neurosurgery, Duesseldorf (Germany); Stoffels, Gabriele; Shah, Nadim J.; Coenen, Heinz H.; Langen, Karl-Josef [Institute of Neuroscience and Medicine (INM-3,-4,-5), Forschungszentrum Juelich, Juelich (Germany); Juelich-Aachen Research Alliance (JARA) - Section JARA-Brain, Aachen (Germany)

    2013-01-15

    To investigate prospectively the potential of O-(2-[{sup 18}F]fluoroethyl)-l-tyrosine ({sup 18}F-FET) PET in comparison to MRI for the assessment of the response of patients with recurrent high-grade glioma (rHGG) to antiangiogenic treatment. Ten patients with rHGG were treated biweekly with bevacizumab/irinotecan (BEV/IR). MR images and dynamic {sup 18}F-FET PET scans were obtained at baseline and at follow-up after the start of treatment (median 4.9 weeks). Using MRI treatment response was evaluated according to RANO (Response Assessment in Neuro-Oncology) criteria. For {sup 18}F-FET PET evaluation, a reduction >45 % of the metabolically active tumour volume was considered as a treatment response, with the metabolically active tumour being defined as a tumour-to-brain ratio (TBR) of {>=}1.6. The results of the treatment assessments were related to progression-free survival (PFS) and overall survival (OS). For further evaluation of PET data, maximum and mean TBR were calculated using region-of-interest analysis at baseline and at follow-up. Additionally, {sup 18}F-FET uptake kinetic studies were performed at baseline and at follow-up in all patients. Time-activity curves were generated and the times to peak (TTP) uptake (in minutes from the beginning of the dynamic acquisition to the maximum uptake) were calculated. At follow-up, MRI showed a complete response according to RANO criteria in one of the ten patients (10 %), a partial response in five patients (50 %), and stable disease in four patients (40 %). Thus, MRI did not detect tumour progression. In contrast, {sup 18}F-FET PET revealed six metabolic responders (60 %) and four nonresponders (40 %). In the univariate survival analyses, a response detected by {sup 18}F-FET PET predicted a significantly longer PFS (median PFS, 9 vs. 3 months; P = 0.001) and OS (median OS 23.0 months vs. 3.5 months; P = 0.001). Furthermore, in four patients (40 %), diagnosis according to RANO criteria and by {sup 18}F-FET PET was

  20. Highly Efficient Thermostable DSM Cellulases: Why & How?

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Manoj [DSM Innovation, Inc., San Francisco, CA (United States)

    2011-04-26

    These are the slides from this presentation. Lignocellulosic biomass is the most abundant, least expensive renewable natural biological resource for the production of biobased products and bioenergy is important for the sustainable development of human civilization in 21st century. For making the fermentable sugars from lignocellulosic biomass, a reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. In this paper, we will provide DSM's efforts in cellulase research and developments and focus on limitations. Cellulase improvement strategies based on directed evolution using screening on relevant substrates, screening for higher thermal tolerance based on activity screening approaches such as continuous culture using insoluble cellulosic substrates as a powerful selection tool for enriching beneficial cellulase mutants from the large library. We will illustrate why and how thermostable cellulases are vital for economic delivery of bioproducts from cellulosic biomass using biochemical conversion approach.

  1. Consistent mutational paths predict eukaryotic thermostability

    Directory of Open Access Journals (Sweden)

    van Noort Vera

    2013-01-01

    Full Text Available Abstract Background Proteomes of thermophilic prokaryotes have been instrumental in structural biology and successfully exploited in biotechnology, however many proteins required for eukaryotic cell function are absent from bacteria or archaea. With Chaetomium thermophilum, Thielavia terrestris and Thielavia heterothallica three genome sequences of thermophilic eukaryotes have been published. Results Studying the genomes and proteomes of these thermophilic fungi, we found common strategies of thermal adaptation across the different kingdoms of Life, including amino acid biases and a reduced genome size. A phylogenetics-guided comparison of thermophilic proteomes with those of other, mesophilic Sordariomycetes revealed consistent amino acid substitutions associated to thermophily that were also present in an independent lineage of thermophilic fungi. The most consistent pattern is the substitution of lysine by arginine, which we could find in almost all lineages but has not been extensively used in protein stability engineering. By exploiting mutational paths towards the thermophiles, we could predict particular amino acid residues in individual proteins that contribute to thermostability and validated some of them experimentally. By determining the three-dimensional structure of an exemplar protein from C. thermophilum (Arx1, we could also characterise the molecular consequences of some of these mutations. Conclusions The comparative analysis of these three genomes not only enhances our understanding of the evolution of thermophily, but also provides new ways to engineer protein stability.

  2. Influence of high temperature and ethanol on thermostable lignocellulolytic enzymes

    DEFF Research Database (Denmark)

    Skovgaard, Pernille Anastasia; Jørgensen, Henning

    2013-01-01

    the influence of temperature and ethanol on enzyme activity and stability in the distillation step, where most enzymes are inactivated due to high temperatures. Two enzyme mixtures, a mesophilic and a thermostable mixture, were exposed to typical process conditions [temperatures from 55 to 65 °C and up to 5...... % ethanol (w/v)] followed by specific enzyme activity analyses and SDS-PAGE. The thermostable and mesophilic mixture remained active at up to 65 and 55 °C, respectively. When the enzyme mixtures reached their maximum temperature limit, ethanol had a remarkable influence on enzyme activity, e.g., the more...... ethanol, the faster the inactivation. The reason could be the hydrophobic interaction of ethanol on the tertiary structure of the enzyme protein. The thermostable mixture was more tolerant to temperature and ethanol and could therefore be a potential candidate for recycling after distillation....

  3. Thermostability of biological systems: fundamentals, challenges, and quantification.

    Science.gov (United States)

    He, Xiaoming

    2011-01-01

    This review examines the fundamentals and challenges in engineering/understanding the thermostability of biological systems over a wide temperature range (from the cryogenic to hyperthermic regimen). Applications of the bio-thermostability engineering to either destroy unwanted or stabilize useful biologicals for the treatment of diseases in modern medicine are first introduced. Studies on the biological responses to cryogenic and hyperthermic temperatures for the various applications are reviewed to understand the mechanism of thermal (both cryo and hyperthermic) injury and its quantification at the molecular, cellular and tissue/organ levels. Methods for quantifying the thermophysical processes of the various applications are then summarized accounting for the effect of blood perfusion, metabolism, water transport across cell plasma membrane, and phase transition (both equilibrium and non-equilibrium such as ice formation and glass transition) of water. The review concludes with a summary of the status quo and future perspectives in engineering the thermostability of biological systems.

  4. Thermostability in rubredoxin and its relationship to mechanical rigidity

    Science.gov (United States)

    Rader, A. J.

    2010-03-01

    The source of increased stability in proteins from organisms that thrive in extreme thermal environments is not well understood. Previous experimental and theoretical studies have suggested many different features possibly responsible for such thermostability. Many of these thermostabilizing mechanisms can be accounted for in terms of structural rigidity. Thus a plausible hypothesis accounting for this remarkable stability in thermophilic enzymes states that these enzymes have enhanced conformational rigidity at temperatures below their native, functioning temperature. Experimental evidence exists to both support and contradict this supposition. We computationally investigate the relationship between thermostability and rigidity using rubredoxin as a case study. The mechanical rigidity is calculated using atomic models of homologous rubredoxin structures from the hyperthermophile Pyrococcus furiosus and mesophile Clostridium pasteurianum using the FIRST software. A global increase in structural rigidity (equivalently a decrease in flexibility) corresponds to an increase in thermostability. Locally, rigidity differences (between mesophilic and thermophilic structures) agree with differences in protection factors.

  5. Thermostability in rubredoxin and its relationship to mechanical rigidity

    International Nuclear Information System (INIS)

    Rader, A J

    2010-01-01

    The source of increased stability in proteins from organisms that thrive in extreme thermal environments is not well understood. Previous experimental and theoretical studies have suggested many different features possibly responsible for such thermostability. Many of these thermostabilizing mechanisms can be accounted for in terms of structural rigidity. Thus a plausible hypothesis accounting for this remarkable stability in thermophilic enzymes states that these enzymes have enhanced conformational rigidity at temperatures below their native, functioning temperature. Experimental evidence exists to both support and contradict this supposition. We computationally investigate the relationship between thermostability and rigidity using rubredoxin as a case study. The mechanical rigidity is calculated using atomic models of homologous rubredoxin structures from the hyperthermophile Pyrococcus furiosus and mesophile Clostridium pasteurianum using the FIRST software. A global increase in structural rigidity (equivalently a decrease in flexibility) corresponds to an increase in thermostability. Locally, rigidity differences (between mesophilic and thermophilic structures) agree with differences in protection factors

  6. Comparison of the thermostability of cellulases from various thermophilic fungi

    Energy Technology Data Exchange (ETDEWEB)

    Wojtczak, G; Breuil, C; Yamada, J; Saddler, J N

    1987-10-01

    The cellulase activities of six thermophilic fungi were compared. Although the thermophilic fungi grew at relatively high temperatures (> 45/sup 0/C) the optimum temperatures for assaying the various cellulase activities were only slightly higher than the optimum temperatures for the mesophilic fungi, Trichoderma harzianum. Over prolonged incubation (> 24 h) the thermophilic strains demonstrated a higher hydrolytic potential as a result of the greater thermostability of the cellulase components. Although the extracellular cellulase activities had similar pH and temperature optima, in some cases the thermostability of the extracellular components were considerably lower.

  7. Prediction of Protein Thermostability by an Efficient Neural Network Approach

    Directory of Open Access Journals (Sweden)

    Jalal Rezaeenour

    2016-10-01

    Full Text Available Introduction: Manipulation of protein stability is important for understanding the principles that govern protein thermostability, both in basic research and industrial applications. Various data mining techniques exist for prediction of thermostable proteins. Furthermore, ANN methods have attracted significant attention for prediction of thermostability, because they constitute an appropriate approach to mapping the non-linear input-output relationships and massive parallel computing. Method: An Extreme Learning Machine (ELM was applied to estimate thermal behavior of 1289 proteins. In the proposed algorithm, the parameters of ELM were optimized using a Genetic Algorithm (GA, which tuned a set of input variables, hidden layer biases, and input weights, to and enhance the prediction performance. The method was executed on a set of amino acids, yielding a total of 613 protein features. A number of feature selection algorithms were used to build subsets of the features. A total of 1289 protein samples and 613 protein features were calculated from UniProt database to understand features contributing to the enzymes’ thermostability and find out the main features that influence this valuable characteristic. Results:At the primary structure level, Gln, Glu and polar were the features that mostly contributed to protein thermostability. At the secondary structure level, Helix_S, Coil, and charged_Coil were the most important features affecting protein thermostability. These results suggest that the thermostability of proteins is mainly associated with primary structural features of the protein. According to the results, the influence of primary structure on the thermostabilty of a protein was more important than that of the secondary structure. It is shown that prediction accuracy of ELM (mean square error can improve dramatically using GA with error rates RMSE=0.004 and MAPE=0.1003. Conclusion: The proposed approach for forecasting problem

  8. Production of thermostable and organic solvent-tolerant alkaline ...

    African Journals Online (AJOL)

    An alkaliphilic bacterium producing organic solvent-tolerant and thermostable alkaline protease was isolated from poultry litter site and identified as Bacillus coagulans PSB-07. Protease production under different submerged fermentation conditions were investigated with the aim of optimizing yield of enzyme. B. coagulans ...

  9. Engineering Thermostable Microbial Xylanases Toward its Industrial Applications.

    Science.gov (United States)

    Kumar, Vishal; Dangi, Arun Kumar; Shukla, Pratyoosh

    2018-03-01

    Xylanases are one of the important hydrolytic enzymes which hydrolyze the β-1, 4 xylosidic linkage of the backbone of the xylan polymeric chain which consists of xylose subunits. Xylanases are mainly found in plant cell walls and are produced by several kinds of microorganisms such as fungi, bacteria, yeast, and some protozoans. The fungi are considered as most potent xylanase producers than that of yeast and bacteria. There is a broad series of industrial applications for the thermostable xylanase as an industrial enzyme. Thermostable xylanases have been used in a number of industries such as paper and pulp industry, biofuel industry, food and feed industry, textile industry, etc. The present review explores xylanase-substrate interactions using gene-editing tools toward the comprehension in improvement in industrial stability of xylanases. The various protein-engineering and metabolic-engineering methods have also been explored to improve operational stability of xylanase. Thermostable xylanases have also been used for improvement in animal feed nutritional value. Furthermore, they have been used directly in bakery and breweries, including a major use in paper and pulp industry as a biobleaching agent. This present review envisages some of such applications of thermostable xylanases for their bioengineering.

  10. Thermophilic growth and enzymatic thermostability are polyphyletic traits within Chaetomiaceae.

    Science.gov (United States)

    van den Brink, Joost; Facun, Kryss; de Vries, Michel; Stielow, J Benjamin

    2015-12-01

    Thermophilic fungi have the potential to produce industrial-relevant thermostable enzymes, in particular for the degradation of plant biomass. Sordariales is one of the few fungal orders containing several thermophilic taxa, of which many have been associated with the production of thermostable enzymes. The evolutionary affiliation of Sordariales fungi, especially between thermophiles and non-thermophilic relatives, is however poorly understood. Phylogenetic analysis within the current study was based on sequence data, derived from a traditional Sanger and highly multiplexed targeted next generation sequencing approach of 45 isolates. The inferred phylogeny and detailed growth analysis rendered the trait 'thermophily' as polyphyletic within Chaetomiaceae (Sordariales, Sordariomycetes), and characteristic to: Myceliophthora spp., Thielavia terrestris, Chaetomium thermophilum, and Mycothermus thermophilus. Compared to mesophiles, the isolates within thermophilic taxa produced enzyme mixtures with the highest thermostability of known cellulase activities. Temperature profiles of the enzyme activities correlated strongly with the optimal growth temperatures of the isolates but not with their phylogenetic relationships. This strong correlation between growth and enzyme characteristics indicated that detailed analysis of growth does give predictive information on enzyme physiology. The variation in growth and enzyme characteristics reveals these fungi as an excellent platform to better understand fungal thermophily and enzyme thermostability. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  11. Thermostable endoglucanases in the liquefaction of hydrothermally pretreated wheat straw

    Directory of Open Access Journals (Sweden)

    Siika-aho Matti

    2011-01-01

    Full Text Available Abstract Background Thermostable enzymes have several benefits in lignocellulose processing. In particular, they potentially allow the use of increased substrate concentrations (because the substrate viscosity decreases as the temperature increases, resulting in improved product yields and reduced capital and processing costs. A short pre-hydrolysis step at an elevated temperature using thermostable enzymes aimed at rapid liquefaction of the feedstock is seen as an attractive way to overcome the technical problems (such as poor mixing and mass transfer properties connected with high initial solid loadings in the lignocellulose to ethanol process. Results The capability of novel thermostable enzymes to reduce the viscosity of high-solid biomass suspensions using a real-time viscometric measurement method was investigated. Heterologously expressed enzymes from various thermophilic organisms were compared for their ability to liquefy the lignocellulosic substrate, hydrothermally pretreated wheat straw. Once the best enzymes were identified, the optimal temperatures for these enzymes to decrease substrate viscosity were compared. The combined hydrolytic properties of the thermostable preparations were tested in hydrolysis experiments. The studied mixtures were primarily designed to have good liquefaction potential, and therefore contained an enhanced proportion of the key liquefying enzyme, EGII/Cel5A. Conclusions Endoglucanases were shown to have a superior ability to rapidly reduce the viscosity of the 15% (w/w; dry matter hydrothermally pretreated wheat straw. Based on temperature profiling studies, Thermoascus aurantiacus EGII/Cel5A was the most promising enzyme for biomass liquefaction. Even though they were not optimized for saccharification, many of the thermostable enzyme mixtures had superior hydrolytic properties compared with the commercial reference enzymes at 55°C.

  12. Development of a thermostable microneedle patch for influenza vaccination

    Science.gov (United States)

    Mistilis, Matthew; Bommarius, Andreas S; Prausnitz, Mark R.

    2017-01-01

    The goal of this study is to develop thermostable microneedle patch formulations for influenza vaccine that can be partially or completely removed from the cold chain. During vaccine drying associated with microneedle patch manufacturing, ammonium acetate and HEPES buffer salts stabilized influenza vaccine, surfactants had little effect during drying, drying temperature had weak effects on vaccine stability, and drying on polydimethylsiloxane led to increased stability compared to drying on stainless steel. A number of excipients, mostly polysaccharides and some amino acids, further stabilized the influenza vaccine during drying. Over longer time scales of storage, combinations of stabilizers preserved the most vaccine activity. Finally, dissolving microneedle patches formulated with arginine and calcium heptagluconate had no significant activity loss for all three strains of seasonal influenza vaccine during storage at room temperature for six months. We conclude that appropriately formulated microneedle patches can exhibit remarkable thermostability that could enable storage and distribution of influenza vaccine outside the cold chain. PMID:25448542

  13. Thermostable Subunit Vaccines for Pulmonary Delivery: How Close Are We?

    DEFF Research Database (Denmark)

    Foged, Camilla

    2016-01-01

    , such as influenza, tuberculosis, and Ebola, for which no good universal vaccines exist. At least two pharmaceutical improvements are expected to help filling this gap: i) The development of thermostable vaccine dosage forms, and ii) the full exploitation of the adjuvant technology for subunit vaccines to potentiate...... strong immune responses. This review highlights the status and recent advances in formulation and pulmonary delivery of thermostable human subunit vaccines. Such vaccines are very appealing from compliance, distribution and immunological point of view: Being non-invasive, inhalable vaccines are self...... immunity. Here, I review state of the art and perspectives in formulation design and processing methods for powder-based subunit vaccines intended for pulmonary administration, and present dry powder inhaler technologies suitable for translating these vaccines into clinical trials....

  14. Thermostable cellulases, and mutants thereof, capable of hydrolyzing cellulose in ionic liquid

    Science.gov (United States)

    Sapra, Rajat; Datta, Supratim; Chen, Zhiwei; Holmes, Bradley M.; Simmons, Blake A.; Blanch, Harvey W.

    2016-04-26

    The present invention provides for a composition comprising an ionic liquid and a thermostable cellulose, and a method of hydrolyzing a cellulose, comprising: (a) providing a composition comprising a solution comprising an ionic liquid and a cellulose, and (b) introducing a thermostable cellulase to the solution, such that the cellulose is hydrolyzed by the cellulase. The present invention also provides for a Thermatoga maritima thermostable cellulase mutant with increased cellulase activity.

  15. Uptake of 3-[125I]iodo-α-methyl-L-tyrosine into colon cancer DLD-1 cells: characterization and inhibitory effect of natural amino acids and amino acid-like drugs

    International Nuclear Information System (INIS)

    Shikano, Naoto; Ogura, Masato; Okudaira, Hiroyuki; Nakajima, Syuichi; Kotani, Takashi; Kobayashi, Masato; Nakazawa, Shinya; Baba, Takeshi; Yamaguchi, Naoto; Kubota, Nobuo; Iwamura, Yukio; Kawai, Keiichi

    2010-01-01

    Introduction: We examined 3-[ 123 I]iodo-α-methyl-L-tyrosine ([ 123 I]IMT) uptake and inhibition by amino acids and amino acid-like drugs in the human DLD-1 colon cancer cell line, to discuss correlation between the inhibition effect and structure. Methods: Expression of relevant neutral amino acid transporters was examined by real-time PCR with DLD-1 cells. The time course of [ 125 I]IMT uptake, contributions of transport systems, concentration dependence and inhibition effects by amino acids and amino acid-like drugs (1 mM) on [ 125 I]IMT uptake were examined. Results: Expression of system L (4F2hc, LAT1 and LAT2), system A (ATA1, ATA2) and system ASC (ASCT1) was strongly detected; system L (LAT3, LAT4) and MCT8 were weakly detected; and B 0 AT was not detected. [ 125 I]IMT uptake in DLD-1 cells involved Na + -independent system L primarily and Na + -dependent system(s). Uptake of [ 125 I]IMT in Na + -free buffer followed Michaelis-Menten kinetics, with a K m of 78 μM and V max of 333 pmol/10 6 cells per minute. Neutral D- and L-amino acids with branched or aromatic large side chains inhibited [ 125 I]IMT uptake. Tyrosine analogues, tryptophan analogues, L-phenylalanine and p-halogeno-L-phenylalanines, and gamma amino acids [including 3,4-dihydroxy-L-phenylalanine (L-DOPA), DL-threo-β-(3,4-dihydroxyphenyl)serine (DOPS), 4-[bis(2-chloroethyl)amino]-L-phenylalanine and 1-(aminomethyl)-cyclohexaneacetic acid] strongly inhibited [ 125 I]IMT uptake, but L-tyrosine methyl ester and R(+)/S(-)-baclofen weakly inhibited uptake. The substrates of system ASC and A did not inhibit [ 125 I]IMT uptake except L-serine and D/L-cysteine. Conclusions: [ 125 I]IMT uptake in DLD-1 cells involves mostly LAT1 and its substrates' (including amino acid-like drugs derived from tyrosine, tryptophan and phenylalanine) affinity to transport via LAT1. Whether transport of gamma amino acid analogues is involved in LAT1 depends on the structure of the group corresponding to the amino acid

  16. Spectroscopic analyses on interaction of o-Vanillin- D-Phenylalanine, o-Vanillin- L-Tyrosine and o-Vanillin- L-Levodopa Schiff Bases with bovine serum albumin (BSA)

    Science.gov (United States)

    Gao, Jingqun; Guo, Yuwei; Wang, Jun; Wang, Zhiqiu; Jin, Xudong; Cheng, Chunping; Li, Ying; Li, Kai

    2011-04-01

    In this work, three o-Vanillin Schiff Bases (o-VSB: o-Vanillin- D-Phenylalanine (o-VDP), o-Vanillin- L-Tyrosine (o-VLT) and o-Vanillin- L-Levodopa (o-VLL)) with alanine constituent were synthesized by direct reflux method in ethanol solution, and then were used to study the interaction to bovine serum albumin (BSA) molecules by fluorescence spectroscopy. Based on the fluorescence quenching calculation, the bimolecular quenching constant ( Kq), apparent quenching constant ( Ksv), effective binding constant ( KA) and corresponding dissociation constant ( KD) as well as binding site number ( n) were obtained. In addition, the binding distance ( r) was also calculated according to Foster's non-radioactive energy transfer theory. The results show that these three o-VSB can efficiently bind to BSA molecules, but the binding array order is o-VDP-BSA > o-VLT-BSA > o-VLL-BSA. Synchronous fluorescence spectroscopy indicates that the o-VDP is more accessibility to tryptophan (Trp) residues of BSA molecules than to tyrosine (Tyr) residues. Nevertheless, the o-VLT and o-VLL are more accessibility to Tyr residues than to Trp residues.

  17. Hydrogen Peroxide Treatment and the Phenylpropanoid Pathway Precursors Feeding Improve Phenolics and Antioxidant Capacity of Quinoa Sprouts via an Induction of L-Tyrosine and L-Phenylalanine Ammonia-Lyases Activities

    Directory of Open Access Journals (Sweden)

    Michał Świeca

    2016-01-01

    Full Text Available Hydrogen peroxide treatment and the phenylpropanoid pathway precursors feeding affected the antioxidant capacity of quinoa sprouts. Compared to the control, total phenolics content was significantly increased by treatment of control sprouts with 50 mM and 200 mM H2O2—an elevation of about 24% and 28%, respectively. The highest increase of flavonoids content was found for the sprouts treated with 200 mM H2O2 obtained from seeds fed with shikimic acid. All the studied modifications increased the antioxidant potential of sprouts (at least by 50% compared to control. The highest reducing power was found for the sprouts treated with 200 mM H2O2 obtained by phenylalanine feeding (5.03 mg TE/g DW and those obtained from the seeds fed with tyrosine (5.26 mg TE/g DW. The activities of L-tyrosine (TAL and L-phenylalanine (PAL ammonia-lyases were strongly affected by germination time as well as the applied modification of sprouting. On the 3rd day the highest PAL activity was determined for both untreated and induced with 50 mM H2O2 sprouts obtained by phenylalanine feeding. H2O2 induced TAL activity; the highest TAL activity was determined for 3-day-old sprouts induced with 200 mM H2O2 obtained from seeds fed with phenylalanine.

  18. The Impact of Making Vaccines Thermostable in Niger’s Vaccine Supply Chain

    Science.gov (United States)

    Lee, Bruce Y.; Cakouros, Brigid E.; Assi, Tina-Marie; Connor, Diana L.; Welling, Joel; Kone, Souleymane; Djibo, Ali; Wateska, Angela R.; Pierre, Lionel; Brown, Shawn T.

    2012-01-01

    Objective Determine the effects on the vaccine cold chain of making different types of World Health Organization (WHO) Expanded Program on Immunizations (EPI) vaccines thermostable. Methods Utilizing a detailed computational, discrete-event simulation model of the Niger vaccine supply chain, we simulated the impact of making different combinations of the six current EPI vaccines thermostable. Findings Making any EPI vaccine thermostable relieved existing supply chain bottlenecks (especially at the lowest levels), increased vaccine availability of all EPI vaccines, and decreased cold storage and transport capacity utilization. By far, the most substantial impact came from making the pentavalent vaccine thermostable, increasing its own vaccine availability from 87% to 97% and the vaccine availabilities of all other remaining non-thermostable EPI vaccines to over 93%. By contrast, making each of the other vaccines thermostable had considerably less effect on the remaining vaccines, failing to increase the vaccine availabilities of other vaccines to more than 89%. Making tetanus toxoid vaccine along with the pentavalent thermostable further increased the vaccine availability of all EPI vaccines by at least 1–2%. Conclusion Our study shows the potential benefits of making any of Niger’s EPI vaccines thermostable and therefore supports further development of thermostable vaccines. Eliminating the need for refrigerators and freezers should not necessarily be the only benefit and goal of vaccine thermostability. Rather, making even a single vaccine (or some subset of the vaccines) thermostable could free up significant cold storage space for other vaccines, and thereby help alleviate supply chain bottlenecks that occur throughout the world. PMID:22789507

  19. The impact of making vaccines thermostable in Niger's vaccine supply chain.

    Science.gov (United States)

    Lee, Bruce Y; Cakouros, Brigid E; Assi, Tina-Marie; Connor, Diana L; Welling, Joel; Kone, Souleymane; Djibo, Ali; Wateska, Angela R; Pierre, Lionel; Brown, Shawn T

    2012-08-17

    Determine the effects on the vaccine cold chain of making different types of World Health Organization (WHO) Expanded Program on Immunizations (EPI) vaccines thermostable. Utilizing a detailed computational, discrete-event simulation model of the Niger vaccine supply chain, we simulated the impact of making different combinations of the six current EPI vaccines thermostable. Making any EPI vaccine thermostable relieved existing supply chain bottlenecks (especially at the lowest levels), increased vaccine availability of all EPI vaccines, and decreased cold storage and transport capacity utilization. By far, the most substantial impact came from making the pentavalent vaccine thermostable, increasing its own vaccine availability from 87% to 97% and the vaccine availabilities of all other remaining non-thermostable EPI vaccines to over 93%. By contrast, making each of the other vaccines thermostable had considerably less effect on the remaining vaccines, failing to increase the vaccine availabilities of other vaccines to more than 89%. Making tetanus toxoid vaccine along with the pentavalent thermostable further increased the vaccine availability of all EPI vaccines by at least 1-2%. Our study shows the potential benefits of making any of Niger's EPI vaccines thermostable and therefore supports further development of thermostable vaccines. Eliminating the need for refrigerators and freezers should not necessarily be the only benefit and goal of vaccine thermostability. Rather, making even a single vaccine (or some subset of the vaccines) thermostable could free up significant cold storage space for other vaccines, and thereby help alleviate supply chain bottlenecks that occur throughout the world. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. EFFECTS OF CHANGING THE INTERACTION BETWEEN SUBDOMAINS ON THE THERMOSTABILITY OF BACILLUS NEUTRAL PROTEASES

    NARCIS (Netherlands)

    EIJSINK, VGH; VRIEND, G; VANDERVINNE, B; HAZES, B; VANDENBURG, B; VENEMA, G

    1992-01-01

    Variants of the thermolabile neutral protease (Npr) of B. subtilis (Npr-sub) and the thermostable neutral protease of B. stearothermophilus (Npr-ste) were produced by means of site-directed mutagenesis and the effects of the mutations on thermostability were determined. Mutations were designed to

  1. EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA); Scientific Opinion on the substantiation of a health claim related to L-tyrosine and contribution to normal synthesis of dopamine pursuant to Article 13(5) of Regulation (EC) No 1924/2006

    DEFF Research Database (Denmark)

    Tetens, Inge

    Following an application from Vitabiotics Ltd. pursuant to Article 13(5) of Regulation (EC) No 1924/2006 via the Competent Authority of the United Kingdom, the Panel on Dietetic Products, Nutrition and Allergies was asked to deliver an opinion on the scientific substantiation of a health claim...... relationship has been established between the consumption of L-tyrosine in a protein adequate diet and contribution to normal synthesis of dopamine. However, no evidence has been provided that the protein supply in the diet of the European population is not sufficient to fulfil this function of the amino acid....... The following wording reflects the scientific evidence: “L-tyrosine contributes to normal synthesis of dopamine”. In order to bear the claim a food should be at least a source of protein as per Annex to Regulation (EC) No 1924/2006. Such amounts can be easily consumed as part of a balanced diet. The target...

  2. Useful halophilic, thermostable and ionic liquids tolerant cellulases

    Science.gov (United States)

    Zhang, Tao; Datta, Supratim; Simmons, Blake A.; Rubin, Edward M.

    2016-06-28

    The present invention provides for an isolated or recombinant polypeptide comprising an amino acid sequence having at least 70% identity with the amino acid sequence of a Halorhabdus utahensis cellulase, such as Hu-CBH1, wherein said amino acid sequence has a halophilic thermostable and/or thermophilic cellobiohydrolase (CBH) activity. In some embodiments, the polypeptide has a CBH activity that is resistant to up to about 20% of ionic liquids. The present invention also provides for compositions comprising and methods using the isolated or recombinant polypeptide.

  3. Understanding thermostability and pH dependent properties of proteins

    DEFF Research Database (Denmark)

    Galberg, Pernille

    The work performed in this thesis is part of a larger project (“Computational design of stable enzymes”) involving several research teams, which aimed to improve PROPKA (http://propka.ki.ku.dk) and to provide the scientific community with a computational protocol and associated PROPKA program......, which could be used for predicting mutations with expectation of increased thermostability at a certain pH value or a shifted pH activity optimum. The ability of a Bacillus circulans xylanase (BCX) mutant (N35D/A115E) to induce a decrease in pH activity optimum was evaluated by a pH dependent xylanase...

  4. An Interindividual Comparison of O-(2- [18F]Fluoroethyl)-L-Tyrosine (FET)– and L-[Methyl-11C]Methionine (MET)–PET in Patients With Brain Gliomas and Metastases

    International Nuclear Information System (INIS)

    Grosu, Anca-Ligia; Astner, Sabrina T.; Riedel, Eva; Nieder, Carsten; Wiedenmann, Nicole; Heinemann, Felix; Schwaiger, Markus

    2011-01-01

    Purpose: L-[methyl- 11 C]methionine (MET)–positron emission tomography (PET) has a high sensitivity and specificity for imaging of gliomas and metastatic brain tumors. The short half-life of 11 C (20 minutes) limits the use of MET-PET to institutions with onsite cyclotron. O-(2- [ 18 F]fluoroethyl)-L-tyrosine (FET) is labeled with 18 F (half-life, 120 minutes) and could be used much more broadly. This study compares the uptake of FET and MET in gliomas and metastases, as well as treatment-induced changes. Furthermore, it evaluates the gross tumor volume (GTV) of gliomas defined on PET and magnetic resonance imaging (MRI). Methods and Materials: We examined 42 patients with pretreated gliomas (29 patients) or brain metastases (13 patients) prospectively by FET- and MET-PET on the same day. Uptake of FET and MET was quantified by standardized uptake values. Imaging contrast was assessed by calculating lesion–to–gray matter ratios. Tumor extension was quantified by contouring GTV in 17 patients with brain gliomas. Gross tumor volume on PET was compared with GTV on MRI. Sensitivity and specificity of MET- and FET-PET for differentiation of viable tumor from benign changes were evaluated by comparing the PET result with histology or clinical follow-up. Results: There was a strong linear correlation between standardized uptake values calculated for both tracers in cortex and lesions: r = 0.78 (p = 0.001) and r = 0.84 (p 18 F]fluoroethyl)-L-tyrosine–PET and MET-PET provide comparable diagnostic information on gliomas and brain metastases. Like MET-PET, FET-PET can be used for differentiation of residual or recurrent tumor from treatment-related changes/pseudoprogression, as well as for delineation of gliomas.

  5. Molecular Dynamics Approach in Designing Thermostable Aspergillus niger Xylanase

    Science.gov (United States)

    Malau, N. D.; Sianturi, M.

    2017-03-01

    Molecular dynamics methods we have applied as a tool in designing thermostable Aspergillus niger Xylanase, by examining Root Mean Square Deviation (RMSD) and The Stability of the Secondary Structure of enzymes structure at its optimum temperature and compare with its high temperature behavior. As RMSD represents structural fluctuation at a particular temperature, a better understanding of this factor will suggest approaches to bioengineer these enzymes to enhance their thermostability. In this work molecular dynamic simulations of Aspergillus niger xylanase (ANX) have been carried at 400K (optimum catalytic temperature) for 2.5 ns and 500K (ANX reported inactive temperature) for 2.5 ns. Analysis have shown that the Root Mean Square Deviation (RMSD) significant increase at higher temperatures compared at optimum temperature and some of the secondary structures of ANX that have been damaged at high temperature. Structural analysis revealed that the fluctuations of the α-helix and β-sheet regions are larger at higher temperatures compared to the fluctuations at optimum temperature.

  6. Potential and utilization of thermophiles and thermostable enzymes in biorefining

    Directory of Open Access Journals (Sweden)

    Karlsson Eva

    2007-03-01

    Full Text Available Abstract In today's world, there is an increasing trend towards the use of renewable, cheap and readily available biomass in the production of a wide variety of fine and bulk chemicals in different biorefineries. Biorefineries utilize the activities of microbial cells and their enzymes to convert biomass into target products. Many of these processes require enzymes which are operationally stable at high temperature thus allowing e.g. easy mixing, better substrate solubility, high mass transfer rate, and lowered risk of contamination. Thermophiles have often been proposed as sources of industrially relevant thermostable enzymes. Here we discuss existing and potential applications of thermophiles and thermostable enzymes with focus on conversion of carbohydrate containing raw materials. Their importance in biorefineries is explained using examples of lignocellulose and starch conversions to desired products. Strategies that enhance thermostablity of enzymes both in vivo and in vitro are also assessed. Moreover, this review deals with efforts made on developing vectors for expressing recombinant enzymes in thermophilic hosts.

  7. Thermostability promotes the cooperative function of split adenylate kinases.

    Science.gov (United States)

    Nguyen, Peter Q; Liu, Shirley; Thompson, Jeremy C; Silberg, Jonathan J

    2008-05-01

    Proteins can often be cleaved to create inactive polypeptides that associate into functional complexes through non-covalent interactions, but little is known about what influences the cooperative function of the ensuing protein fragments. Here, we examine whether protein thermostability affects protein fragment complementation by characterizing the function of split adenylate kinases from the mesophile Bacillus subtilis (AKBs) and the hyperthermophile Thermotoga neapolitana (AKTn). Complementation studies revealed that the split AKTn supported the growth of Escherichia coli with a temperature-sensitive AK, but not the fragmented AKBs. However, weak complementation occurred when the AKBs fragments were fused to polypeptides that strongly associate, and this was enhanced by a Q16L mutation that thermostabilizes the full-length protein. To examine how the split AK homologs differ in structure and function, their catalytic activity, zinc content, and circular dichroism spectra were characterized. The reconstituted AKTn had higher levels of zinc, greater secondary structure, and >10(3)-fold more activity than the AKBs pair, albeit 17-fold less active than full-length AKTn. These findings provide evidence that the design of protein fragments that cooperatively function can be improved by choosing proteins with the greatest thermostability for bisection, and they suggest that this arises because hyperthermophilic protein fragments exhibit greater residual structure compared to their mesophilic counterparts.

  8. A novel non-thermostable deuterolysin from Aspergillus oryzae.

    Science.gov (United States)

    Maeda, Hiroshi; Katase, Toru; Sakai, Daisuke; Takeuchi, Michio; Kusumoto, Ken-Ichi; Amano, Hitoshi; Ishida, Hiroki; Abe, Keietsu; Yamagata, Youhei

    2016-09-01

    Three putative deuterolysin (EC 3.4.24.29) genes (deuA, deuB, and deuC) were found in the Aspergillus oryzae genome database ( http://www.bio.nite.go.jp/dogan/project/view/AO ). One of these genes, deuA, was corresponding to NpII gene, previously reported. DeuA and DeuB were overexpressed by recombinant A. oryzae and were purified. The degradation profiles against protein substrates of both enzymes were similar, but DeuB showed wider substrate specificity against peptidyl MCA-substrates compared with DeuA. Enzymatic profiles of DeuB except for thermostability also resembled those of DeuA. DeuB was inactivated by heat treatment above 80° C, different from thermostable DeuA. Transcription analysis in wild type A. oryzae showed only deuB was expressed in liquid culture, and the addition of the proteinous substrate upregulated the transcription. Furthermore, the NaNO3 addition seems to eliminate the effect of proteinous substrate for the transcription of deuB.

  9. Comparison of the uptake of [123/125I]-2-iodo-D-tyrosine and [123/125I]-2-iodo-L-tyrosine in R1M rhabdomyosarcoma cells in vitro and in R1M tumor-bearing Wag/Rij rats in vivo

    International Nuclear Information System (INIS)

    Bauwens, Matthias; Lahoutte, Tony; Kersemans, Ken; Gallez, Carol; Bossuyt, Axel; Mertens, John

    2006-01-01

    Introduction: Recently, promising results concerning uptake in vivo in tumors of D-amino acids have been published. Therefore, we decided to evaluate the tumor uptake of the D-analogue of [ 123 I]-2-iodo-L-tyrosine, a tracer recently introduced by our group into clinical trials. The uptake of 2-amino-3-(4-hydroxy-2-[ 123/125 I]iodophenyl)-D-propanoic acid (2-iodo-D-tyrosine) was studied in vitro in LAT1-expressing R1M rat rhabdomyosarcoma cells and in vivo in R1M tumor-bearing Wag/Rij rats. Methods: The uptake of [ 125 I]-2-iodo-L-tyrosine and [ 125 I]-2-iodo-D-tyrosine into R1M cells was determined in appropriate buffers, allowing the study of the involved transport systems. In vivo, the biodistribution in R1M-bearing rats of [ 123 I]-2-iodo-L-tyrosine and [ 123 I]-2-iodo-D-tyrosine was performed by both dynamic and static planar imaging with a gamma camera. Results: In in vitro conditions, the uptake of both [ 125 I]-2-iodo-L-tyrosine and [ 125 I]-2-iodo-D-tyrosine in the HEPES buffer was 25% higher in the presence of Na + ions. In the absence of Na + ions, [ 125 I]-2-iodo-D-tyrosine was taken up reversibly in the R1M cells, with an apparent accumulation, probably for the larger part by the LAT1 system. Dynamic planar imaging showed that the uptake in the tumors of [ 123 I]-2-iodo-D-tyrosine was somewhat lower than that of [ 123 I]-2-iodo-L-tyrosine. At 30 min postinjection, the mean differential uptake ratio values of the L- and D-enantiomers are 2.5±0.7 and 1.7±0.6, respectively. Although the uptake of the D-isomer is lower, probably due to a faster clearance from the blood, the tumor-background ratio is the same as that of the L-analogue. Conclusion: A large part (75%) of [ 125 I]-2-iodo-D-tyrosine in vitro and [ 123 I]-2-iodo-D-tyrosine in vivo is reversibly highly taken up in R1M tumor cells by Na + -independent LAT transport systems, more likely by the LAT1. The clearance from the blood of [ 123 I]-2-iodo-D-tyrosine in the rats is faster than that of the

  10. Cooperation between catalytic and DNA binding domains enhances thermostability and supports DNA synthesis at higher temperatures by thermostable DNA polymerases.

    Science.gov (United States)

    Pavlov, Andrey R; Pavlova, Nadejda V; Kozyavkin, Sergei A; Slesarev, Alexei I

    2012-03-13

    We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases [Pavlov, A. R., et al. (2002) Proc. Natl. Acad. Sci. U.S.A.99, 13510-13515]. The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various sequence-nonspecific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting helix-hairpin-helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of Topo V HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105 °C by maintaining processivity of DNA synthesis at high temperatures. We found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding of templates to DNA polymerases.

  11. Phosphate and arsenate removal efficiency by thermostable ferritin enzyme from Pyrococcus furiosus using radioisotopes

    KAUST Repository

    Sevcenco, Ana-Maria

    2015-03-13

    Oxo-anion binding properties of the thermostable enzyme ferritin from Pyrococcus furiosus were characterized with radiography. Radioisotopes 32P and 76As present as oxoanions were used to measure the extent and the rate of their absorption by the ferritin. Thermostable ferritin proved to be an excellent system for rapid phosphate and arsenate removal from aqueous solutions down to residual concentrations at the picomolar level. These very low concentrations make thermostable ferritin a potential tool to considerably mitigate industrial biofouling by phosphate limitation or to remove arsenate from drinking water.

  12. Pretreatment evaluation of carcinomas of the hypopharynx and larynx with {sup 18}F-fluorodeoxyglucose, {sup 123}I-{alpha}-methyl-L-tyrosine and {sup 99m}Tc-hexakis-2-methoxyisobutylisonitrile

    Energy Technology Data Exchange (ETDEWEB)

    Henze, Marcus; Eisenhut, Michael; Haberkorn, Uwe [Department of Nuclear Medicine, University of Heidelberg (Germany); German Cancer Research Center, Heidelberg (Germany); Mohammed, Ashour; Mier, Walter [Department of Nuclear Medicine, University of Heidelberg (Germany); Rudat, Volker [Department of Radiotherapy, University of Heidelberg (Germany); Dietz, Andreas; Nollert, Joerg [Department of Otolaryngology, Head and Neck Surgery, University of Heidelberg (Germany)

    2002-03-01

    While fluorine-18 2-fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET) is helpful in the pretherapeutic evaluation of head and neck cancer, it is only available in selected centres. Therefore, single-photon emission tomography (SPET) tracers would be desirable if they were to demonstrate tumour uptake reliably. This multitracer study was performed to evaluate the pretherapeutic uptake of the SPET tracers iodine-123 {alpha}-methyl-L-tyrosine (IMT) and technetium-99m hexakis-2-methoxyisobutylisonitrile ({sup 99m}Tc-MIBI) in primary carcinomas of the hypopharynx and larynx and to compare the results with those of FDG PET. We examined 22 fasted patients (20 male, 2 female, mean age 60.5{+-}10.2 years) with histologically confirmed carcinoma of the hypopharynx (n=9) or larynx (n=13), within 1 week before therapy. In 20 patients a cervical PET scan was acquired after intravenous injection of 232{+-}43 MBq {sup 18}F-FDG. Data analysis was semiquantitative, being based on standardised uptake values (SUVs) obtained at 60-90 min after injection. After injection of 570{+-}44 MBq {sup 99m}Tc-MIBI, cervical SPET scans (high-resolution collimator, 64 x 64 matrix, 64 steps, 40 s each) were obtained in 19 patients, 15 and 60 min after tracer injection. Finally, 15 min after injection of 327{+-}93 MBq {sup 123}I-IMT (medium-energy collimator, 64 x 64 matrix, 64 steps, 40 s each) SPET scans were acquired in 15 patients. All images were analysed visually and by calculating the tumour to nuchal muscle ratio. Eighteen of 20 (90%) carcinomas showed an increased glucose metabolism, with a mean SUV of 8.7 and a mean carcinoma to muscle ratio of 7.3. The IMT uptake was increased in 13 of 15 (87%) patients, who had a mean carcinoma to muscle ratio of 2.9. Only 13 of 19 (68%) carcinomas revealed pathological MIBI uptake, with a mean tumour to muscle ratio of 2.2 and no significant difference between early and late MIBI SPET images (P=0.23). In conclusion, in the diagnosis of

  13. Pretreatment evaluation of carcinomas of the hypopharynx and larynx with 18F-fluorodeoxyglucose, 123I-α-methyl-L-tyrosine and 99mTc-hexakis-2-methoxyisobutylisonitrile

    International Nuclear Information System (INIS)

    Henze, Marcus; Eisenhut, Michael; Haberkorn, Uwe; Mohammed, Ashour; Mier, Walter; Rudat, Volker; Dietz, Andreas; Nollert, Joerg

    2002-01-01

    While fluorine-18 2-fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET) is helpful in the pretherapeutic evaluation of head and neck cancer, it is only available in selected centres. Therefore, single-photon emission tomography (SPET) tracers would be desirable if they were to demonstrate tumour uptake reliably. This multitracer study was performed to evaluate the pretherapeutic uptake of the SPET tracers iodine-123 α-methyl-L-tyrosine (IMT) and technetium-99m hexakis-2-methoxyisobutylisonitrile ( 99m Tc-MIBI) in primary carcinomas of the hypopharynx and larynx and to compare the results with those of FDG PET. We examined 22 fasted patients (20 male, 2 female, mean age 60.5±10.2 years) with histologically confirmed carcinoma of the hypopharynx (n=9) or larynx (n=13), within 1 week before therapy. In 20 patients a cervical PET scan was acquired after intravenous injection of 232±43 MBq 18 F-FDG. Data analysis was semiquantitative, being based on standardised uptake values (SUVs) obtained at 60-90 min after injection. After injection of 570±44 MBq 99m Tc-MIBI, cervical SPET scans (high-resolution collimator, 64 x 64 matrix, 64 steps, 40 s each) were obtained in 19 patients, 15 and 60 min after tracer injection. Finally, 15 min after injection of 327±93 MBq 123 I-IMT (medium-energy collimator, 64 x 64 matrix, 64 steps, 40 s each) SPET scans were acquired in 15 patients. All images were analysed visually and by calculating the tumour to nuchal muscle ratio. Eighteen of 20 (90%) carcinomas showed an increased glucose metabolism, with a mean SUV of 8.7 and a mean carcinoma to muscle ratio of 7.3. The IMT uptake was increased in 13 of 15 (87%) patients, who had a mean carcinoma to muscle ratio of 2.9. Only 13 of 19 (68%) carcinomas revealed pathological MIBI uptake, with a mean tumour to muscle ratio of 2.2 and no significant difference between early and late MIBI SPET images (P=0.23). In conclusion, in the diagnosis of primary carcinomas of the

  14. Pretreatment evaluation of carcinomas of the hypopharynx and larynx with 18F-fluorodeoxyglucose, 123I-alpha-methyl-L-tyrosine and 99mTc-hexakis-2-methoxyisobutylisonitrile.

    Science.gov (United States)

    Henze, Marcus; Mohammed, Ashour; Mier, Walter; Rudat, Volker; Dietz, Andreas; Nollert, Jörg; Eisenhut, Michael; Haberkorn, Uwe

    2002-03-01

    While fluorine-18 2-fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET) is helpful in the pretherapeutic evaluation of head and neck cancer, it is only available in selected centres. Therefore, single-photon emission tomography (SPET) tracers would be desirable if they were to demonstrate tumour uptake reliably. This multitracer study was performed to evaluate the pretherapeutic uptake of the SPET tracers iodine-123 alpha-methyl-L-tyrosine (IMT) and technetium-99m hexakis-2-methoxyisobutylisonitrile (99mTc-MIBI) in primary carcinomas of the hypopharynx and larynx and to compare the results with those of FDG PET. We examined 22 fasted patients (20 male, 2 female, mean age 60.5+/-10.2 years) with histologically confirmed carcinoma of the hypopharynx (n=9) or larynx (n=13), within 1 week before therapy. In 20 patients a cervical PET scan was acquired after intravenous injection of 232+/-43 MBq 18F-FDG. Data analysis was semiquantitative, being based on standardised uptake values (SUVs) obtained at 60-90 min after injection. After injection of 570+/-44 MBq 99mTc-MIBI, cervical SPET scans (high-resolution collimator, 64x64 matrix, 64 steps, 40 s each) were obtained in 19 patients, 15 and 60 min after tracer injection. Finally, 15 min after injection of 327+/-93 MBq 123I-IMT (medium-energy collimator, 64x64 matrix, 64 steps, 40 s each) SPET scans were acquired in 15 patients. All images were analysed visually and by calculating the tumour to nuchal muscle ratio. Eighteen of 20 (90%) carcinomas showed an increased glucose metabolism, with a mean SUV of 8.7 and a mean carcinoma to muscle ratio of 7.3. The IMT uptake was increased in 13 of 15 (87%) patients, who had a mean carcinoma to muscle ratio of 2.9. Only 13 of 19 (68%) carcinomas revealed pathological MIBI uptake, with a mean tumour to muscle ratio of 2.2 and no significant difference between early and late MIBI SPET images (P=0.23). In conclusion, in the diagnosis of primary carcinomas of the

  15. Thermostating extended Lagrangian Born-Oppenheimer molecular dynamics.

    Science.gov (United States)

    Martínez, Enrique; Cawkwell, Marc J; Voter, Arthur F; Niklasson, Anders M N

    2015-04-21

    Extended Lagrangian Born-Oppenheimer molecular dynamics is developed and analyzed for applications in canonical (NVT) simulations. Three different approaches are considered: the Nosé and Andersen thermostats and Langevin dynamics. We have tested the temperature distribution under different conditions of self-consistent field (SCF) convergence and time step and compared the results to analytical predictions. We find that the simulations based on the extended Lagrangian Born-Oppenheimer framework provide accurate canonical distributions even under approximate SCF convergence, often requiring only a single diagonalization per time step, whereas regular Born-Oppenheimer formulations exhibit unphysical fluctuations unless a sufficiently high degree of convergence is reached at each time step. The thermostated extended Lagrangian framework thus offers an accurate approach to sample processes in the canonical ensemble at a fraction of the computational cost of regular Born-Oppenheimer molecular dynamics simulations.

  16. Phosphate and arsenate removal efficiency by thermostable ferritin enzyme from Pyrococcus furiosus using radioisotopes

    KAUST Repository

    Sevcenco, Ana-Maria; Paravidino, Monica; Vrouwenvelder, Johannes S.; Wolterbeek, Hubert Th.; van Loosdrecht, Mark C.M.; Hagen, Wilfred R.

    2015-01-01

    Oxo-anion binding properties of the thermostable enzyme ferritin from Pyrococcus furiosus were characterized with radiography. Radioisotopes 32P and 76As present as oxoanions were used to measure the extent and the rate of their absorption

  17. Exogenous cellulases of thermophilic micromycetes. Pt. 2. Thermostability of enzyme preparations

    Energy Technology Data Exchange (ETDEWEB)

    Kvesitadze, G; Gogilashvili, L; Svanidze, R; Buachidze, T; Chirgadze, L; Nizharadze, D

    1986-01-01

    The ability of a large number of higher fungi to form extracellular cellulases is investigated. Some representatives of these fungi grow at 40-50/sup 0/C, and form extracellular cellulases exceeding cellulases of mesophilic fungi in thermostability. It is shown that cellulases of higher thermophilic fungi differ by their thermostability. The temperature optimum of cellulase action of higher fungi occurs within 60-62/sup 0/C.

  18. Purification and Characterization of a Thermostable Lipase from Geobacillus thermodenitrificans IBRL-nra

    Directory of Open Access Journals (Sweden)

    Anuradha Balan

    2012-01-01

    Full Text Available Thermostable lipase from Geobacillus thermodenitrificans IBRL-nra was purified and characterized. The production of thermostable lipase from Geobacillus thermodenitrificans IBRL-nra was carried out in a shake-flask system at 65°C in cultivation medium containing; glucose 1.0% (w/v; yeast extract 1.25% (w/v; NaCl 0.45% (w/v olive oil 0.1% (v/v with agitation of 200 rpm for 24 hours. The extracted extracellular crude thermostable lipase was purified to homogeneity by using ultrafiltration, Heparin-affinity chromatography, and Sephadex G-100 gel-filtration chromatography by 34 times with a final yield of 9%. The molecular weight of the purified enzyme was estimated to be 30 kDa after SDS-PAGE analysis. The optimal temperature for thermostable lipase was 65°C and it retained its initial activity for 3 hours. Thermostable lipase activity was highest at pH 7.0 and stable for 16 hours at this pH at 65°C. Thermostable lipase showed elevated activity when pretreated with BaCl2, CaCl2, and KCl with 112%, 108%, and 106%, respectively. Lipase hydrolyzed tripalmitin (C16 and olive oil with optimal activity (100% compared to other substrates.

  19. Thermostable cross-protective subunit vaccine against Brucella species.

    Science.gov (United States)

    Cherwonogrodzky, John W; Barabé, Nicole D; Grigat, Michelle L; Lee, William E; Poirier, Robert T; Jager, Scott J; Berger, Bradley J

    2014-12-01

    A subunit vaccine candidate was produced from Brucella suis 145 (biovar 4; expressing both the A antigen of Brucella abortus and the M antigen of Brucella melitensis). The preparation consisted mostly of polysaccharide (PS; >90% [wt/wt]; both cell-associated PS and exo-PS were combined) and a small amount of protein (1 to 3%) with no apparent nucleic acids. Vaccinated mice were protected (these had a statistically significant reduction in bacterial colonization compared to that of unvaccinated controls) when challenged with representative strains of three Brucella species most pathogenic for humans, i.e., B. abortus, B. melitensis, and B. suis. As little as 1 ng of the vaccine, without added adjuvant, protected mice against B. suis 145 infection (5 × 10(5) CFU), and a single injection of 1 μg of this subunit vaccine protected mice from B. suis 145 challenge for at least 14 months. A single immunization induced a serum IgG response to Brucella antigens that remained elevated for up to 9 weeks. The use of heat (i.e., boiling-water bath, autoclaving) in the vaccine preparation showed that it was thermostable. This method also ensured safety and security. The vaccine produced was immunogenic and highly protective against multiple strains of Brucella and represents a promising candidate for further evaluation. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  20. Structure Based Thermostability Prediction Models for Protein Single Point Mutations with Machine Learning Tools.

    Directory of Open Access Journals (Sweden)

    Lei Jia

    Full Text Available Thermostability issue of protein point mutations is a common occurrence in protein engineering. An application which predicts the thermostability of mutants can be helpful for guiding decision making process in protein design via mutagenesis. An in silico point mutation scanning method is frequently used to find "hot spots" in proteins for focused mutagenesis. ProTherm (http://gibk26.bio.kyutech.ac.jp/jouhou/Protherm/protherm.html is a public database that consists of thousands of protein mutants' experimentally measured thermostability. Two data sets based on two differently measured thermostability properties of protein single point mutations, namely the unfolding free energy change (ddG and melting temperature change (dTm were obtained from this database. Folding free energy change calculation from Rosetta, structural information of the point mutations as well as amino acid physical properties were obtained for building thermostability prediction models with informatics modeling tools. Five supervised machine learning methods (support vector machine, random forests, artificial neural network, naïve Bayes classifier, K nearest neighbor and partial least squares regression are used for building the prediction models. Binary and ternary classifications as well as regression models were built and evaluated. Data set redundancy and balancing, the reverse mutations technique, feature selection, and comparison to other published methods were discussed. Rosetta calculated folding free energy change ranked as the most influential features in all prediction models. Other descriptors also made significant contributions to increasing the accuracy of the prediction models.

  1. Engineering increased thermostability in the GH-10 endo-1,4-ß-xylanase from Thermoascus aurantiacus CBMAI 756

    Science.gov (United States)

    The GH10 endo-xylanase from Thermoascus aurantiacus CBMAI 756 (XynA) is industrially attractive due to its considerable thermostability and high specific activity. Considering the possibility of a further improvement in thermostability, eleven mutants were created in the present study via site-direc...

  2. Structurally stabilized organosilane-templated thermostable mesoporous titania.

    Science.gov (United States)

    Amoli, Vipin; Tiwari, Rashmi; Dutta, Arghya; Bhaumik, Asim; Sinha, Anil Kumar

    2014-01-13

    Structurally thermostable mesoporous anatase TiO2 (m-TiO2) nanoparticles, uniquely decorated with atomically dispersed SiO2, is reported for the first time. The inorganic Si portion of the novel organosilane template, used as a mesopores-directing agent, is found to be incorporated in the pore walls of the titania aggregates, mainly as isolated sites. This is evident by transmission electron microscopy and high-angle annular dark field scanning transmission electron microscopy, combined with electron dispersive X-ray spectroscopy. This type of unique structure provides exceptional stability to this new material against thermal collapse of the mesoporous structure, which is reflected in its high surface area (the highest known for anatase titania), even after high-temperature (550 °C) calcination. Control of crystallite size, pore diameter, and surface area is achieved by varying the molar ratios of the titanium precursor and the template during synthesis. These mesoporous materials retain their porosity and high surface area after template removal and further NaOH/HCl treatment to remove silica. We investigate their performance for dye-sensitized solar cells (DSSCs) with bilayer TiO2 electrodes, which are prepared by applying a coating of m-TiO2 onto a commercial titania (P25) film. The high surface area of the upper mesoporous layer in the P25-m-TiO2 DSSC significantly increases the dye loading ability of the photoanode. The photocurrent and fill factor for the DSSC with the bilayer TiO2 electrode are greatly improved. The large increase in photocurrent current (ca. 56%) in the P25-m-TiO2 DSSC is believed to play a significant role in achieving a remarkable increase in the photovoltaic efficiency (60%) of the device, compared to DSSCs with a monolayer of P25 as the electrode. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. A comparative molecular dynamics study on thermostability of human and chicken prion proteins

    International Nuclear Information System (INIS)

    Ji, Hong-Fang; Zhang, Hong-Yu

    2007-01-01

    To compare the thermostabilities of human and chicken normal cellular prion proteins (HuPrP C and CkPrP C ), molecular dynamics (MD) simulations were performed for both proteins at an ensemble level (10 parallel simulations at 400 K and 5 parallel simulations at 300 K as a control). It is found that the thermostability of HuPrP C is comparable with that of CkPrP C , which implicates that the non-occurrence of prion diseases in non-mammals cannot be completely attributed to the thermodynamic properties of non-mammalian PrP C

  4. In Vitro Characterization of Thermostable CAM Rubisco Activase Reveals a Rubisco Interacting Surface Loop1[OPEN

    Science.gov (United States)

    Shivhare, Devendra

    2017-01-01

    To maintain metabolic flux through the Calvin-Benson-Bassham cycle in higher plants, dead-end inhibited complexes of Rubisco must constantly be engaged and remodeled by the molecular chaperone Rubisco activase (Rca). In C3 plants, the thermolability of Rca is responsible for the deactivation of Rubisco and reduction of photosynthesis at moderately elevated temperatures. We reasoned that crassulacean acid metabolism (CAM) plants must possess thermostable Rca to support Calvin-Benson-Bassham cycle flux during the day when stomata are closed. A comparative biochemical characterization of rice (Oryza sativa) and Agave tequilana Rca isoforms demonstrated that the CAM Rca isoforms are approximately10°C more thermostable than the C3 isoforms. Agave Rca also possessed a much higher in vitro biochemical activity, even at low assay temperatures. Mixtures of rice and agave Rca form functional hetero-oligomers in vitro, but only the rice isoforms denature at nonpermissive temperatures. The high thermostability and activity of agave Rca mapped to the N-terminal 244 residues. A Glu-217-Gln amino acid substitution was found to confer high Rca activity to rice Rca. Further mutational analysis suggested that Glu-217 restricts the flexibility of the α4-β4 surface loop that interacts with Rubisco via Lys-216. CAM plants thus promise to be a source of highly functional, thermostable Rca candidates for thermal fortification of crop photosynthesis. Careful characterization of their properties will likely reveal further protein-protein interaction motifs to enrich our mechanistic model of Rca function. PMID:28546437

  5. Ligand binding and thermostability of different allosteric states of the insulin zinc-hexamer

    DEFF Research Database (Denmark)

    Huus, Kasper; Havelund, Svend; Olsen, Helle B

    2006-01-01

    The influence of ligand binding and conformation state on the thermostability of hexameric zinc-insulin was studied by differential scanning calorimetry (DSC). The insulin hexamer exists in equilibrium between the forms T6, T3R3, and R6. Phenolic ligands induce and stabilize the T3R3- and R6-stat...

  6. Thermostability enhancement of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus by site-directed mutagenesis

    Science.gov (United States)

    Cellobiose 2-epimerase from the thermophile Caldicellulosiruptor saccharolyticus (CsCE) catalyzes the isomerization of lactose into lactulose, a non-digestible disaccharide widely used in food and pharmaceutical industries. Semi-rational approaches were applied to enhance the thermostability of CsCE...

  7. CHARACTERIZATION OF A NEW BACILLUS-STEAROTHERMOPHILUS ISOLATE - A HIGHLY THERMOSTABLE ALPHA-AMYLASE-PRODUCING STRAIN

    NARCIS (Netherlands)

    WIND, RD; BUITELAAR, RM; EGGINK, G; HUIZING, HJ; DIJKHUIZEN, L

    A novel strain of Bacillus stearothermophilus was isolated from samples of a potato-processing industry. Compared to known alpha-amylases from other B. stearothermophilus strains, the isolate was found to produce a highly thermostable alpha-amylase. The half-time of inactivation of this

  8. Enhanced Thermostability of Arabidopsis Rubisco activase improves photosynthesis and growth rates under moderate heat stress.

    Science.gov (United States)

    Kurek, Itzhak; Chang, Thom Kai; Bertain, Sean M; Madrigal, Alfredo; Liu, Lu; Lassner, Michael W; Zhu, Genhai

    2007-10-01

    Plant photosynthesis declines when the temperature exceeds its optimum range. Recent evidence indicates that the reduction in photosynthesis is linked to ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) deactivation due to the inhibition of Rubisco activase (RCA) under moderately elevated temperatures. To test the hypothesis that thermostable RCA can improve photosynthesis under elevated temperatures, we used gene shuffling technology to generate several Arabidopsis thaliana RCA1 (short isoform) variants exhibiting improved thermostability. Wild-type RCA1 and selected thermostable RCA1 variants were introduced into an Arabidopsis RCA deletion (Deltarca) line. In a long-term growth test at either constant 26 degrees C or daily 4-h 30 degrees C exposure, the transgenic lines with the thermostable RCA1 variants exhibited higher photosynthetic rates, improved development patterns, higher biomass, and increased seed yields compared with the lines expressing wild-type RCA1 and a slight improvement compared with untransformed Arabidopsis plants. These results provide clear evidence that RCA is a major limiting factor in plant photosynthesis under moderately elevated temperatures and a potential target for genetic manipulation to improve crop plants productivity under heat stress conditions.

  9. Not so monofunctional-a case of thermostable Thermobifida fusca catalase with peroxidase activity

    NARCIS (Netherlands)

    Lončar, Nikola; Fraaije, Marco W

    Thermobifida fusca is a mesothermophilic organism known for its ability to degrade plant biomass and other organics, and it was demonstrated that it represents a rich resource of genes encoding for potent enzymes for biocatalysis. The thermostable catalase from T. fusca has been cloned and

  10. Hot or not? Discovery and characterization of a thermostable alditol oxidase from Acidothermus cellulolyticus 11B

    NARCIS (Netherlands)

    Winter, Remko T.; Heuts, Dominic P. H. M.; Rijpkema, Egon M. A.; van Bloois, Edwin; Wijma, Hein J.; Fraaije, Marco W.

    We describe the discovery, isolation and characterization of a highly thermostable alditol oxidase from Acidothermus cellulolyticus 11B. This protein was identified by searching the genomes of known thermophiles for enzymes homologous to Streptomyces coelicolor A3(2) alditol oxidase (AldO). A gene

  11. Evaluating the value proposition for improving vaccine thermostability to increase vaccine impact in low and middle-income countries.

    Science.gov (United States)

    Karp, Christopher L; Lans, Deborah; Esparza, José; Edson, Eleanore B; Owen, Katey E; Wilson, Christopher B; Heaton, Penny M; Levine, Orin S; Rao, Raja

    2015-07-09

    The need to keep vaccines cold in the face of high ambient temperatures and unreliable access to electricity is a challenge that limits vaccine coverage in low and middle-income countries (LMICs). Greater vaccine thermostability is generally touted as the obvious solution. Despite conventional wisdom, comprehensive analysis of the value proposition for increasing vaccine thermostability has been lacking. Further, while significant investments have been made in increasing vaccine thermostability in recent years, no vaccine products have been commercialized as a result. We analyzed the value proposition for increasing vaccine thermostability, grounding the analysis in specific vaccine use cases (e.g., use in routine immunization [RI] programs, or in campaigns) and in the broader context of cold chain technology and country level supply chain system design. The results were often surprising. For example, cold chain costs actually represent a relatively small fraction of total vaccine delivery system costs. Further, there are critical, vaccine use case-specific temporal thresholds that need to be overcome for significant benefits to be reaped from increasing vaccine thermostability. We present a number of recommendations deriving from this analysis that suggest a rational path toward unlocking the value (maximizing coverage, minimizing total system costs) of increased vaccine thermostability, including: (1) the full range of thermostability of existing vaccines should be defined and included in their labels; (2) for new vaccines, thermostability goals should be addressed up-front at the level of the target product profile; (3) improving cold chain infrastructure and supply chain system design is likely to have the largest impact on total system costs and coverage in the short term-and will influence the degree of thermostability required in the future; (4) in the long term, there remains value in monitoring the emergence of disruptive technologies that could remove the

  12. Highly thermostable xylanase production from a thermophilic Geobacillus sp. strain WSUCF1 utilizing lignocellulosic biomass

    Directory of Open Access Journals (Sweden)

    Aditya eBhalla

    2015-06-01

    Full Text Available AbstractEfficient enzymatic hydrolysis of lignocellulose to fermentable sugars requires a complete repertoire of biomass deconstruction enzymes. Hemicellulases play an important role in hydrolyzing hemicellulose component of lignocellulose to xylo-oligosaccharides and xylose. Thermostable xylanases have been a focus of attention as industrially important enzymes due to their long shelf life at high temperatures. Geobacillus sp. strain WSUCF1 produced thermostable xylanase activity (crude xylanase cocktail when grown on xylan or various inexpensive untreated and pretreated lignocellulosic biomasses such as prairie cord grass and corn stover. The optimum pH and temperature for the crude xylanase cocktail were 6.5 and 70ºC, respectively. The WSUCF1 crude xylanase was found to be highly thermostable with half-lives of 18 and 12 days at 60 and 70ºC, respectively. At 70ºC, rates of xylan hydrolysis were also found to be better with the WSUCF1 secretome than those with commercial enzymes, i.e., for WSUCF1 crude xylanase, CellicHTec2, and AccelleraseXY, the percent xylan conversions were 68.9, 49.4, and 28.92, respectively. To the best of our knowledge, WSUCF1 crude xylanase cocktail is among the most thermostable xylanases produced by thermophilic Geobacillus spp. and other thermophilic microbes (optimum growth temperature ≤70ºC. High thermostability, activity over wide range of temperatures, and better xylan hydrolysis than commercial enzymes make WSUCF1 crude xylanase suitable for thermophilic lignocellulose bioconversion processes.

  13. Molecular characterization of a novel thermostable laccase PPLCC2 from the brown rot fungus Postia placenta MAD-698-R

    Directory of Open Access Journals (Sweden)

    Hongde An

    2015-11-01

    Conclusions: This is the first identified thermo activated and thermostable laccase in brown rot fungi. This investigation will contribute to understanding the roles played by laccases in brown rot fungi.

  14. Screening of strains with the high activity and thermostability nattokinase by {sup 60}Co γ-ray irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Shuying, Li; Ying, Nie; Huan, Du; Xuanming, Tang [Institute of Agro-products Processing Science and Technology, Chinese Academy of Agricultural Sciences, Beijing (China); Zhonglin, Zhao [College of Sciences, Henan Agricultural University, Zhengzhou (China); Xin, Ma [Agricultural Information Institute, Chinese Academic of Agricultural Sciences, Beijing (China); Yan, Li [School of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou (China)

    2013-06-15

    In this study, Bacillus natto was irradiated by {sup 60}Co γ-ray, and activity was determined by Casein plate method in order to get high activity and thermostability strains. 60 strains with high activity were obtained through irradiation by 800 Gy {sup 60}Co γ-ray. In this dose, the positive mutation rate was 45%. Then 60 strains was treated by different tempreture and 11 strains showed thermostability at 65℃. (authors)

  15. Screening of strains with the high activity and thermostability nattokinase by "6"0Co γ-ray irradiation

    International Nuclear Information System (INIS)

    Li Shuying; Nie Ying; Du Huan; Tang Xuanming; Zhao Zhonglin; Ma Xin; Li Yan

    2013-01-01

    In this study, Bacillus natto was irradiated by "6"0Co γ-ray, and activity was determined by Casein plate method in order to get high activity and thermostability strains. 60 strains with high activity were obtained through irradiation by 800 Gy "6"0Co γ-ray. In this dose, the positive mutation rate was 45%. Then 60 strains was treated by different tempreture and 11 strains showed thermostability at 65℃. (authors)

  16. FireProt: web server for automated design of thermostable proteins

    Science.gov (United States)

    Musil, Milos; Stourac, Jan; Brezovsky, Jan; Prokop, Zbynek; Zendulka, Jaroslav; Martinek, Tomas

    2017-01-01

    Abstract There is a continuous interest in increasing proteins stability to enhance their usability in numerous biomedical and biotechnological applications. A number of in silico tools for the prediction of the effect of mutations on protein stability have been developed recently. However, only single-point mutations with a small effect on protein stability are typically predicted with the existing tools and have to be followed by laborious protein expression, purification, and characterization. Here, we present FireProt, a web server for the automated design of multiple-point thermostable mutant proteins that combines structural and evolutionary information in its calculation core. FireProt utilizes sixteen tools and three protein engineering strategies for making reliable protein designs. The server is complemented with interactive, easy-to-use interface that allows users to directly analyze and optionally modify designed thermostable mutants. FireProt is freely available at http://loschmidt.chemi.muni.cz/fireprot. PMID:28449074

  17. Thermostability of bovine submaxillary mucin (BSM) in bulk solution and at a sliding interface

    DEFF Research Database (Denmark)

    Madsen, Jan Busk; Pakkanen, Kirsi I.; Lee, Seunghwan

    2014-01-01

    Thermostability of bovine submaxillary mucin (BSM) was studied in terms of its structure, hydrodynamic size, surface adsorption, and lubricating properties in the temperature range of 5-85°C. The overall random coil structure of BSM showed a gradual loosening with increasing temperature as charac......Thermostability of bovine submaxillary mucin (BSM) was studied in terms of its structure, hydrodynamic size, surface adsorption, and lubricating properties in the temperature range of 5-85°C. The overall random coil structure of BSM showed a gradual loosening with increasing temperature...... as characterized by circular dichroism (CD) spectroscopy, but this change was fully reversible upon lowering temperature. Extended heating up to 120min at 80°C did not make any appreciable changes in the structure of BSM when it was cooled to room temperature. The hydrodynamic size of BSM, as studied by dynamic...

  18. First isolation of a novel thermostable antifungal peptide secreted by Aspergillus clavatus.

    Science.gov (United States)

    Skouri-Gargouri, Houda; Gargouri, Ali

    2008-11-01

    A novel antifungal peptide produced by an indigenous fungal strain (VR) of Aspergillus clavatus was purified. The antifungal peptide was enriched in the supernatant after heat treatment at 70 degrees C. The thermostable character was exploited in the first purification step, as purified peptide was obtained after ultrafiltration and reverse phase-HPLC on C18 column application. The purified peptide named "AcAFP" for A. clavatus antifungal peptide, has molecular mass of 5773Da determined by MALDI-ToF spectrometry. The N-terminal sequence showed a notable identity to the limited family of antifungal peptides produced by ascomycetes fungi. The AcAFP activity remains intact even after heat treatment at 100 degrees C for 1h confirming its thermostability. It exhibits a strong inhibitory activity against mycelial growth of several serious human and plant pathogenic fungi: Fusariuym oxysporum, Fusarium solani, Aspergillus niger, Botrytis cinerea, Alternaria solani, whereas AcAFP did not affect yeast and bacterial growth.

  19. An optimized formulation of a thermostable spray dried virus-like particles vaccine against human papillomavirus

    Science.gov (United States)

    Saboo, Sugandha; Tumban, Ebenezer; Peabody, Julianne; Wafula, Denis; Peabody, David S.; Chackerian, Bryce; Muttil, Pavan

    2016-01-01

    Existing vaccines against human papillomavirus (HPV) require continuous cold-chain storage. Previously, we developed a bacteriophage virus-like particle (VLP) based vaccine for Human Papillomavirus (HPV) infection, which elicits broadly neutralizing antibodies against diverse HPV types. Here, we formulated these VLPs into a thermostable dry powder using a multi-component excipient system and by optimizing the spray drying parameters using a half-factorial design approach. Dry powder VLPs were stable after spray drying and after long-term storage at elevated temperatures. Immunization of mice with a single dose of reconstituted dry powder VLPs that were stored at 37°C for more than a year elicited high anti-L2 IgG antibody titers. Spray dried thermostable, broadly protective L2 bacteriophage VLPs vaccine could be accessible to remote regions of the world (where ~84% of cervical cancer patients reside) by eliminating the cold-chain requirement during transportation and storage. PMID:27019231

  20. MOLECULAR CLONING AND CHARACTERIZATION OF NOVEL THERMOSTABLE LIPASE FROM SHEWANELLA PUTREFACIENS AND USING ENZYMATIC BIODIESEL PRODUCTION

    Directory of Open Access Journals (Sweden)

    Fahri Akbas

    2015-02-01

    Full Text Available A novel thermostable lipase from Shewanella putrefaciens was identified, expressed in Escherichia coli, characterized and used in biodiesel production. Enzyme characterization was carried out by enzyme assay, SDS-PAGE and other biochemical reactions. The recombinant lipase was found to have a molecular mass of 29 kDa and exhibited lipase activity when Tween 80 was used as the substrate. The purified enzyme showed maximum activity at pH 5.0 and at 80°C. The recombinant lipase was used for the transesterification of canola oil and waste oil. The enzyme retains 50% of its activity at 90°C for 30 minutes. It is also able to retain 20% of its activity even at 100 °C for 20 minutes. These properties of the obtained new recombinant thermostable lipase make it promising as a biocatalyst for industrial processes.

  1. In Vitro Characterization of Thermostable CAM Rubisco Activase Reveals a Rubisco Interacting Surface Loop.

    Science.gov (United States)

    Shivhare, Devendra; Mueller-Cajar, Oliver

    2017-07-01

    To maintain metabolic flux through the Calvin-Benson-Bassham cycle in higher plants, dead-end inhibited complexes of Rubisco must constantly be engaged and remodeled by the molecular chaperone Rubisco activase (Rca). In C3 plants, the thermolability of Rca is responsible for the deactivation of Rubisco and reduction of photosynthesis at moderately elevated temperatures. We reasoned that crassulacean acid metabolism (CAM) plants must possess thermostable Rca to support Calvin-Benson-Bassham cycle flux during the day when stomata are closed. A comparative biochemical characterization of rice ( Oryza sativa ) and Agave tequilana Rca isoforms demonstrated that the CAM Rca isoforms are approximately10°C more thermostable than the C3 isoforms. Agave Rca also possessed a much higher in vitro biochemical activity, even at low assay temperatures. Mixtures of rice and agave Rca form functional hetero-oligomers in vitro, but only the rice isoforms denature at nonpermissive temperatures. The high thermostability and activity of agave Rca mapped to the N-terminal 244 residues. A Glu-217-Gln amino acid substitution was found to confer high Rca activity to rice Rca Further mutational analysis suggested that Glu-217 restricts the flexibility of the α4-β4 surface loop that interacts with Rubisco via Lys-216. CAM plants thus promise to be a source of highly functional, thermostable Rca candidates for thermal fortification of crop photosynthesis. Careful characterization of their properties will likely reveal further protein-protein interaction motifs to enrich our mechanistic model of Rca function. © 2017 American Society of Plant Biologists. All Rights Reserved.

  2. Tyr51: Key Determinant of the Low Thermostability of the Colwellia psychrerythraea Cold-Shock Protein.

    Science.gov (United States)

    Lee, Yeongjoon; Kwak, Chulhee; Jeong, Ki-Woong; Durai, Prasannavenkatesh; Ryu, Kyoung-Seok; Kim, Eun-Hee; Cheong, Chaejoon; Ahn, Hee-Chul; Kim, Hak Jun; Kim, Yangmee

    2018-05-18

    Cold-shock proteins (Csps) are expressed at lower-than-optimum temperatures, and they function as RNA chaperones; however, no structural studies on psychrophilic Csps have been reported. Here, we aimed to investigate the structure and dynamics of the Csp of psychrophile Colwellia psychrerythraea 34H, ( Cp-Csp). Although Cp-Csp shares sequence homology, common folding patterns, and motifs, including a five β-stranded barrel, with its thermophilic counterparts, its thermostability (37 °C) was markedly lower than those of other Csps. Cp-Csp binds heptathymidine with an affinity of 10 -7 M, thereby increasing its thermostability to 50 °C. Nuclear magnetic resonance spectroscopic analysis of the Cp-Csp structure and backbone dynamics revealed a flexible structure with only one salt bridge and 10 residues in the hydrophobic cavity. Notably, Cp-Csp contains Tyr51 instead of the conserved Phe in the hydrophobic core, and its phenolic hydroxyl group projects toward the surface. The Y51F mutation increased the stability of hydrophobic packing and may have allowed for the formation of a K3-E21 salt bridge, thereby increasing its thermostability to 43 °C. Cp-Csp exhibited conformational exchanges in its ribonucleoprotein motifs 1 and 2 (754 and 642 s -1 ), and heptathymidine binding markedly decreased these motions. Cp-Csp lacks salt bridges and has longer flexible loops and a less compact hydrophobic cavity resulting from Tyr51 compared to mesophilic and thermophilic Csps. These might explain the low thermostability of Cp-Csp. The conformational flexibility of Cp-Csp facilitates its accommodation of nucleic acids at low temperatures in polar oceans and its function as an RNA chaperone for cold adaptation.

  3. Design and Testing of a Thermostable Platform for Multimerization of Single Domain Antibodies

    Science.gov (United States)

    2012-08-01

    H.J. Properties , production, and applications of camelid single domain antibody fragments. Appl. Microbiol. Biot. 2007, 77, 13‒22. 2. Goldman...Conway, J.; Sherwood, L.J.; Fech, M.; Vo, B.; Liu, J.L.; Hayhurst, A. Thermostable llama single domain antibodies for detection of Botulinum A...antiparallel coiled-coil inserted. J. Mol. Bio. 2001, 306, 25‒35. 9. Liu, J.L.; Anderson, G.P.; Goldman, E.R. Isolation of anti- toxin single domain

  4. Site-saturation mutagenesis of Glomerella cingulata cutinase gene for enhanced enzyme thermostability

    Science.gov (United States)

    Hanapi, Wan Nurhidayah Wan; Iuan-Sheau, Chin; Mahadi, Nor Muhammad; Murad, Abdul Munir Abdul; Bakar, Farah Diba Abu

    2015-09-01

    Cutinase is an important biocatalyst for various industrial applications. This enzyme which has dual functionality comparable to esterases and lipases, is efficient in the hydrolysis of soluble esters and emulsified triacylglycerols. Naturally-occurring enzymes usually have disadvantages when applied in non-natural catalysis due to Glomerella cingulata cutinase enzyme thermostability. It is postulated that by increasing the rigidity at certain amino acid positions showing high mobility based on the three-dimensional structure of G. cingulata cutinase, the improvement in thermostability will be achieved. The amino acid N82 of G. cingulata cutinase was selected based on its high B-factor value determined via the B-FITTER program. Megaprimer PCR was employed to introduce mutations at the chosen site by randomization using NNK degenerate primers. About 300 transformants were selected for screening of positive cutinase variants. The N82_V14 cutinase variant was observed to be more thermostable at an almost 2-fold increase when exposed at 50°C for 1 hr as compared to the wild-type enzyme. This study may provide valuable information regarding thermal stability of cutinases denaturation at high temperatures.

  5. Expression and characterization of thermostable glycogen branching enzyme from Geobacillus mahadia Geo-05

    Directory of Open Access Journals (Sweden)

    Nur Syazwani Mohtar

    2016-12-01

    Full Text Available The glycogen branching enzyme (EC 2.4.1.18, which catalyses the formation of α-1,6-glycosidic branch points in glycogen structure, is often used to enhance the nutritional value and quality of food and beverages. In order to be applicable in industries, enzymes that are stable and active at high temperature are much desired. Using genome mining, the nucleotide sequence of the branching enzyme gene (glgB was extracted from the Geobacillus mahadia Geo-05 genome sequence provided by the Malaysia Genome Institute. The size of the gene is 2013 bp, and the theoretical molecular weight of the protein is 78.43 kDa. The gene sequence was then used to predict the thermostability, function and the three dimensional structure of the enzyme. The gene was cloned and overexpressed in E. coli to verify the predicted result experimentally. The purified enzyme was used to study the effect of temperature and pH on enzyme activity and stability, and the inhibitory effect by metal ion on enzyme activity. This thermostable glycogen branching enzyme was found to be most active at 55 °C, and the half-life at 60 °C and 70 °C was 24 h and 5 h, respectively. From this research, a thermostable glycogen branching enzyme was successfully isolated from Geobacillus mahadia Geo-05 by genome mining together with molecular biology technique.

  6. 'THERMOST' for analysing thermo-structural behaviour of LWR fuel rods under PCI conditions

    International Nuclear Information System (INIS)

    Nuno, H.; Ogawa, S.; Kobayashi, H.

    1983-01-01

    As a method for evaluating fuel rod performance under power ramping or load following operations, the combined FROST/ THERMOST system has been developed and brought into practical use. FROST was presented at the IAEA Blackpool Meeting in 1978, and THERMOST is the subject of this paper. The major purpose of THERMOST is to analyse very detailed thermal and structural fuel behaviour in a rather localised part of the fuel rod whereas FROST deals with whole rod general performance. The code handles two-dimensional thermal and structural analyses simultaneously by using a finite element method, in axial section or in lateral section. It consists of a fundamental FEM system of generalised constitution, and a surrounding subroutine system which characterises fuel behaviour, such as temperature distribution, thermal expansion, elastoplasticity, creep, cracking, swelling, growth, etc. Thermal analysis is handled by heat conduction and heat transfer element (six kinds), and structural analysis by axisymmetric ring and lateral plane element (six kinds). Boundary problems such as contact, friction and cracking are treated by gap and crack elements. A sample calculation of PCI performance on a PWR fuel rod under ramping conditions is presented with some in-pile test data. (author)

  7. Structural characterization of the thermostable Bradyrhizobium japonicumD-sorbitol dehydrogenase.

    Science.gov (United States)

    Fredslund, Folmer; Otten, Harm; Gemperlein, Sabrina; Poulsen, Jens Christian N; Carius, Yvonne; Kohring, Gert Wieland; Lo Leggio, Leila

    2016-11-01

    Bradyrhizobium japonicum sorbitol dehydrogenase is NADH-dependent and is active at elevated temperatures. The best substrate is D-glucitol (a synonym for D-sorbitol), although L-glucitol is also accepted, giving it particular potential in industrial applications. Crystallization led to a hexagonal crystal form, with crystals diffracting to 2.9 Å resolution. In attempts to phase the data, a molecular-replacement solution based upon PDB entry 4nbu (33% identical in sequence to the target) was found. The solution contained one molecule in the asymmetric unit, but a tetramer similar to that found in other short-chain dehydrogenases, including the search model, could be reconstructed by applying crystallographic symmetry operations. The active site contains electron density consistent with D-glucitol and phosphate, but there was not clear evidence for the binding of NADH. In a search for the features that determine the thermostability of the enzyme, the T m for the orthologue from Rhodobacter sphaeroides, for which the structure was already known, was also determined, and this enzyme proved to be considerably less thermostable. A continuous β-sheet is formed between two monomers in the tetramer of the B. japonicum enzyme, a feature not generally shared by short-chain dehydrogenases, and which may contribute to thermostability, as may an increased Pro/Gly ratio.

  8. Expression of Acidothermus cellulolyticus thermostable cellulases in tobacco and rice plants

    Directory of Open Access Journals (Sweden)

    Xiran Jiang

    2017-01-01

    Full Text Available The production of cellulases in plants is an economical method for the conversion of lignocellulosic biomass into fuels. Herein we report the expressions of two thermostable Acidothermus cellulolyticus cellulases, endo-1,4-β-D-glucanase (E1 and exoglucanase (Gux1, in tobacco and rice. To evaluate the expression of these recombinant cellulases, we expressed the full-length E1, the catalytic domains of E1 (E1cd and Gux1 (Gux1cd, as well as an E1–Gux1cd fusion enzyme in various subcellular compartments. In the case of tobacco, transgenic plants that expressed apoplast-localized E1 showed the highest level of activity, about three times higher than those that expressed the cytosolic E1. In the case of rice, the level of cellulase-specific activity in the transgenic plants ranged from 11 to 20 nmol 4-methylumbelliferone min−1 mg−1 total soluble protein. The recombinant cellulases exhibited good thermostability below 70 °C. Furthermore, transgenic rice leaves that were stored at room temperature for a month lost about 20% of the initial cellulase activity. Taken together, the results suggested that heterologous expression of thermostable cellulases in plants may be a viable option for biomass conversion.

  9. Improvement in the thermostability of chitosanase from Bacillus ehimensis by introducing artificial disulfide bonds.

    Science.gov (United States)

    Sheng, Jun; Ji, Xiaofeng; Zheng, Yuan; Wang, Zhipeng; Sun, Mi

    2016-10-01

    To determine the effects of artificial disulfide bridges on the thermostability and catalytic efficiency of chitosanase EAG1. Five artificial disulfide bridges were designed based on the structural information derived from the three-dimensional (3-D) model of chitosanase EAG1. Two beneficial mutants (G113C/D116C, A207C-L286C) were located in the flexible surface loop region, whereas the similar substitutions introduced in α-helices regions had a negligible effect. Mut5, the most active mutant, had a longer half-life at 50 °C (from 10.5 to 69.3 min) and a 200 % higher catalytic efficiency (K cat/K m) than that of the original EAG1. The contribution of disulfide bridges to enzyme thermostability is mainly dependent on its location within the polypeptide chain. Strategical placement of a disulfide bridge in flexible regions provides a rigid support and creation of a protected microenvironment, which is effective in improving enzyme's thermostability and catalytic efficiency.

  10. Isolation and molecular characterization of thermostable phytase from Bacillus subtilis (BSPhyARRMK33).

    Science.gov (United States)

    Reddy, Chinreddy Subramanyam; Achary, V Mohan Murali; Manna, Mrinalini; Singh, Jitender; Kaul, Tanushri; Reddy, Malireddy K

    2015-03-01

    The thermostable phytase gene was isolated from Bacillus subtilis ARRMK33 (BsPhyARRMK33). The gene has an ORF of 1152 bp and that encodes a protein of 383 amino acids. Sequence analysis showed high homology with Bacillus sp. phytase proteins, but no similarity was found with other phytases. SDS-PAGE analysis exhibited a predicted molecular mass of 42 kDa. Homology modeling of BsPhyARRMK33 protein based on Bacillus amyloliquefaciens crystal structure disclosed its β-propeller structure. BsPhyARRMK33 recombinant plasmid in pET-28a(+) was expressed in Rosetta gami B DE3 cells and the maximum phytase activity 15.3 U mg(-1) obtained. The enzyme exhibits high thermostability at various temperatures and broad pH ranges. The recombinant protein retained 74% of its original activity after incubation at 95 °C for 10 min. In the presence of Ca(2+), the recombinant phytase activity was maximal where as it was inhibited by EDTA. The optimal pH and temperature for the recombinant phytase activity is achieved at 7.0 and 55 °C, respectively. Thermostable nature and wide range of pH are promising features of recombinant BsPhyARRMK33 protein that may be employed as an efficient alternative to commercially known phytases and thereby alleviate environmental eutrophication.

  11. Cooperation between Catalytic and DNA-binding Domains Enhances Thermostability and Supports DNA Synthesis at Higher Temperatures by Thermostable DNA Polymerases

    Science.gov (United States)

    Pavlov, Andrey R.; Pavlova, Nadejda V.; Kozyavkin, Sergei A.; Slesarev, Alexei I.

    2012-01-01

    We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases (Pavlov et. al., (2002) Proc. Natl. Acad. Sci. USA 99, 13510–13515). The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various non-specific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting Helix-hairpin-Helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species, but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of TopoV HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105°C by maintaining processivity of DNA synthesis at high temperatures. We also found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding templates to DNA polymerases. PMID:22320201

  12. Recovery of avirulent, thermostable Newcastle disease virus strain NDV4-C from cloned cDNA and stable expression of an inserted foreign gene

    NARCIS (Netherlands)

    Zhang, X.; Liu, H.; Liu, P.; Peeters, B.P.H.; Zhao, C.; Kong, X.

    2013-01-01

    A reverse genetics system for thermostable Newcastle disease virus (NDV) is not currently available. In this study, we developed a reverse genetics system for the avirulent and thermostable NDV4-C strain. Successful recovery of NDV4-C was achieved by using either T7 RNA polymerase or cellular RNA

  13. A novel engineered interchain disulfide bond in the constant region enhances the thermostability of adalimumab Fab.

    Science.gov (United States)

    Nakamura, Hitomi; Oda-Ueda, Naoko; Ueda, Tadashi; Ohkuri, Takatoshi

    2018-01-01

    We constructed a system for expressing the Fab of the therapeutic human monoclonal antibody adalimumab at a yield of 20 mg/L in the methylotrophic yeast Pichia pastoris. To examine the contribution of interchain disulfide bonds to conformational stability, we prepared adalimumab Fab from which the interchain disulfide bond at the C-terminal region at both the CH 1 and CL domains was deleted by substitution of Cys with Ala (Fab ΔSS ). DSC measurements showed that the Tm values of Fab ΔSS were approximately 5 °C lower than those of wild-type Fab, suggesting that the interchain disulfide bond contributes to conformational thermostability. Using computer simulations, we designed a novel interchain disulfide bond outside the C-terminal region to increase the stability of Fab ΔSS . The resulting Fab (mutSS Fab ΔSS ) had the mutations H:V177C and L:Q160C in Fab ΔSS , confirming the formation of the disulfide bond between CH 1 and CL. The thermostability of mutSS Fab ΔSS was approximately 5 °C higher than that of Fab ΔSS . Therefore, the introduction of the designed interchain disulfide bond enhanced the thermostability of Fab ΔSS and mitigated the destabilization caused by partial reduction of the interchain disulfide bond at the C-terminal region, which occurs in site-specific modification such as PEGylation. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Engineering and introduction of de novo disulphide bridges in organophosphorus hydrolase enzyme for thermostability improvement.

    Science.gov (United States)

    Farnoosh, Gholamreza; Khajeh, Khosro; Latifi, Ali Mohammad; Aghamollaei, Hossein

    2016-12-01

    The organophosphorus hydrolase (OPH) has been used to degrade organophosphorus chemicals, as one of the most frequently used decontamination methods. Under chemical and thermal denaturing conditions, the enzyme has been shown to unfold. To utilize this enzyme in various applications, the thermal stability is of importance. The engineering of de novo disulphide bridges has been explored as a means to increase the thermal stability of enzymes in the rational method of protein engineering. In this study, Disulphide by Design software, homology modelling and molecular dynamics simulations were used to select appropriate amino acid pairs for the introduction of disulphide bridge to improve protein thermostability. The thermostability of the wild-type and three selected mutant enzymes were evaluated by half-life, delta G inactivation (ΔGi) and structural studies (fluorescence and far-UV CD analysis). Data analysis showed that half-life of A204C/T234C and T128C/E153C mutants were increased up to 4 and 24 min, respectively; however, for the G74C/A78C mutant, the half-life was decreased up to 9 min. For the T128C/E124C mutant, both thermal stability and Catalytic efficiency (kcat) were also increased. The half-life and ΔGi results were correlated to the obtained information from structural studies by circular dichroism (CD) spectrometry and extrinsic fluorescence experiments; as rigidity increased in A204C/T2234C and T128C/E153C mutants, half-life and ΔGi also increased. For G74C/A78C mutant, these parameters decreased due to its higher flexibility. The results were submitted a strong evidence for the possibility to improve the thermostability of OPH enzyme by introducing a disulphide bridge after bioinformatics design, even though this design would not be always successful.

  15. Protein thermostability prediction within homologous families using temperature-dependent statistical potentials.

    Directory of Open Access Journals (Sweden)

    Fabrizio Pucci

    Full Text Available The ability to rationally modify targeted physical and biological features of a protein of interest holds promise in numerous academic and industrial applications and paves the way towards de novo protein design. In particular, bioprocesses that utilize the remarkable properties of enzymes would often benefit from mutants that remain active at temperatures that are either higher or lower than the physiological temperature, while maintaining the biological activity. Many in silico methods have been developed in recent years for predicting the thermodynamic stability of mutant proteins, but very few have focused on thermostability. To bridge this gap, we developed an algorithm for predicting the best descriptor of thermostability, namely the melting temperature Tm, from the protein's sequence and structure. Our method is applicable when the Tm of proteins homologous to the target protein are known. It is based on the design of several temperature-dependent statistical potentials, derived from datasets consisting of either mesostable or thermostable proteins. Linear combinations of these potentials have been shown to yield an estimation of the protein folding free energies at low and high temperatures, and the difference of these energies, a prediction of the melting temperature. This particular construction, that distinguishes between the interactions that contribute more than others to the stability at high temperatures and those that are more stabilizing at low T, gives better performances compared to the standard approach based on T-independent potentials which predict the thermal resistance from the thermodynamic stability. Our method has been tested on 45 proteins of known Tm that belong to 11 homologous families. The standard deviation between experimental and predicted Tm's is equal to 13.6°C in cross validation, and decreases to 8.3°C if the 6 worst predicted proteins are excluded. Possible extensions of our approach are discussed.

  16. Enhancing activity and thermostability of lipase A from Serratia marcescens by site-directed mutagenesis.

    Science.gov (United States)

    Mohammadi, Mohsen; Sepehrizadeh, Zargham; Ebrahim-Habibi, Azadeh; Shahverdi, Ahmad Reza; Faramarzi, Mohammad Ali; Setayesh, Neda

    2016-11-01

    Lipases as significant biocatalysts had been widely employed to catalyze various chemical reactions such as ester hydrolysis, ester synthesis, and transesterification. Improving the activity and thermostability of enzymes is desirable for industrial applications. The lipase of Serratia marcescens belonging to family I.3 lipase has a very important pharmaceutical application in production of chiral precursors. In the present study, to achieve improved lipase activity and thermostability, using computational predictions of protein, four mutant lipases of SML (MutG2P, MutG59P, Mut H279K and MutL613WA614P) were constructed by site-directed mutagenesis. The recombinant mutant proteins were over-expressed in E. coli and purified by affinity chromatography on the Ni-NTA system. Circular dichroism spectroscopy, differential scanning calorimetry and kinetic parameters (Km and kcat) were determined. Our results have shown that the secondary structure of all lipases was approximately similar to one another. The MutG2P and MutG59P were more stable than wild type by approximately 2.3 and 2.9 in T 1/2 , respectively. The catalytic efficiency (kcat/Km) of MutH279K was enhanced by 2-fold as compared with the wild type (p<0.05). These results indicate that using protein modeling program and creating mutation, can enhance lipase activity and/or thermostability of SML and it also could be used for improving other properties of enzyme to the desired requirements as well as further mutations. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Identification of thermostable β-xylosidase activities produced by Aspergillus brasiliensis and Aspergillus niger

    DEFF Research Database (Denmark)

    Pedersen, Mads; Lauritzen, Henrik Klitgaard; Frisvad, Jens Christian

    2007-01-01

    Twenty Aspergillus strains were evaluated for production of extracellular cellulolytic and xylanolytic activities. Aspergillus brasiliensis, A. niger and A. japonicus produced the highest xylanase activities with the A. brasiliensis and A. niger strains producing thermostable beta......-xylosidases. The beta-xylosidase activities of the A. brasiliensis and A. niger strains had similar temperature and pH optima at 75 degrees C and pH 5 and retained 62% and 99%, respectively, of these activities over 1 h at 60 degrees C. At 75 degrees C, these values were 38 and 44%, respectively. Whereas A. niger...

  18. Thermal Degradation Mechanism of a Thermostable Polyester Stabilized with an Open-Cage Oligomeric Silsesquioxane

    Directory of Open Access Journals (Sweden)

    Yolanda Bautista

    2017-12-01

    Full Text Available A polyester composite was prepared through the polymerization of an unsaturated ester resin with styrene and an open-cage oligomeric silsesquioxane with methacrylate groups. The effect of the open-cage oligomeric silsesquioxane on the thermal stability of the thermostable polyester was studied using both thermogravimetric analysis and differential thermal analysis. The results showed that the methacryl oligomeric silsesquioxane improved the thermal stability of the polyester. The decomposition mechanism of the polyester/oligomer silsesquioxane composite was proposed by Fourier transform infrared spectroscopy (FTIR analysis of the volatiles.

  19. Methods of hydrolyzing a cellulose using halophilic, thermostable and ionic liquids tolerant cellulases

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Tao; Datta, Supratim; Simmons, Blake A.; Rubin, Edward M.

    2018-01-09

    The present invention provides for an isolated or recombinant polypeptide comprising an amino acid sequence having at least 70% identity with the amino acid sequence of a Halorhabdus utahensis cellulase, such as Hu-CBH1, wherein said amino acid sequence has a halophilic thermostable and/or thermophilic cellobiohydrolase (CBH) activity. In some embodiments, the polypeptide has a CBH activity that is resistant to up to about 20% of ionic liquids. The present invention also provides for compositions comprising and methods using the isolated or recombinant polypeptide.

  20. Evaluations on power ramp data of PWR fuels by FROST and THERMOST codes

    International Nuclear Information System (INIS)

    Murai, K.; Ogawa, S.; Nuno, H.; Kondo, Y.

    1987-01-01

    An evaluation is presented of power ramp data of Mitsubishi's PWR fuel rods tested in R-2, Studsvik, which was analysed by FROST and THERMOST codes. The analyses give good predictions for measured diameter changes and on-power rod elongations. The work indicates that FROST is capable of analysing both radial and axial pellet-cladding mechanism interaction (PCMI) appropriately, and that predicted states of PCMI (i.e. stress and strain which cannot be measured directly) are considered to be reliable. The ramp data used in the present analyses were obtained in two joint programmes with five Japanese PWR utilities (KEPCO, KYEPCO, SEPCO, HEPCO, and JAPCO). (UK)

  1. Protein features as determinants of wild-type glycoside hydrolase thermostability

    DEFF Research Database (Denmark)

    Geertz-Hansen, Henrik Marcus; Kiemer, Lars; Nielsen, Morten

    2017-01-01

    -silico methods guiding the discovery process would be of high value. To develop such an in-silico method and provide the data foundation of it, we determined the melting temperatures of 602 fungal glycoside hydrolases from the families GH5, 6, 7, 10, 11, 43 and AA9 (formerly GH61). We, then used sequence...... and homology modeled structure information of these enzymes to develop the ThermoP melting temperature prediction method. Futhermore, in the context of thermostability, we determined the relative importance of 160 molecular features, such as amino acid frequencies and spatial interactions, and exemplified...

  2. Problems of increasing of thermostability of highly permeable Ni-Zn ferrites and relative materials for telecommunications

    Energy Technology Data Exchange (ETDEWEB)

    Gonchar, A. E-mail: letyuk@mail.ru; Andreev, V.; Letyuk, L.; Shishkanov, A.; Maiorov, V

    2003-01-01

    The work considers ways of increasing of thermostability of ferrites of the basic systems NiO-ZnO-Fe{sub 2}O{sub 3} and MgO-ZnO-Fe{sub 2}O{sub 3} and relative materials for telecommunication. Sufficient results in increasing of the thermostability were achieved by doping Cu ions and controlling rejection of Fe{sub 2}O{sub 3} content from equimolar composition. These results allow to increase the Curie temperature to 130-140 deg. C for Ni-Zn ferrites with initial permeability 2000.

  3. The ''THERMOST'' for analysing thermo-structural behaviour of LWR fuel rod under PCI conditions

    International Nuclear Information System (INIS)

    Nuno, H.; Ogawa, S.; Kobayashi, H.

    1983-01-01

    As one of the methods for evaluating the fuel rod performances under power ramping or load following operations, the combined ''FROST'' and ''THERMOST'' system has been developed and being brought into practical use. The former had already been presented at Blackpool Meeting in 1978, and the latter is going to be presented in this paper. The major purpose of the THERMOST is to analyse very detailed thermal and structural fuel behaviours in a rather localized part of fuel rod whereas the FROST deals with whole-rod-wide general performances. The code handles 2-dimensional thermal and structural analyses simultaneously by using finite element method, in axial section wide or in lateral section wide. It consists of a fundamental FEM system of generalized constitution and its surrounding subroutine system which characterizes fuel behaviours such as temperature distribution, thermal expansion, elastoplasticity, creep, cracking, swelling, growth, etc. Thermal analysis is handled by heat conduction and heat transfer elements (6 kinds) and structural analysis by axisymmetric ring and lateral plane elements (6 kinds). Boundary problems such as contact, friction and cracking are treated by gap and crack elements. A sample calculation of PCI performance on a PWR fuel rod under ramping condition is presented with some inpile test data. (author)

  4. Molecular Dynamics Simulation of Barnase: Contribution of Noncovalent Intramolecular Interaction to Thermostability

    Directory of Open Access Journals (Sweden)

    Zhiguo Chen

    2013-01-01

    Full Text Available Bacillus amyloliquefaciens ribonuclease Barnase (RNase Ba is a 12 kD (kilodalton small extracellular ribonuclease. It has broad application prospects in agriculture, clinical medicine, pharmaceutical, and so forth. In this work, the thermal stability of Barnase has been studied using molecular dynamics simulation at different temperatures. The present study focuses on the contribution of noncovalent intramolecular interaction to protein stability and how they affect the thermal stability of the enzyme. Profiles of root mean square deviation and root mean square fluctuation identify thermostable and thermosensitive regions of Barnase. Analyses of trajectories in terms of secondary structure content, intramolecular hydrogen bonds and salt bridge interactions indicate distinct differences in different temperature simulations. In the simulations, Four three-member salt bridge networks (Asp8-Arg110-Asp12, Arg83-Asp75-Arg87, Lys66-Asp93-Arg69, and Asp54-Lys27-Glu73 have been identified as critical salt bridges for thermostability which are maintained stably at higher temperature enhancing stability of three hydrophobic cores. The study may help enlighten our knowledge of protein structural properties, noncovalent interactions which can stabilize secondary peptide structures or promote folding, and also help understand their actions better. Such an understanding is required for designing efficient enzymes with characteristics for particular applications at desired working temperatures.

  5. High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases

    Science.gov (United States)

    Qin, Yidan; Yao, Jun; Wu, Douglas C.; Nottingham, Ryan M.; Mohr, Sabine; Hunicke-Smith, Scott; Lambowitz, Alan M.

    2016-01-01

    Next-generation RNA-sequencing (RNA-seq) has revolutionized transcriptome profiling, gene expression analysis, and RNA-based diagnostics. Here, we developed a new RNA-seq method that exploits thermostable group II intron reverse transcriptases (TGIRTs) and used it to profile human plasma RNAs. TGIRTs have higher thermostability, processivity, and fidelity than conventional reverse transcriptases, plus a novel template-switching activity that can efficiently attach RNA-seq adapters to target RNA sequences without RNA ligation. The new TGIRT-seq method enabled construction of RNA-seq libraries from RNA in RNA in 1-mL plasma samples from a healthy individual revealed RNA fragments mapping to a diverse population of protein-coding gene and long ncRNAs, which are enriched in intron and antisense sequences, as well as nearly all known classes of small ncRNAs, some of which have never before been seen in plasma. Surprisingly, many of the small ncRNA species were present as full-length transcripts, suggesting that they are protected from plasma RNases in ribonucleoprotein (RNP) complexes and/or exosomes. This TGIRT-seq method is readily adaptable for profiling of whole-cell, exosomal, and miRNAs, and for related procedures, such as HITS-CLIP and ribosome profiling. PMID:26554030

  6. Crystallization and preliminary X-ray characterization of two thermostable DNA nucleases

    International Nuclear Information System (INIS)

    Kuettner, E. Bartholomeus; Pfeifer, Sven; Keim, Antje; Greiner-Stöffele, Thomas; Sträter, Norbert

    2006-01-01

    Two thermostable DNA nucleases from archaea were crystallized in different space groups; the crystals were suitable for X-ray analysis. Temperature-tolerant organisms are an important source to enhance the stability of enzymes used in biotechnological processes. The DNA-cleaving enzyme exonuclease III from Escherichia coli is used in several applications in gene technology. A thermostable variant could expand the applicability of the enzyme in these methods. Two homologous nucleases from Archaeoglobus fulgidus (ExoAf) and Methanothermobacter thermoautrophicus (ExoMt) were studied for this purpose. Both enzymes were crystallized in different space groups using (poly)ethylene glycols, 2,4-methyl pentandiol, dioxane, ethanol or 2-propanol as precipitants. The addition of a 10-mer DNA oligonucleotide was important to obtain monoclinic crystals of ExoAf and ExoMt that diffracted to resolutions better than 2 Å using synchrotron radiation. The crystal structures of the homologous proteins can serve as templates for genetic engineering of the E. coli exonuclease III and will aid in understanding the different catalytic properties of the enzymes

  7. Stabilizing salt-bridge enhances protein thermostability by reducing the heat capacity change of unfolding.

    Directory of Open Access Journals (Sweden)

    Chi-Ho Chan

    Full Text Available Most thermophilic proteins tend to have more salt bridges, and achieve higher thermostability by up-shifting and broadening their protein stability curves. While the stabilizing effect of salt-bridge has been extensively studied, experimental data on how salt-bridge influences protein stability curves are scarce. Here, we used double mutant cycles to determine the temperature-dependency of the pair-wise interaction energy and the contribution of salt-bridges to ΔC(p in a thermophilic ribosomal protein L30e. Our results showed that the pair-wise interaction energies for the salt-bridges E6/R92 and E62/K46 were stabilizing and insensitive to temperature changes from 298 to 348 K. On the other hand, the pair-wise interaction energies between the control long-range ion-pair of E90/R92 were negligible. The ΔC(p of all single and double mutants were determined by Gibbs-Helmholtz and Kirchhoff analyses. We showed that the two stabilizing salt-bridges contributed to a reduction of ΔC(p by 0.8-1.0 kJ mol⁻¹ K⁻¹. Taken together, our results suggest that the extra salt-bridges found in thermophilic proteins enhance the thermostability of proteins by reducing ΔC(p, leading to the up-shifting and broadening of the protein stability curves.

  8. Crystallization and X-ray structure analysis of a thermostable penicillin G acylase from Alcaligenes faecalis

    International Nuclear Information System (INIS)

    Varshney, Nishant Kumar; Suresh Kumar, R.; Ignatova, Zoya; Prabhune, Asmita; Pundle, Archana; Dodson, Eleanor; Suresh, C. G.

    2012-01-01

    A thermostable penicillin G acylase from A. faecalis has been crystallized in two space groups: C222 1 and P4 1 2 1 2. X-ray diffraction data were collected to 3.3 and 3.5 Å resolution, respectively. The enzyme penicillin G acylase (EC 3.5.1.11) catalyzes amide-bond cleavage in benzylpenicillin (penicillin G) to yield 6-aminopenicillanic acid, an intermediate chemical used in the production of semisynthetic penicillins. A thermostable penicillin G acylase from Alcaligenes faecalis (AfPGA) has been crystallized using the hanging-drop vapour-diffusion method in two different space groups: C222 1 , with unit-cell parameters a = 72.9, b = 86.0, c = 260.2 Å, and P4 1 2 1 2, with unit-cell parameters a = b = 85.6, c = 298.8 Å. Data were collected at 293 K and the structure was determined using the molecular-replacement method. Like other penicillin acylases, AfPGA belongs to the N-terminal nucleophilic hydrolase superfamily, has undergone post-translational processing and has a serine as the N-terminal residue of the β-chain. A disulfide bridge has been identified in the structure that was not found in the other two known penicillin G acylase structures. The presence of the disulfide bridge is perceived to be one factor that confers higher stability to this enzyme

  9. Thermostable 𝜶-Amylase Activity from Thermophilic Bacteria Isolated from Bora Hot Spring, Central Sulawesi

    Science.gov (United States)

    Gazali, F. M.; Suwastika, I. N.

    2018-03-01

    α-Amylase is one of the most important enzyme in biotechnology field, especially in industrial application. Thermostability of α-Amylase produced by thermophilic bacteria improves industrial process of starch degradation in starch industry. The present study were concerned to the characterization of α-Amylase activity from indigenous thermophilic bacteria isolated from Bora hot spring, Central Sulawesi. There were 18 isolates which had successfully isolated from 90°C sediment samples of Bora hot spring and 13 of them showed amylolytic activity. The α-Amylase activity was measured qualitatively at starch agar and quantitatively based on DNS (3,5-Dinitrosalicylic acid) methods, using maltose as standard solution. Two isolates (out of 13 amylolytic bacteria), BR 002 and BR 015 showed amylolytic index of 0.8 mm and 0.5 mm respectively, after being incubated at 55°C in the 0.002% Starch Agar Medium. The α-Amylase activity was further characterized quantitatively which includes the optimum condition of pH and temperature of α-Amylase crude enzyme from each isolate. To our knowledge, this is the first report on isolation and characterization of a thermostable α-Amylase from thermophilic bacteria isolated from Central Sulawesi particularly from Bora hot spring.

  10. Structure of the Aeropyrum pernix L7Ae multifunctional protein and insight into its extreme thermostability

    International Nuclear Information System (INIS)

    Bhuiya, Mohammad Wadud; Suryadi, Jimmy; Zhou, Zholi; Brown, Bernard Andrew II

    2013-01-01

    The crystal structure of A. pernix L7Ae is reported, providing insight into the extreme thermostability of this protein. Archaeal ribosomal protein L7Ae is a multifunctional RNA-binding protein that directs post-transcriptional modification of archaeal RNAs. The L7Ae protein from Aeropyrum pernix (Ap L7Ae), a member of the Crenarchaea, was found to have an extremely high melting temperature (>383 K). The crystal structure of Ap L7Ae has been determined to a resolution of 1.56 Å. The structure of Ap L7Ae was compared with the structures of two homologs: hyperthermophilic Methanocaldococcus jannaschii L7Ae and the mesophilic counterpart mammalian 15.5 kD protein. The primary stabilizing feature in the Ap L7Ae protein appears to be the large number of ion pairs and extensive ion-pair network that connects secondary-structural elements. To our knowledge, Ap L7Ae is among the most thermostable single-domain monomeric proteins presently observed

  11. Improving specific activity and thermostability of Escherichia coli phytase by structure-based rational design.

    Science.gov (United States)

    Wu, Tzu-Hui; Chen, Chun-Chi; Cheng, Ya-Shan; Ko, Tzu-Ping; Lin, Cheng-Yen; Lai, Hui-Lin; Huang, Ting-Yung; Liu, Je-Ruei; Guo, Rey-Ting

    2014-04-10

    Escherichia coli phytase (EcAppA) which hydrolyzes phytate has been widely applied in the feed industry, but the need to improve the enzyme activity and thermostability remains. Here, we conduct rational design with two strategies to enhance the EcAppA performance. First, residues near the substrate binding pocket of EcAppA were modified according to the consensus sequence of two highly active Citrobacter phytases. One out of the eleven mutants, V89T, exhibited 17.5% increase in catalytic activity, which might be a result of stabilized protein folding. Second, the EcAppA glycosylation pattern was modified in accordance with the Citrobacter phytases. An N-glycosylation motif near the substrate binding site was disrupted to remove spatial hindrance for phytate entry and product departure. The de-glycosylated mutants showed 9.6% increase in specific activity. On the other hand, the EcAppA mutants that adopt N-glycosylation motifs from CbAppA showed improved thermostability that three mutants carrying single N-glycosylation motif exhibited 5.6-9.5% residual activity after treatment at 80°C (1.8% for wild type). Furthermore, the mutant carrying all three glycosylation motifs exhibited 27% residual activity. In conclusion, a successful rational design was performed to obtain several useful EcAppA mutants with better properties for further applications. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Thermostable Alcohol Dehydrogenase from Thermococcus kodakarensis KOD1 for Enantioselective Bioconversion of Aromatic Secondary Alcohols

    Science.gov (United States)

    Wu, Xi; Zhang, Chong; Orita, Izumi; Imanaka, Tadayuki

    2013-01-01

    A novel thermostable alcohol dehydrogenase (ADH) showing activity toward aromatic secondary alcohols was identified from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkADH). The gene, tk0845, which encodes an aldo-keto reductase, was heterologously expressed in Escherichia coli. The enzyme was found to be a monomer with a molecular mass of 31 kDa. It was highly thermostable with an optimal temperature of 90°C and a half-life of 4.5 h at 95°C. The apparent Km values for the cofactors NAD(P)+ and NADPH were similar within a range of 66 to 127 μM. TkADH preferred secondary alcohols and accepted various ketones and aldehydes as substrates. Interestingly, the enzyme could oxidize 1-phenylethanol and its derivatives having substituents at the meta and para positions with high enantioselectivity, yielding the corresponding (R)-alcohols with optical purities of greater than 99.8% enantiomeric excess (ee). TkADH could also reduce 2,2,2-trifluoroacetophenone to (R)-2,2,2-trifluoro-1-phenylethanol with high enantioselectivity (>99.6% ee). Furthermore, the enzyme showed high resistance to organic solvents and was particularly highly active in the presence of H2O–20% 2-propanol and H2O–50% n-hexane or n-octane. This ADH is expected to be a useful tool for the production of aromatic chiral alcohols. PMID:23354700

  13. Cytoplasmic expression of a thermostable invertase from Thermotoga maritima in Lactococcus lactis.

    Science.gov (United States)

    Pek, Han Bin; Lim, Pei Yu; Liu, Chengcheng; Lee, Dong-Yup; Bi, Xuezhi; Wong, Fong Tian; Ow, Dave Siak-Wei

    2017-05-01

    To evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis. The thermostable invertase from T. maritima was cloned with and without the USP45 secretory peptide into the pNZ8148 vector for nisin-inducible expression in L. lactis. The introduction of an USP45 secretion peptide at the N-terminal of the enzyme led to a loss of protein solubility. Computational homology modeling and hydrophobicity studies indicated that the USP45 peptide exposes a stretch of hydrophobic amino acids on the protein surface resulting in lower solubility. Removal of the USP45 secretion peptide allowed a soluble and functional invertase to be expressed intracellularly in L. lactis. Immobilized metal affinity chromatography purification of the cell lysate with nickel-NTA gave a single protein band on SDS-PAGE, while E. coli-expressed invertase consistently co-purified with an additional band. The yields of the purified invertase from E. coli and L. lactis were 14.1 and 6.3 mg/l respectively. Invertase can be expressed in L. lactis and purified in a functional form. L. lactis is a suitable host for the production of food-grade invertase for use in the food and biotechnology industries.

  14. Characterization of an extremely salt-tolerant and thermostable phytase from Bacillus amyloliquefaciens US573.

    Science.gov (United States)

    Boukhris, Ines; Farhat-Khemakhem, Ameny; Blibech, Monia; Bouchaala, Kameleddine; Chouayekh, Hichem

    2015-09-01

    The extracellular phytase produced by the Bacillus amyloliquefaciens US573 strain, isolated from geothermal soil located in Southern Tunisia was purified and characterized. This calcium-dependent and bile-stable enzyme (PHY US573) was optimally active at pH 7.5 and 70 °C. It showed a good stability at pH ranging from 4 to 10, and especially, an exceptional thermostability as it recovered 50 and 62% of activity after heating for 10 min at 100 and 90 °C, respectively. In addition, PHY US573 was found to be extremely salt-tolerant since it preserved 80 and 95% of activity in the presence of 20 g/l of NaCl and LiCl, respectively. The gene corresponding to PHY US573 was cloned. It encodes a 383 amino acids polypeptide exhibiting 99% identity with the highly thermostable phytases from Bacillus sp. MD2 and B. amyloliquefaciens DS11 (3 and 5 residues difference, respectively), suggesting the existence of common molecular determinants responsible for their remarkable heat stability. Overall, our findings illustrated that in addition to its high potential for application in feed industry, the salt tolerance of the PHY US573 phytase, may represent an exciting new avenue for improvement of phosphorus-use efficiency of salt-tolerant plants in soils with high salt and phytate content. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Enhanced Thermostability of Arabidopsis Rubisco Activase Improves Photosynthesis and Growth Rates under Moderate Heat Stress[OA

    Science.gov (United States)

    Kurek, Itzhak; Chang, Thom Kai; Bertain, Sean M.; Madrigal, Alfredo; Liu, Lu; Lassner, Michael W.; Zhu, Genhai

    2007-01-01

    Plant photosynthesis declines when the temperature exceeds its optimum range. Recent evidence indicates that the reduction in photosynthesis is linked to ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) deactivation due to the inhibition of Rubisco activase (RCA) under moderately elevated temperatures. To test the hypothesis that thermostable RCA can improve photosynthesis under elevated temperatures, we used gene shuffling technology to generate several Arabidopsis thaliana RCA1 (short isoform) variants exhibiting improved thermostability. Wild-type RCA1 and selected thermostable RCA1 variants were introduced into an Arabidopsis RCA deletion (Δrca) line. In a long-term growth test at either constant 26°C or daily 4-h 30°C exposure, the transgenic lines with the thermostable RCA1 variants exhibited higher photosynthetic rates, improved development patterns, higher biomass, and increased seed yields compared with the lines expressing wild-type RCA1 and a slight improvement compared with untransformed Arabidopsis plants. These results provide clear evidence that RCA is a major limiting factor in plant photosynthesis under moderately elevated temperatures and a potential target for genetic manipulation to improve crop plants productivity under heat stress conditions. PMID:17933901

  16. Protein engineering of Bacillus acidopullulyticus pullulanase for enhanced thermostability using in silico data driven rational design methods.

    Science.gov (United States)

    Chen, Ana; Li, Yamei; Nie, Jianqi; McNeil, Brian; Jeffrey, Laura; Yang, Yankun; Bai, Zhonghu

    2015-10-01

    Thermostability has been considered as a requirement in the starch processing industry to maintain high catalytic activity of pullulanase under high temperatures. Four data driven rational design methods (B-FITTER, proline theory, PoPMuSiC-2.1, and sequence consensus approach) were adopted to identify the key residue potential links with thermostability, and 39 residues of Bacillus acidopullulyticus pullulanase were chosen as mutagenesis targets. Single mutagenesis followed by combined mutagenesis resulted in the best mutant E518I-S662R-Q706P, which exhibited an 11-fold half-life improvement at 60 °C and a 9.5 °C increase in Tm. The optimum temperature of the mutant increased from 60 to 65 °C. Fluorescence spectroscopy results demonstrated that the tertiary structure of the mutant enzyme was more compact than that of the wild-type (WT) enzyme. Structural change analysis revealed that the increase in thermostability was most probably caused by a combination of lower stability free-energy and higher hydrophobicity of E518I, more hydrogen bonds of S662R, and higher rigidity of Q706P compared with the WT. The findings demonstrated the effectiveness of combined data-driven rational design approaches in engineering an industrial enzyme to improve thermostability. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. INCREASING THE THERMOSTABILITY OF THE NEUTRAL PROTEINASE OF BACILLUS-STEAROTHERMOPHILUS BY IMPROVEMENT OF INTERNAL HYDROGEN-BONDING

    NARCIS (Netherlands)

    EIJSINK, VGH; VRIEND, G; VANDERZEE, [No Value; VANDENBURG, B; VENEMA, G

    1992-01-01

    In an attempt to increase the thermostability of the neutral proteinase of Bacillus stearothermophilus the buried Ala-170 was replaced by serine. Molecular-dynamics simulations showed that Ser-170 stabilizes the enzyme by formation of an internal hydrogen bond. In addition, the hydroxy group of

  18. Synergistic function of four novel thermostable glycoside hydrolases from a long-term enriched thermophilic methanogenic digester

    Directory of Open Access Journals (Sweden)

    Meng eWang

    2015-05-01

    Full Text Available In biofuel production from lignocellulose, low thermostability and product inhibition strongly restrict the enzyme activities and production process. Application of multiple thermostable glycoside hydrolases, forming an enzyme cocktail, can result in a synergistic action and therefore improve production efficiency and reduce operational costs. Therefore, increasing enzyme thermostabilities and compatibility are important for the biofuel industry. In this study, we reported the screening, cloning and biochemical characterization of four novel thermostable lignocellulose hydrolases from a metagenomic library of a long-term dry thermophilic methanogenic digester community, which were highly compatible with optimal conditions and specific activities. The optimal temperatures of the four enzymes, β-xylosidase, xylanase, β-glucosidase, and cellulase ranged from 60°C to 75°C, and over 80% residual activities were observed after 2 h incubation at 50°C. Mixtures of these hydrolases retained high residual synergistic activities after incubation with cellulose, xylan, and steam-exploded corncob at 50°C for 72 h. In addition, about 55% dry weight of steam-exploded corncob was hydrolyzed to glucose and xylose by the synergistic action of the four enzymes at 50°C for 48 h. This work suggested that since different enzymes from a same ecosystem could be more compatible, screening enzymes from a long-term enriching community could be a favorable strategy.

  19. Structure of a thermostable serralysin from Serratia sp. FS14 at 1.1 Å resolution.

    Science.gov (United States)

    Wu, Dongxia; Ran, Tinting; Wang, Weiwu; Xu, Dongqing

    2016-01-01

    Serralysin is a well studied metalloprotease, and typical serralysins are not thermostable. The serralysin isolated from Serratia sp. FS14 was found to be thermostable, and in order to reveal the mechanism responsible for its thermostability, the crystal structure of serralysin from Serratia sp. FS14 was solved to a crystallographic R factor of 0.1619 at 1.10 Å resolution. Similar to its homologues, it mainly consists of two domains: an N-terminal catalytic domain and a `parallel β-roll' C-terminal domain. Comparative studies show that the shape of the catalytic active-site cavity is more open owing to the 189-198 loop, with a short 310-helix protruding further from the molecular surface, and that the β-sheets comprising the `parallel β-roll' are longer than those in its homologues. The formation of hydrogen bonds from one of the nonconserved residues (Asn200) to Lys27 may contribute to the thermostability.

  20. Virus-like particle nanoreactors: programmed en capsulation of the thermostable CelB glycosidase inside the P22 capsid

    NARCIS (Netherlands)

    Patterson, D.P.; Schwarz, B.; El-Boubbou, K.; Oost, van der J.; Prevelige, P.E.; Douglas, T.

    2012-01-01

    Self-assembling biological systems hold great potential for the synthetic construction of new active soft nanomaterials. Here we demonstrate the hierarchical bottom-up assembly of bacteriophage P22 virus-like particles (VLPs) that encapsulate the thermostable CelB glycosidase creating catalytically

  1. Identification and characterization of a novel thermostable pyrethroid-hydrolyzing enzyme isolated through metagenomic approach

    Directory of Open Access Journals (Sweden)

    Fan Xinjiong

    2012-03-01

    Full Text Available Abstract Background Pyrethroid pesticides are broad-spectrum pest control agents in agricultural production. Both agricultural and residential usage is continuing to grow, leading to the development of insecticide resistance in the pest and toxic effects on a number of nontarget organisms. Thus, it is necessary to hunt suitable enzymes including hydrolases for degrading pesticide residues, which is an efficient "green" solution to biodegrade polluting chemicals. Although many pyrethroid esterases have consistently been purified and characterized from various resources including metagenomes and organisms, the thermostable pyrethroid esterases have not been reported up to the present. Results In this study, we identified a novel pyrethroid-hydrolyzing enzyme Sys410 belonging to familyV esterases/lipases with activity-based functional screening from Turban Basin metagenomic library. Sys410 contained 280 amino acids with a predicted molecular mass (Mr of 30.8 kDa and was overexpressed in Escherichia coli BL21 (DE3 in soluble form. The optimum pH and temperature of the recombinant Sys410 were 6.5 and 55°C, respectively. The enzyme was stable in the pH range of 4.5-8.5 and at temperatures below 50°C. The activity of Sys410 decreased a little when stored at 4°C for 10 weeks, and the residual activity reached 94.1%. Even after incubation at 25°C for 10 weeks, it kept 68.3% of its activity. The recombinant Sys410 could hydrolyze a wide range of ρ-nitrophenyl esters, but its best substrate is ρ-nitrophenyl acetate with the highest activity (772.9 U/mg. The enzyme efficiently degraded cyhalothrin, cypermethrin, sumicidin, and deltamethrin under assay conditions of 37°C for 15 min, with exceeding 95% hydrolysis rate. Conclusion This is the first report to construct metagenomic libraries from Turban Basin to obtain the thermostable pyrethroid-hydrolyzing enzyme. The recombinant Sys410 with broad substrate specificities and high activity was the most

  2. Engineering improved thermostability of the GH11 xylanase from Neocallimastix patriciarum via computational library design.

    Science.gov (United States)

    Bu, Yifan; Cui, Yinglu; Peng, Ying; Hu, Meirong; Tian, Yu'e; Tao, Yong; Wu, Bian

    2018-04-01

    Xylanases, which cleave the β-1,4-glycosidic bond between xylose residues to release xylooligosaccharides (XOS), are widely used as food additives, animal feeds, and pulp bleaching agents. However, the thermally unstable nature of xylanases would hamper their industrial application. In this study, we used in silico design in a glycoside hydrolase family (GH) 11 xylanase to stabilize the enzyme. A combination of the best mutations increased the apparent melting temperature by 14 °C and significantly enhanced thermostability and thermoactivation. The variant also showed an upward-shifted optimal temperature for catalysis without compromising its activity at low temperatures. Moreover, a 10-fold higher XOS production yield was obtained at 70 °C, which compensated the low yield obtained with the wild-type enzyme. Collectively, the variant constructed by the computational strategy can be used as an efficient biocatalyst for XOS production at industrially viable conditions.

  3. Roles of thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) in Vibrio parahaemolyticus.

    Science.gov (United States)

    Raghunath, Pendru

    2014-01-01

    Vibrio parahaemolyticus is the leading cause of seafood borne bacterial gastroenteritis in the world, often associated with the consumption of raw or undercooked seafood. However, not all strains of V. parahaemolyticus are pathogenic. The thermostable direct hemolysin (TDH) or TDH-related hemolysin (TRH) encoded by tdh and trh genes, respectively, are considered major virulence factors in V. parahaemolyticus. However, about 10% of clinical strains do not contain tdh and/or trh. Environmental isolates of V. parahaemolyticus lacking tdh and/or trh are also highly cytotoxic to human gastrointestinal cells. Even in the absence of these hemolysins, V. parahaemolyticus remains pathogenic indicating other virulence factors exist. This mini review aims at discussing the possible roles of tdh and trh genes in clinical and environmental isolates of V. parahaemolyticus.

  4. Characterization of thermostable cellulase produced by Bacillus strains isolated from solid waste of carrageenan

    Science.gov (United States)

    Listyaningrum, N. P.; Sutrisno, A.; Wardani, A. K.

    2018-03-01

    Cellulase-producing bacteria was isolated from solid waste of carrageenan and identified as Bacillus licheniformis C55 by 16S rRNA sequencing. The optimum condition for cellulase production was obtained at pH and temperature of 8.0 and 50°C, respectively in a medium containing glucose as carbon source and 1.0% carboxymethyl cellulose (CMC) to stimulate the cellulase production. Most remarkably, the enzyme retained its relative activity over 50% after incubation at 50°C for 90 minutes. Substrate specificity suggested that the enzyme is an endoglucanase. The molecular mass of Bacillus licheniformis C55 crude cellulase was found about 18 kDa by SDS-PAGE analysis. This thermostable enzyme would facilitate development of more efficient and cost-effective forms of the process to convert lignocellulosic biomass into high-value products.

  5. Chemoenzymatic Synthesis of Oligo(L-cysteine) for Use as a Thermostable Bio-Based Material.

    Science.gov (United States)

    Ma, Yinan; Sato, Ryota; Li, Zhibo; Numata, Keiji

    2016-01-01

    Oligomerization of thiol-unprotected L-cysteine ethyl ester (Cys-OEt) catalyzed by proteinase K in aqueous solution has been used to synthesize oligo(L-cysteine) (OligoCys) with a well-defined chemical structure and relatively large degree of polymerization (DP) up to 16-17 (average 8.8). By using a high concentration of Cys-OEt, 78.0% free thiol content was achieved. The thermal properties of OligoCys are stable, with no glass transition until 200 °C, and the decomposition temperature could be increased by oxidation. Chemoenzymatically synthesized OligoCys has great potential for use as a thermostable bio-based material with resistance to oxidation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Improved lignocellulose conversion to biofuels with thermophilic bacteria and thermostable enzymes.

    Science.gov (United States)

    Bhalla, Aditya; Bansal, Namita; Kumar, Sudhir; Bischoff, Kenneth M; Sani, Rajesh K

    2013-01-01

    Second-generation feedstock, especially nonfood lignocellulosic biomass is a potential source for biofuel production. Cost-intensive physical, chemical, biological pretreatment operations and slow enzymatic hydrolysis make the overall process of lignocellulosic conversion into biofuels less economical than available fossil fuels. Lignocellulose conversions carried out at ≤ 50 °C have several limitations. Therefore, this review focuses on the importance of thermophilic bacteria and thermostable enzymes to overcome the limitations of existing lignocellulosic biomass conversion processes. The influence of high temperatures on various existing lignocellulose conversion processes and those that are under development, including separate hydrolysis and fermentation, simultaneous saccharification and fermentation, and extremophilic consolidated bioprocess are also discussed. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Thermostability and photophysical properties of mixed-ligand carboxylate/benzimidazole Zn(II)-coordination polymers

    International Nuclear Information System (INIS)

    Barros, Bráulio Silva; Chojnacki, Jaroslaw; Macêdo Soares, Antonia Alice; Kulesza, Joanna; Lourenço da Luz, Leonis; Júnior, Severino Alves

    2015-01-01

    The reaction between Zn(NO 3 ) 2 ·6H 2 O or Zn(CH 3 COO) 2 ·2H 2 O and isophthalic acid (1,3-H 2 bdc) in the presence of benzimidazole (Hbzim) in dimethylformamide (DMF)/ethanol (EtOH)/H 2 O solvent mixture at room temperature yielded two structurally different coordination polymers: [Zn 2 (1,3-bdc) 2 (Hbzim) 2 ] (1) and [Zn 2 (1,3-bdc)(bzim) 2 ] (2). (1) is a 2D-layered framework with a molecule of benzimidazole coordinated to the Zn center, whereas (2) is a 3D framework with benzimidazolate species acting as a co-ligand and bridging two Zn(II) ions. Reactions performed at 90 °C led to the formation of coordination polymers structurally similar to (2), independently of the Zn(II) source used. In the absence of benzimidazole, the reaction between ZnAc 2 .2H 2 O and 1,3-H 2 bdc at 90 °C resulted in the formation of (3), a 3D coordination polymer Zn(HCOO) 3 (Me 2 NH 2 + ). It was observed that the thermostability and the photophysical properties of (1) and (2) are strongly dependent on the coordination modes and packing of benzimidazole in the solid state. These materials present photoluminescence in the wide range of the spectrum, from UV to IR. A full understanding of a physical process occurring in these intriguing systems, including complete energy level diagrams with possible transitions were provided. - Graphical abstract: Display Omitted - Highlights: • Structurally different Zn(II)-coordination polymers were prepared. • The formation of frameworks was counter anion and temperature dependent. • Photoluminescence in the wide range of the spectrum, from UV to IR was observed. • Thermostability and luminescence depended on bzim packing in the structure

  8. Iterative key-residues interrogation of a phytase with thermostability increasing substitutions identified in directed evolution.

    Science.gov (United States)

    Shivange, Amol V; Roccatano, Danilo; Schwaneberg, Ulrich

    2016-01-01

    Bacterial phytases have attracted industrial interest as animal feed supplement due to their high activity and sufficient thermostability (required for feed pelleting). We devised an approach named KeySIDE,  an iterative Key-residues interrogation of the wild type with Substitutions Identified in Directed Evolution for improving Yersinia mollaretii phytase (Ymphytase) thermostability by combining key beneficial substitutions and elucidating their individual roles. Directed evolution yielded in a discovery of nine positions in Ymphytase and combined iteratively to identify key positions. The "best" combination (M6: T77K, Q154H, G187S, and K289Q) resulted in significantly improved thermal resistance; the residual activity improved from 35 % (wild type) to 89 % (M6) at 58 °C and 20-min incubation. Melting temperature increased by 3 °C in M6 without a loss of specific activity. Molecular dynamics simulation studies revealed reduced flexibility in the loops located next to helices (B, F, and K) which possess substitutions (Helix-B: T77K, Helix-F: G187S, and Helix-K: K289E/Q). Reduced flexibility in the loops might be caused by strengthened hydrogen bonding network (e.g., G187S and K289E/K289Q) and a salt bridge (T77K). Our results demonstrate a promising approach to design phytases in food research, and we hope that the KeySIDE might become an attractive approach for understanding of structure-function relationships of enzymes.

  9. Enzimas termoestáveis: fontes, produção e aplicação industrial Thermostable enzymes: sources, production and industrial applications

    Directory of Open Access Journals (Sweden)

    Eleni Gomes

    2007-02-01

    Full Text Available REVIEW: Living organisms encountered in hostile environments that are characterized by extreme temperatures rely on novel molecular mechanisms to enhance the thermal stability of their proteins, nucleic acids, lipids and cell membranes. Proteins isolated from thermophilic organisms usually exhibit higher intrinsic thermal stabilities than their counterparts isolated from mesophilic organisms. Although the molecular basis of protein thermostability is only partially understood, structural studies have suggested that the factors that may contribute to enhance protein thermostability mainly include hydrophobic packing, enhanced secondary structure propensity, helix dipole stabilization, absence of residues sensitive to oxidation or deamination, and increased electrostatic interactions. Thermostable enzymes such as amylases, xylanases and pectinases isolated from thermophilic organisms are potentially of interest in the optimization of industrial processes due to their enhanced stability. In the present review, an attempt is made to delineate the structural factors that increase enzyme thermostability and to document the research results in the production of these enzymes.

  10. Engineering of an Extremely Thermostable Alpha/Beta Barrel Scaffold to Serve as a High Affinity Molecular Recognition Element for Use in Sensor Applications

    Science.gov (United States)

    2015-12-23

    Molecular Recognition Element For Use in Sensor Applications Report Title The overall goal of the project was to evolve a highly thermostable enzyme ( alcohol ...SECURITY CLASSIFICATION OF: The overall goal of the project was to evolve a highly thermostable enzyme ( alcohol dehydrogenase D (AdhD) from Pyrococcus...furiosus) to bind an explosive molecule, RDX. The enzyme naturally catalyzes the nicotinamide cofactor-dependent oxidation or reduction of alcohols

  11. Enhancement of thermostability and kinetic efficiency of Aspergillus niger PhyA phytase by site-directed mutagenesis.

    Science.gov (United States)

    Hesampour, Ardeshir; Siadat, Seyed Ehsan Ranaei; Malboobi, Mohammad Ali; Mohandesi, Nooshin; Arab, Seyed Shahriar; Ghahremanpour, Mohammad Mehdi

    2015-03-01

    Phytase efficiently catalyzes the hydrolysis of phytate to phosphate; it can be utilized as an animal supplement to provide animals their nutrient requirements for phosphate and to mitigate environmental pollution caused by unutilized feed phosphate. Owing to animal feed being commonly pelleted at 70 to 90 °C, phytase with a sufficiently high thermal stability is desirable. Based on the crystal structure of PhyA and bioinformatics analysis at variant heat treatments, 12 single and multiple mutants were introduced by site-directed mutagenesis in order to improve phytase thermostability. Mutated constructs were expressed in Pichia pastoris. The manipulated phytases were purified; their biochemical and kinetic investigation revealed that while the thermostability of six mutants was improved, P9 (T314S Q315R V62N) and P12 (S205N S206A T151A T314S Q315R) showed the highest heat stability (P phytase to be used as an animal feed supplement.

  12. Crystallization and preliminary X-ray diffraction studies of the precursor protein of a thermostable variant of papain

    International Nuclear Information System (INIS)

    Roy, Sumana; Choudhury, Debi; Chakrabarti, Chandana; Biswas, Sampa; Dattagupta, J. K.

    2011-01-01

    The crystallization of the precursor of a thermostable variant of papain and the collection of diffraction data to 2.6 Å resolution are reported. The crystallization of a recombinant thermostable variant of pro-papain has been carried out. The mutant pro-enzyme was expressed in Escherichia coli as inclusion bodies, refolded, purified and crystallized. The crystals belonged to space group P2 1 , with unit-cell parameters a = 42.9, b = 74.8, c = 116.5 Å, β = 93.0°, and diffracted to 2.6 Å resolution using synchrotron radiation. Assuming the presence of two molecules in the asymmetric unit, the calculated Matthews coefficient is 2.28 Å 3 Da −1 , corresponding to a solvent content of 46%. Initial attempts to solve the structure using molecular-replacement techniques were successful

  13. Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties

    Directory of Open Access Journals (Sweden)

    Koh Eunhee

    2007-07-01

    Full Text Available Abstract Background EstE1 is a hyperthermophilic esterase belonging to the hormone-sensitive lipase family and was originally isolated by functional screening of a metagenomic library constructed from a thermal environmental sample. Dimers and oligomers may have been evolutionally selected in thermophiles because intersubunit interactions can confer thermostability on the proteins. The molecular mechanisms of thermostabilization of this extremely thermostable esterase are not well understood due to the lack of structural information. Results Here we report for the first time the 2.1-Å resolution crystal structure of EstE1. The three-dimensional structure of EstE1 exhibits a classic α/β hydrolase fold with a central parallel-stranded beta sheet surrounded by alpha helices on both sides. The residues Ser154, Asp251, and His281 form the catalytic triad motif commonly found in other α/β hydrolases. EstE1 exists as a dimer that is formed by hydrophobic interactions and salt bridges. Circular dichroism spectroscopy and heat inactivation kinetic analysis of EstE1 mutants, which were generated by structure-based site-directed mutagenesis of amino acid residues participating in EstE1 dimerization, revealed that hydrophobic interactions through Val274 and Phe276 on the β8 strand of each monomer play a major role in the dimerization of EstE1. In contrast, the intermolecular salt bridges contribute less significantly to the dimerization and thermostability of EstE1. Conclusion Our results suggest that intermolecular hydrophobic interactions are essential for the hyperthermostability of EstE1. The molecular mechanism that allows EstE1 to endure high temperature will provide guideline for rational design of a thermostable esterase/lipase using the lipolytic enzymes showing structural similarity to EstE1.

  14. Thermostability of Multidomain Proteins: Elongation Factors EF-Tu from Escherichia coli and Bacillus stearothermophilus and Their Chimeric Forms

    Czech Academy of Sciences Publication Activity Database

    Šanderová, Hana; Hůlková, Marta; Maloň, Petr; Kepková, M.; Jonák, Jiří

    2004-01-01

    Roč. 13, č. 1 (2004), s. 89-99 ISSN 0961-8368 R&D Projects: GA AV ČR IPP1050128; GA ČR GA204/98/0863; GA ČR GA303/02/0689 Institutional research plan: CEZ:AV0Z4055905; CEZ:AV0Z5052915 Keywords : elongation factor EF-Tu, thermostability, chimeric protein Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.116, year: 2004

  15. Bilirubin oxidase-like proteins from Podospora anserina: promising thermostable enzymes for application in transformation of plant biomass.

    Science.gov (United States)

    Xie, Ning; Ruprich-Robert, Gwenaël; Silar, Philippe; Chapeland-Leclerc, Florence

    2015-03-01

    Plant biomass degradation by fungi is a critical step for production of biofuels, and laccases are common ligninolytic enzymes envisioned for ligninolysis. Bilirubin oxidases (BODs)-like are related to laccases, but their roles during lignocellulose degradation have not yet been fully investigated. The two BODs of the ascomycete fungus Podospora anserina were characterized by targeted gene deletions. Enzymatic assay revealed that the bod1(Δ) and bod2(Δ) mutants lost partly a thermostable laccase activity. A triple mutant inactivated for bod1, bod2 and mco, a previously investigated multicopper oxidase gene distantly related to laccases, had no thermostable laccase activity. The pattern of fruiting body production in the bod1(Δ) bod2(Δ) double mutant was changed. The bod1(Δ) and bod2(Δ) mutants were reduced in their ability to grow on ligneous and cellulosic materials. Furthermore, bod1(Δ) and bod2(Δ) mutants were defective towards resistance to phenolic substrates and H2 O2 , which may also impact lignocellulose breakdown. Double and triple mutants were more affected than single mutants, evidencing redundancy of function among BODs and mco. Overall, the data show that bod1, bod2 and mco code for non-canonical thermostable laccases that participate in the degradation of lignocellulose. Thanks to their thermal stability, these enzymes may be more promising candidate for biotechnological application than canonical laccases. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  16. Enhancement of thermo-stability and product tolerance of Pseudomonas putida nitrile hydratase by fusing with self-assembling peptide.

    Science.gov (United States)

    Liu, Yi; Cui, Wenjing; Liu, Zhongmei; Cui, Youtian; Xia, Yuanyuan; Kobayashi, Michihiko; Zhou, Zhemin

    2014-09-01

    Self-assembling amphipathic peptides (SAPs) are the peptides that can spontaneously assemble into ordered nanostructures. It has been reported that the attachment of SAPs to the N- or C-terminus of an enzyme can benefit the thermo-stability of the enzyme. Here, we discovered that the thermo-stability and product tolerance of nitrile hydratase (NHase) were enhanced by fusing with two of the SAPs (EAK16 and ELK16). When the ELK16 was fused to the N-terminus of β-subunit, the resultant NHase (SAP-NHase-2) became an active inclusion body; EAK16 fused NHase in the N-terminus of β-subunit (SAP-NHase-1) and ELK16 fused NHase in the C-terminus of β-subunit (SAP-NHase-10) did not affect NHase solubility. Compared with the deactivation of the wild-type NHase after 30 min incubation at 50°C, SAP-NHase-1, SAP-NHase-2 and SAP-NHase-10 retained 45%, 30% and 50% activity; after treatment in the buffer containing 10% acrylamide, the wild-type retained 30% activity, while SAP-NHase-1, SAP-NHase-2 and SAP-NHase-10 retained 52%, 42% and 55% activity. These SAP-NHases with enhanced thermo-stability and product tolerance would be helpful for further industrial applications of the NHase. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  17. A hyper-thermostable α-amylase from Pyrococcus furiosus accumulates in Nicotiana tabacum as functional aggregates.

    Science.gov (United States)

    Zhu, Hong; Reynolds, L Bruce; Menassa, Rima

    2017-06-19

    Alpha amylase hydrolyzes α-bonds of polysaccharides such as starch and produces malto-oligosaccharides. Its starch saccharification applications make it an essential enzyme in the textile, food and brewing industries. Commercially available α-amylase is mostly produced from Bacillus or Aspergillus. A hyper-thermostable and Ca 2++ independent α-amylase from Pyrococcus furiosus (PFA) expressed in E.coli forms insoluble inclusion bodies and thus is not feasible for industrial applications. We expressed PFA in Nicotiana tabacum and found that plant-produced PFA forms functional aggregates with an accumulation level up to 3.4 g/kg FW (fresh weight) in field conditions. The aggregates are functional without requiring refolding and therefore have potential to be applied as homogenized plant tissue without extraction or purification. PFA can also be extracted from plant tissue upon dissolution in a mild reducing buffer containing SDS. Like the enzyme produced in P. furiosus and in E. coli, plant produced PFA preserves hyper-thermophilicity and hyper-thermostability and has a long shelf life when stored in lyophilized leaf tissue. With tobacco's large biomass and high yield, hyper-thermostable α-amylase was produced at a scale of 42 kg per hectare. Tobacco may be a suitable bioreactor for industrial production of active hyperthermostable alpha amylase.

  18. Development of thermostable Peste des Petits Ruminants (PPR) virus vaccine and assessment of molecular changes in the F gene

    International Nuclear Information System (INIS)

    Palaniswami, K.S.; Thangavelu, A.; Velmurugan, R.

    2005-01-01

    Two Indian PPRV isolates were subjected to thermal hardening procedures to increase the proportion of temperature-resistant virions. Initial infectivity loss was compensated by titre increases on subsequent cell passages at 37 deg C. The immunogenicity of 'thermostable' viruses was assessed by virulent PPRV challenge and for safety by host animal inoculation and antibodies assessment. Vaccine viruses were not found using PCR on ocular and nasal swabs, although virus nucleic acid and antigens were demonstrated in spleen and lymph nodes by FAT and PCR. One vaccine strain (MIB187(T)) giving 100% protection (tested on only a few animals) was freeze dried and the minimum protective dose calculated. Changes in the virus genome after thermo-adaptation were examined using RT-PCR to amplify portions of the F gene, and three base changes were observed in the thermostable PPR strain (compared with the F gene sequence of the Nigerian PPRV strain). At room temperature, the titre and potency of the thermo-adapted vaccine remained constant up to one month at the10 5.5 TCID 50 level, and was 10 4.5 TCID 50 /100 μl after two months. Field trials with over 40 000 doses of the thermostable vaccine under various environmental conditions have given serum neutralization titres exceeding 2 3 and are assumed protective. (author)

  19. Field-acclimated Gossypium hirsutum cultivars exhibit genotypic and seasonal differences in photosystem II thermostability.

    Science.gov (United States)

    Snider, John L; Oosterhuis, Derrick M; Collins, Guy D; Pilon, Cristiane; Fitzsimons, Toby R

    2013-03-15

    Previous investigations have demonstrated that photosystem II (PSII) thermostability acclimates to prior exposure to heat and drought, but contrasting results have been reported for cotton (Gossypium hirsutum). We hypothesized that PSII thermotolerance in G. hirsutum would acclimate to environmental conditions during the growing season and that there would be differences in PSII thermotolerance between commercially-available U.S. cultivars. To this end, three cotton cultivars were grown under dryland conditions in Tifton Georgia, and two under irrigated conditions in Marianna Arkansas. At Tifton, measurements included PSII thermotolerance (T15, the temperature causing a 15% decline in maximum quantum yield), leaf temperatures, air temperatures, midday (1200 to 1400h) leaf water potentials (ΨMD), leaf-air vapor pressure deficit (VPD), actual quantum yield (ΦPSII) and electron transport rate through PSII (ETR) on three sample dates. At Marianna, T15 was measured on two sample dates. Optimal air and leaf temperatures were observed on all sample dates in Tifton, but PSII thermotolerance increased with water deficit conditions (ΨMD=-3.1MPa), and ETR was either unaffected or increased under water-stress. Additionally, T15 for PHY 499 was ∼5°C higher than for the other cultivars examined (DP 0912 and DP 1050). The Marianna site experienced more extreme high temperature conditions (20-30 days Tmax≥35°C), and showed an increase in T15 with higher average Tmax. When average T15 values for each location and sample date were plotted versus average daily Tmax, strong, positive relationships (r(2) from .954 to .714) were observed between Tmax and T15. For all locations T15 was substantially higher than actual field temperature conditions. We conclude that PSII thermostability in G. hirsutum acclimates to pre-existing environmental conditions; PSII is extremely tolerant to high temperature and water-deficit stress; and differences in PSII thermotolerance exist between

  20. Carbohydrate-Based Ice Recrystallization Inhibitors Increase Infectivity and Thermostability of Viral Vectors

    Science.gov (United States)

    Ghobadloo, Shahrokh M.; Balcerzak, Anna K.; Gargaun, Ana; Muharemagic, Darija; Mironov, Gleb G.; Capicciotti, Chantelle J.; Briard, Jennie G.; Ben, Robert N.; Berezovski, Maxim V.

    2014-07-01

    The inability of vaccines to retain sufficient thermostability has been an obstacle to global vaccination programs. To address this major limitation, we utilized carbohydrate-based ice recrystallization inhibitors (IRIs) to eliminate the cold chain and stabilize the potency of Vaccinia virus (VV), Vesicular Stomatitis virus (VSV) and Herpes virus-1 (HSV-1). The impact of these IRIs was tested on the potency of the viral vectors using a plaque forming unit assay following room temperature storage, cryopreservation with successive freeze-thaw cycles and lyophilization. Viral potency after storage with all three conditions demonstrated that N-octyl-gluconamide (NOGlc) recovered the infectivity of shelf stored VV, 5.6 Log10 PFU mL-1 during 40 days, and HSV-1, 2.7 Log10 PFU mL-1 during 9 days. Carbon-linked antifreeze glycoprotein analogue ornithine-glycine-glycine-galactose (OGG-Gal) increases the recovery of VV and VSV more than 1 Log10 PFU mL-1 after 10 freeze-thaw cycles. In VSV, cryostorage with OGG-Gal maintains high infectivity and reduces temperature-induced aggregation of viral particles by 2 times that of the control. In total, OGG-Gal and NOGlc preserve virus potency during cryostorage. Remarkably, NOGlc has potential to eliminate the cold chain and permit room temperature storage of viral vectors.

  1. VapD in Xylella fastidiosa Is a Thermostable Protein with Ribonuclease Activity.

    Science.gov (United States)

    Mendes, Juliano S; Santiago, André da S; Toledo, Marcelo A S; Rosselli-Murai, Luciana K; Favaro, Marianna T P; Santos, Clelton A; Horta, Maria Augusta C; Crucello, Aline; Beloti, Lilian L; Romero, Fabian; Tasic, Ljubica; de Souza, Alessandra A; de Souza, Anete P

    2015-01-01

    Xylella fastidiosa strain 9a5c is a gram-negative phytopathogen that is the causal agent of citrus variegated chlorosis (CVC), a disease that is responsible for economic losses in Brazilian agriculture. The most well-known mechanism of pathogenicity for this bacterial pathogen is xylem vessel occlusion, which results from bacterial movement and the formation of biofilms. The molecular mechanisms underlying the virulence caused by biofilm formation are unknown. Here, we provide evidence showing that virulence-associated protein D in X. fastidiosa (Xf-VapD) is a thermostable protein with ribonuclease activity. Moreover, protein expression analyses in two X. fastidiosa strains, including virulent (Xf9a5c) and nonpathogenic (XfJ1a12) strains, showed that Xf-VapD was expressed during all phases of development in both strains and that increased expression was observed in Xf9a5c during biofilm growth. This study is an important step toward characterizing and improving our understanding of the biological significance of Xf-VapD and its potential functions in the CVC pathosystem.

  2. On-Demand Production of Flow-Reactor Cartridges by 3D Printing of Thermostable Enzymes.

    Science.gov (United States)

    Maier, Manfred; Radtke, Carsten P; Hubbuch, Jürgen; Niemeyer, Christof M; Rabe, Kersten S

    2018-05-04

    The compartmentalization of chemical reactions is an essential principle of life that provides a major source of innovation for the development of novel approaches in biocatalysis. To implement spatially controlled biotransformations, rapid manufacturing methods are needed for the production of biocatalysts that can be applied in flow systems. Whereas three-dimensional (3D) printing techniques offer high-throughput manufacturing capability, they are usually not compatible with the delicate nature of enzymes, which call for physiological processing parameters. We herein demonstrate the utility of thermostable enzymes in the generation of biocatalytic agarose-based inks for a simple temperature-controlled 3D printing process. As examples we utilized an esterase and an alcohol dehydrogenase from thermophilic organisms as well as a decarboxylase that was thermostabilized by directed protein evolution. We used the resulting 3D-printed parts for a continuous, two-step sequential biotransformation in a fluidic setup. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. A thermostable serralysin inhibitor from marine bacterium Flavobacterium sp. YS-80-122

    Science.gov (United States)

    Liang, Pengjuan; Li, Shangyong; Wang, Kun; Wang, Fang; Xing, Mengxin; Hao, Jianhua; Sun, Mi

    2018-03-01

    Serralysin inhibitors have been proposed as potent drugs against many diseases and may help to prevent further development of antibiotic-resistant pathogenic bacteria. In this study, a novel serralysin inhibitor gene, lupI, was cloned from the marine bacterium Flavobacterium sp. YS-80-122 and expressed in Escherichia coli. The deduced serralysin inhibitor, LupI, shows <40% amino acid identity to other reported serralysin inhibitors. Multiple sequence alignment and phylogenetic analysis of LupI with other serralysin inhibitors indicated that LupI was a novel type of serralysin inhibitor. The inhibitory constant for LupI towards its target metalloprotease was 0.64 μmol/L. LupI was thermostable at high temperature, in which 35.6%-90.7% of its inhibitory activity was recovered after treatment at 100°C for 1-60 min followed by incubation at 0°C. This novel inhibitor may represent a candidate drug for the treatment of serralysin-related infections.

  4. Thermostable Bacteriocin BL8 from Bacillus licheniformis isolated from marine sediment.

    Science.gov (United States)

    Smitha, S; Bhat, S G

    2013-03-01

    To isolate and characterize bacteriocin, BL8, from the bacteria identified as Bacillus licheniformis from marine environment. One-hundred and twelve bacterial isolates from sediment and water samples collected off the coast of Cochin, India, were screened for antibacterial activity. Strain BTHT8, identified as Bacillus licheniformis, inhibited the growth of Gram-positive test organisms. The active component labelled as bacteriocin BL8 was partially purified by ammonium sulphate fractionation and was subjected to glycine SDS-PAGE. The band exhibiting antimicrobial activity was electroeluted and analysed using MALDI-TOF mass spectrometry, and the molecular mass was determined as 1.4 kDa. N-terminal amino acid sequencing of BL8 gave a 13 amino acid sequence stretch. Bacteriocin BL8 was stable even after boiling at 100 °C for 30 min and over a wide pH range of 1-12. A novel, pH-tolerant and thermostable bacteriocin BL8, active against the tested Gram-positive bacteria, was isolated from Bacillus licheniformis. This study reports a stable, low molecular weight bacteriocin from Bacillus licheniformis. This bacteriocin can be used to address two important applications: as a therapeutic agent and as a biopreservative in food processing industry. © 2012 The Society for Applied Microbiology.

  5. High yield recombinant thermostable α-amylase production using an improved Bacillus licheniformis system

    Directory of Open Access Journals (Sweden)

    Shi Gui-Yang

    2009-10-01

    Full Text Available Abstract Background Some strains of Bacillus licheniformis have been improved by target-directed screening as well as by classical genetic manipulation and used in commercial thermostable α-amylase and alkaline protease production for over 40 years. Further improvements in production of these enzymes are desirable. Results A new strain of B. licheniformis CBBD302 carrying a recombinant plasmid pHY-amyL for Bacillus licheniformis α-amylase (BLA production was constructed. The combination of target-directed screening and genetic recombination led to an approximately 26-fold improvement of BLA production and export in B. licheniformis. Furthermore, a low-cost fermentation medium containing soybean meal and cottonseed meal for BLA production in shake-flasks and in a 15 liter bioreactor was developed and a BLA concentration of up to 17.6 mg per ml growth medium was attained. Conclusion This production level of BLA by B. licheniformis CBBD302(pHY-amyL is amongst the highest levels in Gram-positive bacteria reported so far.

  6. Geometric Simulation Approach for Grading and Assessing the Thermostability of CALBs

    Directory of Open Access Journals (Sweden)

    B. Senthilkumar

    2016-01-01

    Full Text Available Candida antarctica lipase B (CALB is a known stable and highly active enzyme used widely in biodiesel synthesis. In this work, the stability of native (4K6G and mutant (4K5Q CALB was studied through various structural parameters using conformational sampling approach. The contours of polar surface area and surface area of mutant CALB were 11357.67 Å2 and 30007.4 Å2, respectively, showing an enhanced stability compared to native CALB with a statistically significant P value of < 0.0001. Moreover, simulated thermal denaturation of CALB, a process involving dilution of hydrogen bond, significantly shielded against different intervals of energy application in mutant CALB revealing its augmentation of structural rigidity against native CALB. Finally, computational docking analysis showed an increase in the binding affinity of CALB and its substrate (triglyceride in mutant CALB with Atomic Contact Energy (ACE of −91.23 kcal/mol compared to native CALB (ACE of −70.3 kcal/mol. The computational observations proposed that the use of mutant CALB (4K5Q could serve as a best template for production of biodiesel in the future. Additionally, it can also be used as a template to identify efficient thermostable lipases through further mutations.

  7. Construction and engineering of a thermostable self-sufficient cytochrome P450

    Energy Technology Data Exchange (ETDEWEB)

    Mandai, Takao; Fujiwara, Shinsuke [Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337 (Japan); Imaoka, Susumu, E-mail: imaoka@kwansei.ac.jp [Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337 (Japan)

    2009-06-19

    CYP175A1 is a thermophilic cytochrome P450 and hydroxylates {beta}-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP{sup +} reductase (FNR): H{sub 2}N-CYP175A1-Fdx-FNR-COOH (175FR) and H{sub 2}N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V{sub max} value for {beta}-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k{sub m} values of these enzymes were similar. 175RF retained 50% residual activity even at 80 {sup o}C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.

  8. FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants.

    Science.gov (United States)

    Bednar, David; Beerens, Koen; Sebestova, Eva; Bendl, Jaroslav; Khare, Sagar; Chaloupkova, Radka; Prokop, Zbynek; Brezovsky, Jan; Baker, David; Damborsky, Jiri

    2015-11-01

    There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C) by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.

  9. FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants.

    Directory of Open Access Journals (Sweden)

    David Bednar

    2015-11-01

    Full Text Available There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.

  10. VapD in Xylella fastidiosa Is a Thermostable Protein with Ribonuclease Activity.

    Directory of Open Access Journals (Sweden)

    Juliano S Mendes

    Full Text Available Xylella fastidiosa strain 9a5c is a gram-negative phytopathogen that is the causal agent of citrus variegated chlorosis (CVC, a disease that is responsible for economic losses in Brazilian agriculture. The most well-known mechanism of pathogenicity for this bacterial pathogen is xylem vessel occlusion, which results from bacterial movement and the formation of biofilms. The molecular mechanisms underlying the virulence caused by biofilm formation are unknown. Here, we provide evidence showing that virulence-associated protein D in X. fastidiosa (Xf-VapD is a thermostable protein with ribonuclease activity. Moreover, protein expression analyses in two X. fastidiosa strains, including virulent (Xf9a5c and nonpathogenic (XfJ1a12 strains, showed that Xf-VapD was expressed during all phases of development in both strains and that increased expression was observed in Xf9a5c during biofilm growth. This study is an important step toward characterizing and improving our understanding of the biological significance of Xf-VapD and its potential functions in the CVC pathosystem.

  11. Thermostable, haloalkaline cellulase from Bacillus halodurans CAS 1 by conversion of lignocellulosic wastes.

    Science.gov (United States)

    Annamalai, Neelamegam; Rajeswari, Mayavan Veeramuthu; Elayaraja, Sivaramasamy; Balasubramanian, Thangavel

    2013-04-15

    An extracellular thermostable, haloalkaline cellulase by bioconversion of lignocellulosic wastes from Bacillus halodurans CAS 1 was purified to homogeneity with recovery of 12.54% and purity fold 7.96 with the molecular weight of 44 kDa. The optimum temperature, pH and NaCl for enzyme activity was determined as 60°C, 9.0 and 30% and it retained 80% of activity even at 80°C, 12 and 35% respectively. The activity was greatly inhibited by EDTA, indicating that it was a metalloenzyme and significant inhibition by PMSF revealed that serine residue was essential for catalytic activity. The purified cellulase hydrolyzed CMC, cellobiose and xylan, but not avicel, cellulose and PNPG. Furthermore, the cellulase was highly stable in the presence of detergents and organic solvents such as acetone, n-hexane and acetonitrile. Thus, the purified cellulase from B. halodurans utilizing lignocellulosic biomass could be greatly useful to develop industrial processes. Published by Elsevier Ltd.

  12. Thermostable phycocyanin from the red microalga Cyanidioschyzon merolae, a new natural blue food colorant.

    Science.gov (United States)

    Rahman, D Y; Sarian, F D; van Wijk, A; Martinez-Garcia, M; van der Maarel, M J E C

    2017-01-01

    The demand for natural food colorants is growing as consumers question the use of artificial colorants more and more. The phycobiliprotein C-phycocyanin of Arthospira platensis is used as a natural blue colorant in certain food products. The thermoacidophilic red microalga Cyanidioschyzon merolae might provide an alternative source of phycocyanin. Cyanidioschyzon merolae belongs to the order Cyanidiophyceae of the phylum Rhodophyta. Its natural habitat are sulfuric hot springs and geysers found near volcanic areas in, e.g., Yellowstone National Park in the USA and in Java, Indonesia. It grows optimally at a pH between 0.5 and 3.0 and at temperatures up to 56 °C. The low pH at which C . merolae grows minimizes the risk of microbial contamination and could limit production loss. As C . merolae lacks a cell wall, phycocyanin with a high purity number of 9.9 could be extracted by an osmotic shock using a simple ultrapure water extraction followed by centrifugation. The denaturation midpoint at pH 5 was 83 °C, being considerably higher than the A . platensis phycocyanin (65 °C). The C . merolae phycocyanin was relatively stable at pH 4 and 5 up to 80 °C. The high thermostability at slightly acidic pH makes the C . merolae phycocyanin an interesting alternative to A . platensis phycocyanin as a natural blue food colorant.

  13. Reconstitution of β-carotene hydroxylase activity of thermostable CYP175A1 monooxygenase

    International Nuclear Information System (INIS)

    Momoi, Kyoko; Hofmann, Ute; Schmid, Rolf D.; Urlacher, Vlada B.

    2006-01-01

    CYP175A1 is a thermostable P450 Monooxygenase from Thermus thermophilus HB27, demonstrating in vivo activity towards β-carotene. Activity of CYP175A1 was reconstituted in vitro using artificial electron transport proteins. First results were obtained in the mixture with a crude Escherichia coli cell extract at 37 o C. In this system, β-carotene was hydroxylated to β-cryptoxanthin. The result indicated the presence of electron transport enzymes among the E. coli proteins, which are suitable for CYP175A1. However, upon in vitro reconstitution of CYP175A1 activity with purified recombinant flavodoxin and flavodoxin reductase from E. coli, only very low β-cryptoxanthin production was observed. Remarkably, with another artificial electron transport system, putidaredoxin and putidaredoxin reductase from Pseudomonas putida, purified CYP175A1 enzyme hydroxylated β-carotene at 3- and also 3'-positions, resulting in β-cryptoxanthin and zeaxanthin. Under the optimal reaction conditions, the turnover rate of the enzyme reached 0.23 nmol β-cryptoxanthin produced per nmol P450 per min

  14. Construction and engineering of a thermostable self-sufficient cytochrome P450

    International Nuclear Information System (INIS)

    Mandai, Takao; Fujiwara, Shinsuke; Imaoka, Susumu

    2009-01-01

    CYP175A1 is a thermophilic cytochrome P450 and hydroxylates β-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP + reductase (FNR): H 2 N-CYP175A1-Fdx-FNR-COOH (175FR) and H 2 N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V max value for β-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k m values of these enzymes were similar. 175RF retained 50% residual activity even at 80 o C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.

  15. Not so monofunctional--a case of thermostable Thermobifida fusca catalase with peroxidase activity.

    Science.gov (United States)

    Lončar, Nikola; Fraaije, Marco W

    2015-03-01

    Thermobifida fusca is a mesothermophilic organism known for its ability to degrade plant biomass and other organics, and it was demonstrated that it represents a rich resource of genes encoding for potent enzymes for biocatalysis. The thermostable catalase from T. fusca has been cloned and overexpressed in Escherichia coli with a yield of 400 mg/L. Heat treatment of disrupted cells at 60 °C for 1 h resulted in enzyme preparation of high purity; hence, no chromatography steps are needed for large-scale production. Except for catalyzing the dismutation of hydrogen peroxide, TfuCat was also found to catalyze oxidations of phenolic compounds. The catalase activity was comparable to other described catalases while peroxidase activity was quite remarkable with a k obs of nearly 1000 s(-1) for catechol. Site directed mutagenesis was used to alter the ratio of peroxidase/catalase activity. Resistance to inhibition by classic catalase inhibitors and an apparent melting temperature of 74 °C classifies this enzyme as a robust biocatalyst. As such, it could compete with other commercially available catalases while the relatively high peroxidase activity also offers new biocatalytic possibilities.

  16. Hepatitis B surface antigen incorporated in dissolvable microneedle array patch is antigenic and thermostable.

    Science.gov (United States)

    Poirier, Danielle; Renaud, Frédéric; Dewar, Vincent; Strodiot, Laurent; Wauters, Florence; Janimak, Jim; Shimada, Toshio; Nomura, Tatsuya; Kabata, Koki; Kuruma, Koji; Kusano, Takayuki; Sakai, Masaki; Nagasaki, Hideo; Oyamada, Takayoshi

    2017-11-01

    Alternatives to syringe-based administration are considered for vaccines. Intradermal vaccination with dissolvable microneedle arrays (MNA) appears promising in this respect, as an easy-to-use and painless method. In this work, we have developed an MNA patch (MNAP) made of hydroxyethyl starch (HES) and chondroitin sulphate (CS). In swines, hepatitis B surface antigen (HBsAg) formulated with the saponin QS-21 as adjuvant, both incorporated in HES-based MNAP, demonstrated the same level of immunogenicity as a commercially available aluminum-adjuvanted HBsAg vaccine, after two immunizations 28 days apart. MNAP application was associated with transient skin reactions (erythema, lump, scab), particularly evident when the antigen was delivered with the adjuvant. The thermostability of the adjuvanted antigen when incorporated in the HES-based matrix was also assessed by storing MNAP at 37, 45 or 50 °C for up to 6 months. We could demonstrate that antigenicity was retained at 37 and 45 °C and only a 10% loss was observed after 6 months at 50 °C. Our results are supportive of MNAP as an attractive alternative to classical syringe-based vaccination. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  17. Heterologous Expression and Characterization of a Thermostable Exo-β-D-Glucosaminidase from Aspergillus oryzae.

    Science.gov (United States)

    Wu, Dingxin; Wang, Linchun; Li, Yuwei; Zhao, Shumiao; Peng, Nan; Liang, Yunxiang

    2016-02-01

    An exo-β-D-glucosaminidase (AorCsxA) from Aspergillus oryzae FL402 was heterologously expressed and purified. The deduced amino acid sequence indicated that AorCsxA belonged to glycoside hydrolase family 2. AorCsxA digested colloid chitosan into glucosamine but not into chitosan oligosaccharides, demonstrating exo-β-D-glucosaminidase (CsxA) activity. AorCsxA exhibited optimal activity at pH 5.5 and 50°C; however, the enzyme expressed in Pichia pastoris (PpAorCsxA) showed much stronger thermostability at 50°C than that expressed in Escherichia coli (EcAorCsxA), which may be related to glycosylation. AorCsxA activity was inhibited by EDTA and most of the tested metal ions. A single amino acid mutation (F769W) in AorCsxA significantly enhanced the specific activity and hydrolysis velocity as revealed by comparison of Vmax and kcat values with those of the wild-type enzyme. The three-dimensional structure suggested the tightened pocket at the active site of F769W enabled efficient substrate binding. The AorCsxA gene was heterologously expressed in P. pastoris, and one transformant was found to produce 222 U/ml activity during the high-cell-density fermentation. This AorCsxA-overexpressing P. pastoris strain is feasible for large-scale production of AorCsxA.

  18. The effect of different stabilizers on the thermostability of electron beam crosslinked polyethylene in hot water

    International Nuclear Information System (INIS)

    Hassanpour, S.; Khoylou, F.

    2003-01-01

    Plastic pipes owing to their flexibility, great lengths, easier handling and absence of corrosion have been used for hot-water installations. Crosslinked high-density polyethylene is one of the best materials, being used for this purpose. The useful lifetime of unstabilized polyethylene is predicted to vary from a few months in hot water (30-40 deg. C) to almost two years in cool water (0-10 deg. C). Polyethylene was mixed with different types of stabilizers, in order to increase its durability. The samples were irradiated at 100-150 kGy. The amount of gel fraction and the changes in mechanical properties were measured. Irradiated samples were immersed in hot water for 1000 h. The thermostability of the specimens and the existence of antioxidants were measured by the induction time technique using differential scanning calorimetry at different time intervals. Furthermore, the changes in chemical structure and mechanical properties of the samples during their immersion in hot water were determined

  19. Rational Design of Disulfide Bonds Increases Thermostability of a Mesophilic 1,3-1,4-β-Glucanase from Bacillus terquilensis.

    Directory of Open Access Journals (Sweden)

    Chengtuo Niu

    Full Text Available 1,3-1,4-β-glucanase is an important biocatalyst in brewing industry and animal feed industry, while its low thermostability often reduces its application performance. In this study, the thermostability of a mesophilic β-glucanase from Bacillus terquilensis was enhanced by rational design and engineering of disulfide bonds in the protein structure. Protein spatial configuration was analyzed to pre-exclude the residues pairs which negatively conflicted with the protein structure and ensure the contact of catalytic center. The changes in protein overall and local flexibility among the wild-type enzyme and the designated mutants were predicted to select the potential disulfide bonds for enhancement of thermostability. Two residue pairs (N31C-T187C and P102C-N125C were chosen as engineering targets and both of them were proved to significantly enhance the protein thermostability. After combinational mutagenesis, the double mutant N31C-T187C/P102C-N125C showed a 48.3% increase in half-life value at 60°C and a 4.1°C rise in melting temperature (Tm compared to wild-type enzyme. The catalytic property of N31C-T187C/P102C-N125C mutant was similar to that of wild-type enzyme. Interestingly, the optimal pH of double mutant was shifted from pH6.5 to pH6.0, which could also increase its industrial application. By comparison with mutants with single-Cys substitutions, the introduction of disulfide bonds and the induced new hydrogen bonds were proved to result in both local and overall rigidification and should be responsible for the improved thermostability. Therefore, the introduction of disulfide bonds for thermostability improvement could be rationally and highly-effectively designed by combination with spatial configuration analysis and molecular dynamics simulation.

  20. Optimization of Thermostable Alpha-Amylase Production Via Mix Agricultural-Residues and Bacillus amyloliquefaciens

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    Shalini RAI

    2014-03-01

    Full Text Available This study reports utilization of mixture of wheat and barley bran (1:1 for the production of thermostable alpha-amylase enzyme through a spore former, heat tolerant strain of Bacillus amyloliquefaciens in solid state fermentation. Maximum yield of alpha-amylase (252.77 U mL-1 was obtained in following optimized conditions, inoculums size 2 mL (2 × 106 CFU/mL, moisture 80%, pH 7±0.02, NaCl (3%, temperature 38±1°C, incubation for 72 h, maltose (1% and tryptone (1%. After SSF crude enzyme was purified via ammonium sulfate precipitation, ion exchange and column chromatography by DEAE Cellulose. Purified protein showed a molecular weight of 42 kDa by SDS-PAGE electrophoresis. After purification, purified enzyme was characterized against several enzymes inhibitors such as temperature, NaCl, pH, metal and surfactants. Pure enzyme was highly active over broad temperature (50-70°C, NaCl concentration (0.5-4 M, and pH (6-10 ranges, indicating it’s a thermoactive and alkali-stable nature. Moreover, CaCl2, MnCl2, =-mercaptoethanol were found to stimulate the amylase activity, whereas FeCl3, sodium dodecyl sulfate (SDS, CuCl3 and ethylenediaminetetraacetic acid (EDTA strongly inhibited the enzyme. Moreover, enzyme specificity and thermal stability conformed by degradation of different soluble starch up to 55°C. Therefore, the present study proved that the extracellular alpha-amylase extracted through wheat flour residues by organism B. amyloliquefaciens MCCB0075, both have considerable potential for industrial application owing to its properties.

  1. Evaluation of enhanced thermostability and operational stability of carbonic anhydrase from Micrococcus species.

    Science.gov (United States)

    Bhattacharya, Abhishek; Shrivastava, Ankita; Sharma, Anjana

    2013-06-01

    Carbonic anhydrase (CA) was purified from Micrococcus lylae and Micrococcus luteus with 49.90 and 53.8 % yield, respectively, isolated from calcium carbonate kilns. CA from M. lylae retained 80 % stability in the pH and temperature range of 6.0-8.0 and 35-45 °C, respectively. However, CA from M. luteus was stable in the pH and temperature range of 7.5-10.0 and 35-55 °C, respectively. Cross-linked enzyme aggregates (CLEAs) raised the transition temperature of M. lylae and M. luteus CA up to 67.5 and 74.0 °C, while the operational stability (T(1/20) of CA at 55 °C was calculated to be 7.7 and 12.0 h, respectively. CA from both the strains was found to be monomeric in nature with subunit molecular weight and molecular mass of 29 kDa. Ethoxozolamide was identified as the most potent inhibitor based on both IC(50) values and inhibitory constant measurement (K(i)). The K(m) and V(max) for M. lylae CA (2.31 mM; 769.23 μmol/mg/min) and M. luteus CA (2.0 mM; 1,000 μmol/mg/min) were calculated from Lineweaver-Burk plots in terms of esterase activity. Enhanced thermostability of CLEAs alleviates its role in operational stability for application at an on-site scrubber. The characteristic profile of purified CA from Micrococcus spp. advocates its effective application in biomimetic CO(2) sequestration.

  2. Production, Purification, and Characterization of Thermostable α-Amylase Produced by Bacillus licheniformis Isolate AI20

    Directory of Open Access Journals (Sweden)

    Yasser R. Abdel-Fattah

    2013-01-01

    Full Text Available An optimization strategy, based on statistical experimental design, is employed to enhance the production of thermostable α-amylase by a thermotolerant B. licheniformis AI20 isolate. Using one variant at time (OVAT method, starch, yeast extract, and CaCl2 were observed to influence the enzyme production significantly. Thereafter, the response surface methodology (RSM was adopted to acquire the best process conditions among the selected variables, where a three-level Box-Behnken design was employed to create a polynomial quadratic model correlating the relationship between the three variables and α-amylase activity. The optimal combination of the major constituents of media for α-amylase production was 1.0% starch, 0.75% yeast extract, and 0.02% CaCl2. The predicted optimum α-amylase activity was 384 U/mL/min, which is two folds more than the basal medium conditions. The produced α-amylase was purified through various chromatographic techniques. The estimated enzyme molecular mass was 55 kDa and the α-amylase had an optimal temperature and pH of 60–80°C and 6–7.5, respectively. Values of Vmax and Km for the purified enzyme were 454 mU/mg and 0.709 mg/mL. The α-amylase enzyme showed great stability against different solvents. Additionally, the enzyme activity was slightly inhibited by detergents, sodium dodecyl sulphate (SDS, or chelating agents such as EDTA and EGTA. On the other hand, great enzyme stability against different divalent metal ions was observed at 0.1 mM concentration, but 10 mM of Cu2+ or Zn2+ reduced the enzyme activity by 25 and 55%, respectively.

  3. Studies for methods to improve thermostability of the functionalized butadiene styrene rubbers

    Directory of Open Access Journals (Sweden)

    A. L. Rumyantseva

    2018-01-01

    Full Text Available It is well known that the tire performance properties can deteriorate in the processes of production, processing, storage and operation. One of the reasons for that is a series of processes occurring in the polymer under the influence of different factors: thermal, mechanical or chemical. This problem is particularly relevant for functionalized polymers, as functional groups can interact with each other, causing side cross linking reactions that lead to a deterioration of consumer properties of the products. The main purpose of this work was to study influence of several key factors on the thermostability of functionalized rubbers in order to find a solution: different types of antioxidants, rubber polymerizate stripping conditions and rubber processing. In accordance with the problem, solutions were found and work was carried out in several directions: changing the pH of the medium in the rubber stripping and using antioxidants containing carbonyl groups located in ?-positions to methylene groups, namely Irganox 1520 and Irganox 1076. As an evaluation factor, thermal treatment was selected in two modes: at 100 °C for 48 hours and after extruder at 130 °C for 5 minutes + 100 °C for 48 hours. At the same time, the following parameters were determined: molecular weight characteristics and Mooney viscosity of the starting polymers and after thermal aging. During the experiments, it was found that the acidity of the medium in the water degasser does not affect the crosslinking of the functionalized rubber during storage. In addition, a study was made of the effect of the type of antioxidant and its quantity on the thermal stability of functionalized styrene butadiene rubbers, as well as the study of the effect of the content of the modifying agent on the thermal stability of the product. It has been found that the use, as antioxidants, of carbonyl compounds containing a methylene group at the ?-position, leads to inhibition of the cross

  4. Cloning, purification and characterization of a thermostable β-galactosidase from Bacillus licheniformis strain KG9.

    Science.gov (United States)

    Matpan Bekler, F; Stougaard, P; Güven, K; Gül Güven, R; Acer, Ö

    2015-06-28

    A thermo— and alkalitolerant Bacillus licheniformis KG9 isolated from Taşlıdere hot water spring in Batman/Turkey was found to produce a thermostable β—galactosidase. Phylogenetic analysis showed that the 16S rRNA gene from B. licheniformis strain KG9 was 99.9% identical to that of the genome sequenced B. licheniformis strain DSM 13. Analysis of the B. licheniformis DSM 13 genomic sequence revealed four putative β—galactosidase genes. PCR primers based on the genome sequence of strain DSM 13 were used to isolate the corresponding β—galactosidase genes from B. licheniformis strain KG9. The calculated molecular weights of the β—galactosidases I, II, III, and IV using sequencing data were 30, 79, 74, and 79 kDa, respectively. The genes were inserted into an expression vector and recombinant β—galactosidase was produced in Escherichia coli. Of the four β—galactosidase genes identified in strain KG9, three of them were expressed as active, intracellular enzymes in E. coli. One of the recombinant enzymes, β—galactosidase III, was purified and characterized. Optimal temperature and pH was determined to be at 60 ºC and pH 6.0, respectively. Km was determined to be 1.3 mM and 13.3 mM with oNPG (ortho—nitrophenyl—β—D—galactopyranoside) and lactose as substrates, respectively, and Vmax was measured to 1.96 μmol/min and 1.55 μmol/min with oNPG and lactose, respectively.

  5. Characterization of thermostable alkaline proteases from Bacillus infantis SKS1 isolated from garden soil.

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    Sandeep Kaur Saggu

    Full Text Available Proteases are one of the largest groups of hydrolytic enzymes constituting about 60% of total worldwide sales of industrial enzymes due to their wide applications in detergent, leather, textile, food and pharmaceutical industry. Microbial proteases have been preferred over animal and plant proteases because of their fundamental features and ease in production. Bacillus infantis SKS1, an alkaline protease producing bacteria has been isolated from garden soil of north India and identified using morphological, biochemical and molecular methods. 16S rDNA sequence amplified using universal primers has 99% sequence identity with corresponding gene sequence of Bacillus infantis strain FM 34 and Bacillus sp. Beige. The bacterial culture and its 16S rDNA gene sequence have been deposited to Microbial Culture Collection (Pune, India with accession number MCC 3035 and GenBank with accession number KR092197 respectively. The partially purified extract of Bacillus infantis SKS1 was thermostable and active in presence of Mg2+, acetyl acetone and laundry detergents implicating its application in industry. Production of these enzymes using this strain was maximized by optimization of various parameters including temperature, pH, media components and other growth conditions. Our results show that fructose and dextrose serve as the best carbon sources for production of these enzymes, highlighting the use of this strain for enzyme production utilizing relatively inexpensive substrates like beet molasses and corn steep liquor. Additionally, this strain showed maximum production of enzymes at 40°C similar to bacterial species used for commercial production of alkaline proteases. Characterization of alkaline proteases from this strain of Bacillus infantis and optimization of parameters for its production would help in understanding its industrial application and large-scale production.

  6. Crystal structure of a thermostable Old Yellow Enzyme from Thermus scotoductus SA-01

    International Nuclear Information System (INIS)

    Opperman, Diederik J.; Sewell, Bryan T.; Litthauer, Derek; Isupov, Mikhail N.; Littlechild, Jennifer A.; Heerden, Esta van

    2010-01-01

    Recent characterization of the chromate reductase (CrS) from the thermophile Thermus scotoductus SA-01 revealed this enzyme to be related to the Old Yellow Enzyme (OYE) family. Here, we report the structure of a thermostable OYE homolog in its holoform at 2.2 A as well as its complex with p-hydroxybenzaldehyde (pHBA). The enzyme crystallized as octamers with the monomers showing a classical TIM barrel fold which upon dimerization yields the biologically active form of the protein. A sulfate ion is bound above the si-side of the non-covalently bound FMN cofactor in the oxidized solved structure but is displaced upon pHBA binding. The active-site architecture is highly conserved as with other members of this enzyme family. The pHBA in the CrS complex is positioned by hydrogen bonding to the two conserved catalytic-site histidines. The most prominent structural difference between CrS and other OYE homologs is the size of the 'capping domain'. Thermostabilization of the enzyme is achieved in part through increased proline content within loops and turns as well as increased intersubunit interactions through hydrogen bonding and complex salt bridge networks. CrS is able to reduce the C=C bonds of α,β-unsaturated carbonyl compounds with a preference towards cyclic substrates however no activity was observed towards β-substituted substrates. Mutational studies have confirmed the role of Tyr177 as the proposed proton donor although reduction could still occur at a reduced rate when this residue was mutated to phenylalanine.

  7. Isolation of a thermostable acid phytase from Aspergillus niger UFV-1 with strong proteolysis resistance

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    Paulo S. Monteiro

    2015-03-01

    Full Text Available An Aspergillus niger UFV-1 phytase was characterized and made available for industrial application. The enzyme was purified via ultrafiltration followed by acid precipitation, ion exchange and gel filtration chromatography. This protein exhibited a molecular mass of 161 kDa in gel filtration and 81 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, indicating that it may be a dimer. It presented an optimum temperature of 60 °C and optimum pH of 2.0. The KM for sodium phytate hydrolysis was 30.9 mM, while the kcat and kcat/KM were 1.46 ×105 s−1 and 4.7 × 106s−1.M−1, respectively. The purified phytase exhibited broad specificity on a range of phosphorylated compounds, presenting activity on sodium phytate, p-NPP, 2- naphthylphosphate, 1- naphthylphosphate, ATP, phenyl-phosphate, glucose-6-phosphate, calcium phytate and other substrates. Enzymatic activity was slightly inhibited by Mg2+, Cd2+, K+ and Ca2+, and it was drastically inhibited by F−. The enzyme displayed high thermostability, retaining more than 90% activity at 60 °C during 120 h and displayed a t1/2 of 94.5 h and 6.2 h at 70 °C and 80 °C, respectively. The enzyme demonstrated strong resistance toward pepsin and trypsin, and it retained more than 90% residual activity for both enzymes after 1 h treatment. Additionally, the enzyme efficiently hydrolyzed phytate in livestock feed, liberating 15.3 μmol phosphate/mL after 2.5 h of treatment.

  8. Partial Purification and Characterization of a Thermostable β-Mannanase from Aspergillus foetidus

    Directory of Open Access Journals (Sweden)

    Juliana da Conceição Infante de Marco

    2015-10-01

    Full Text Available An extracellular β-mannanase was isolated from samples of crude extract of the mesophilic fungus Aspergillus foetidus grown on soybean husk as a carbon source. The induction profile showed that β-mannanase reached a maximum activity level (2.0 IU/mL on the 15th day of cultivation. The enzyme was partially purified by ultrafiltration and gel filtration chromatography procedures and was named Man 58. Sodium dodecyl sulfate-polyacrilamide electrophoresis and zymogram analysis of Man 58 showed two bands of approximately 43 and 45 kDa with β-mannanase activity. Ultrafiltration showed that β-mannanase activity was only detected in the concentrated sample. Man 58 was most active at 60 °C and at pH 4.0. It was thermostable in the temperature range of 40–60 °C for eleven days, and the half-life at 70 °C was ten days. Man 58 showed Km and Vmax values of 3.29 mg/mL and 1.76 IU/mL respectively, with locust bean gum as a substrate. It was mostly activated by FeSO4 and CoCl2 and inhibited by MgSO4, FeCl3, CuSO4, MgCl2, ZnCl2, ZnSO4, CaCl2, CuCl2, KCl and ethylenediaminetetraacetic acid (EDTA. Phenolic compounds did not inhibit the enzyme. On the other hand, auto-hydrolysis liquor showed an inhibitory effect on Man 58 activity.

  9. Experimental study on the thermostable property of aramid fiber reinforced PE-RT pipes

    Directory of Open Access Journals (Sweden)

    Guoquan Qi

    2015-11-01

    Full Text Available Flexible composite pipes are advantageous in ultra high strength, high modulus, pH and corrosion resistance and light weight, but there are still some hidden safety troubles because they are poorer in thermostable capacity. Therefore, test samples of flexible composite pipes were prepared with high-temperature polythene (PE-RT as the neck bush and aramid fiber as the reinforcement layer. Experimental study was conducted by using HPHT vessel and differential thermal scanner for different working conditions, different temperatures, whole-pipe pressure-bearing capacity and 1000 h viability. It is shown by the environmental compatibility test that high temperature has little effect on the weight, Vicat softening temperature, mechanical properties and structures of neck bush PE-RT, but exerts an obvious effect on the tensility and whole-pipe water pressure blasting of the reinforcement aramid fiber. Besides, the drop of whole-pipe pressure-bearing capacity is caused by deformation and breaking of aramid fibers when the reinforcement layer is under the force of internal pressure. Finally, disorientation and crystallization of molecular thermal motion occur with the rise of temperature, so amorphous orientation reduces, crystallinity factor and crystalline orientation factor increase gradually, thus, disorientation of macromolecular chains increases and tensile strength decreases. It is concluded that this type of flexible composite pipe can smoothly pass 1000 h viability test. And it is recommended that it be used in the situations with temperature not higher than 95 °C and internal pressure not higher than 4 MPa.

  10. Isolation of a thermostable acid phytase from Aspergillus niger UFV-1 with strong proteolysis resistance

    Science.gov (United States)

    Monteiro, Paulo S.; Guimarães, Valéria M.; de Melo, Ricardo R.; de Rezende, Sebastião T.

    2015-01-01

    An Aspergillus niger UFV-1 phytase was characterized and made available for industrial application. The enzyme was purified via ultrafiltration followed by acid precipitation, ion exchange and gel filtration chromatography. This protein exhibited a molecular mass of 161 kDa in gel filtration and 81 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), indicating that it may be a dimer. It presented an optimum temperature of 60 °C and optimum pH of 2.0. The K M for sodium phytate hydrolysis was 30.9 mM, while the k cat and k cat / K M were 1.46 ×10 5 s −1 and 4.7 × 10 6 s −1 .M −1 , respectively. The purified phytase exhibited broad specificity on a range of phosphorylated compounds, presenting activity on sodium phytate, p-NPP, 2- naphthylphosphate, 1- naphthylphosphate, ATP, phenyl-phosphate, glucose-6-phosphate, calcium phytate and other substrates. Enzymatic activity was slightly inhibited by Mg 2+ , Cd 2+ , K + and Ca 2+ , and it was drastically inhibited by F − . The enzyme displayed high thermostability, retaining more than 90% activity at 60 °C during 120 h and displayed a t 1/2 of 94.5 h and 6.2 h at 70 °C and 80 °C, respectively. The enzyme demonstrated strong resistance toward pepsin and trypsin, and it retained more than 90% residual activity for both enzymes after 1 h treatment. Additionally, the enzyme efficiently hydrolyzed phytate in livestock feed, liberating 15.3 μmol phosphate/mL after 2.5 h of treatment. PMID:26221114

  11. Design of thermostable rhamnogalacturonan lyase mutants from Bacillus licheniformis by combination of targeted single point mutations

    DEFF Research Database (Denmark)

    da Silva, Ines Isabel Cardoso Rodrigues; Jers, Carsten; Otten, Harm

    2014-01-01

    Rhamnogalacturonan I lyases (RGI lyases) (EC 4.2.2.-) catalyze cleavage of α-1,4 bonds between rhamnose and galacturonic acid in the backbone of pectins by β-elimination. In the present study, targeted improvement of the thermostability of a PL family 11 RGI lyase from Bacillus licheniformis (DSM......, were obtained due to additive stabilizing effects of single amino acid mutations (E434L, G55V, and G326E) compared to the wild type. The crystal structure of the B. licheniformis wild-type RGI lyase was also determined; the structural analysis corroborated that especially mutation of charged amino...

  12. Draft genome sequence of a thermostable, alkaliphilic α-amylase and protease producing Bacillus amyloliquefaciens strain KCP2.

    Science.gov (United States)

    Prajapati, Vimalkumar S; Ray, Sanket; Narayan, Jitendra; Joshi, Chaitanya C; Patel, Kamlesh C; Trivedi, Ujjval B; Patel, R M

    2017-12-01

    Bacillus amyloliquefaciens strain KCP2 was isolated from municipal food waste samples collected in Vallabh Vidyanagar, Gujarat, India. Strain KCP2 is noteworthy due to its ability to produce a thermostable, alkaliphilic α-amylase and a protease. These enzymes have importance in several industrial processes including bread making, brewing, starch processing, pharmacy, and textile industries. Whole genome sequencing of strain KCP2 showed that the estimated genome size was 3.9 Mb, the G + C content was 46%, and it coded for 4113 genes.

  13. Mutation of Aspergillus oryzae for improved production of 3, 4-dihydroxy phenyl-L-alanine (L-DOPA from L-tyrosine Mutação de Aspergillus orizae para produção melhorada de 3,4-dihidroxi fenil-L-alanina (L-DOPA a partir de L-tirosina

    Directory of Open Access Journals (Sweden)

    Ikram-ul Haq

    2006-03-01

    Full Text Available Aspergillus oryzae mutant strain UV-7 was further improved for the production of L-DOPA from L-tyrosine using chemical mutation. Different putative mutant strains of organism were tested for the production of L-DOPA in submerged fermentation. Among these putative mutant strains, mutant designated SI-12 gave maximum production of L-DOPA (300 mg L-DOPA.g-1 cells. The production of L-DOPA from different carbon source solutions (So= 30 g.l-1 by mutant culture was investigated at different nitrogen sources, initial pH and temperature values. At optimum pH (pHo= 5.0, and temperature (t=30ºC, 100% sugars were utilized for production and cell mass formation, corresponding to final L-DOPA product yield of 150 mg.g-1 substrate utilized, and maximum volumetric and specific productivities of 125 mg.l-1.h-1, and 150 mg.g-1 cells. h-1, respectively. There was up to 3-fold enhancement in product formation rate. This enhancement is the highest reported in literature. To explain the kinetic mechanism of L-DOPA formation and thermal inactivation of tyrosinase, the thermodynamic parameters were determined with the application of Arrhenius model: activation enthalpy and entropy for product formation, in case of mutant derivative, were 40 k j/mol and 0.076 k j/mol. K for L-DOPA production and 116 k j/mol and 0.590 k j/mol. K for thermal inactivation, respectively. The respective values for product formation were lower while those for product deactivation were higher than the respective values for the parental culture. Therefore, the mutant strain was thermodynamically more resistant to thermal denaturation.A produção de L-DOPA a partir de tirosina pela cepa mutante de Aspergillus orizae UV-7 foi melhorada através de mutação química. Diferentes cepas foram testadas quanto a produção de L-DOPA por fermentação submersa, observando-se que a cepa denominada SI-12 foi a melhor produtora (300 mg de L-DOPA por g de células. A produção de L-DOPA pela cepa

  14. Improving the Thermostability and Optimal Temperature of a Lipase from the Hyperthermophilic Archaeon Pyrococcus furiosus by Covalent Immobilization

    Directory of Open Access Journals (Sweden)

    Roberta V. Branco

    2015-01-01

    Full Text Available A recombinant thermostable lipase (Pf2001Δ60 from the hyperthermophilic Archaeon Pyrococcus furiosus (PFUL was immobilized by hydrophobic interaction on octyl-agarose (octyl PFUL and by covalent bond on aldehyde activated-agarose in the presence of DTT at pH = 7.0 (one-point covalent attachment (glyoxyl-DTT PFUL and on glyoxyl-agarose at pH 10.2 (multipoint covalent attachment (glyoxyl PFUL. The enzyme’s properties, such as optimal temperature and pH, thermostability, and selectivity, were improved by covalent immobilization. The highest enzyme stability at 70°C for 48 h incubation was achieved for glyoxyl PFUL (around 82% of residual activity, whereas glyoxyl-DTT PFUL maintained around 69% activity, followed by octyl PFUL (27% remaining activity. Immobilization on glyoxyl-agarose improved the optimal temperature to 90°C, while the optimal temperature of octyl PFUL was 70°C. Also, very significant changes in activity with different substrates were found. In general, the covalent bond derivatives were more active than octyl PFUL. The E value also depended substantially on the derivative and the conditions used. It was observed that the reaction of glyoxyl-DTT PFUL using methyl mandelate as a substrate at pH 7 presented the best results for enantioselectivity E=22 and enantiomeric excess (ee (% = 91.

  15. Doubling Power Output of Starch Biobattery Treated by the Most Thermostable Isoamylase from an Archaeon Sulfolobus tokodaii.

    Science.gov (United States)

    Cheng, Kun; Zhang, Fei; Sun, Fangfang; Chen, Hongge; Percival Zhang, Y-H

    2015-08-20

    Biobattery, a kind of enzymatic fuel cells, can convert organic compounds (e.g., glucose, starch) to electricity in a closed system without moving parts. Inspired by natural starch metabolism catalyzed by starch phosphorylase, isoamylase is essential to debranch alpha-1,6-glycosidic bonds of starch, yielding linear amylodextrin - the best fuel for sugar-powered biobattery. However, there is no thermostable isoamylase stable enough for simultaneous starch gelatinization and enzymatic hydrolysis, different from the case of thermostable alpha-amylase. A putative isoamylase gene was mined from megagenomic database. The open reading frame ST0928 from a hyperthermophilic archaeron Sulfolobus tokodaii was cloned and expressed in E. coli. The recombinant protein was easily purified by heat precipitation at 80 (o)C for 30 min. This enzyme was characterized and required Mg(2+) as an activator. This enzyme was the most stable isoamylase reported with a half lifetime of 200 min at 90 (o)C in the presence of 0.5 mM MgCl2, suitable for simultaneous starch gelatinization and isoamylase hydrolysis. The cuvett-based air-breathing biobattery powered by isoamylase-treated starch exhibited nearly doubled power outputs than that powered by the same concentration starch solution, suggesting more glucose 1-phosphate generated.

  16. Directed Evolution of Recombinant C-Terminal Truncated Staphylococcus epidermidis Lipase AT2 for the Enhancement of Thermostability

    Directory of Open Access Journals (Sweden)

    Jiivittha Veno

    2017-11-01

    Full Text Available In the industrial processes, lipases are expected to operate at temperatures above 45 °C and could retain activity in organic solvents. Hence, a C-terminal truncated lipase from Staphylococcus epidermis AT2 (rT-M386 was engineered by directed evolution. A mutant with glycine-to-cysteine substitution (G210C demonstrated a remarkable improvement of thermostability, whereby the mutation enhanced the activity five-fold when compared to the rT-M386 at 50 °C. The rT-M386 and G210C lipases were purified concurrently using GST-affinity chromatography. The biochemical and biophysical properties of both enzymes were investigated. The G210C lipase showed a higher optimum temperature (45 °C and displayed a more prolonged half-life in the range of 40–60 °C as compared to rT-M386. Both lipases exhibited optimal activity and stability at pH 8. The G210C showed the highest stability in the presence of polar organic solvents at 50 °C compared to the rT-M386. Denatured protein analysis presented a significant change in the molecular ellipticity value above 60 °C, which verified the experimental result on the temperature and thermostability profile of G210C.

  17. Glycine in the conserved motif III modulates the thermostability and oxidative stress resistance of peptide deformylase in Mycobacterium tuberculosis.

    Science.gov (United States)

    Narayanan, Sai Shyam; Sokkar, Pandian; Ramachandran, Murugesan; Nampoothiri, Kesavan Madhavan

    2011-07-01

    Peptide deformylase (PDF) catalyses the removal of the N-formyl group from the nascent polypeptide during protein maturation. The PDF of Mycobacterium tuberculosis H37Rv (MtbPDF), overexpressed and purified from Escherichia coli, was characterized as an iron-containing enzyme with stability towards H(2) O(2) and moderate thermostability. Substitution of two conserved residues (G49 and L107) from MtbPDF with the corresponding residues found in human PDF affected its deformylase activity. Among characterized PDFs, glycine (G151) in motif III instead of conserved aspartate is characteristic of M. tuberculosis. Although the G151D mutation in MtbPDF increased its deformylase activity and thermostability, it also affected enzyme stability towards H(2) O(2) . Molecular dynamics and docking results confirmed improved substrate binding and catalysis for the G151D mutant and the study provides another possible molecular basis for the stability of MtbPDF against oxidizing agents. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  18. Effects of cryoprotectants on the structure and thermostability of the human carbonic anhydrase II–acetazolamide complex

    International Nuclear Information System (INIS)

    Aggarwal, Mayank; Boone, Christopher D.; Kondeti, Bhargav; Tu, Chingkuang; Silverman, David N.; McKenna, Robert

    2013-01-01

    Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Protein X-ray crystallography has seen a progressive shift from data collection at cool/room temperature (277–298 K) to data collection at cryotemperature (100 K) because of its ease of crystal preparation and the lessening of the detrimental effects of radiation-induced crystal damage, with 20–25%(v/v) glycerol (GOL) being the preferred choice of cryoprotectant. Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Comparative studies of crystal structure, kinetics, inhibition and thermostability were performed on CA II and its complex with AZM in the presence of either GOL or sucrose. These results suggest that even though the cryoprotectant GOL was previously shown to be directly bound in the active site and to interact with AZM, it affects neither the thermostability of CA II nor the binding of AZM in the crystal structure or in solution. However, addition of GOL does affect the kinetics of CA II, presumably as it displaces the water proton-transfer network in the active site

  19. Effects of cryoprotectants on the structure and thermostability of the human carbonic anhydrase II–acetazolamide complex

    Energy Technology Data Exchange (ETDEWEB)

    Aggarwal, Mayank; Boone, Christopher D.; Kondeti, Bhargav; Tu, Chingkuang; Silverman, David N.; McKenna, Robert, E-mail: rmckenna@ufl.edu [University of Florida, 1600 SW Archer Road, PO Box 100245, Gainesville, FL 32610 (United States)

    2013-05-01

    Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Protein X-ray crystallography has seen a progressive shift from data collection at cool/room temperature (277–298 K) to data collection at cryotemperature (100 K) because of its ease of crystal preparation and the lessening of the detrimental effects of radiation-induced crystal damage, with 20–25%(v/v) glycerol (GOL) being the preferred choice of cryoprotectant. Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Comparative studies of crystal structure, kinetics, inhibition and thermostability were performed on CA II and its complex with AZM in the presence of either GOL or sucrose. These results suggest that even though the cryoprotectant GOL was previously shown to be directly bound in the active site and to interact with AZM, it affects neither the thermostability of CA II nor the binding of AZM in the crystal structure or in solution. However, addition of GOL does affect the kinetics of CA II, presumably as it displaces the water proton-transfer network in the active site.

  20. Improving the thermostability by introduction of arginines on the surface of α-L-rhamnosidase (r-Rha1) from Aspergillus niger.

    Science.gov (United States)

    Li, Lijun; Liao, Hui; Yang, Yan; Gong, Jianye; Liu, Jianan; Jiang, Zedong; Zhu, Yanbing; Xiao, Anfeng; Ni, Hui

    2018-06-01

    To improve the thermostability of α-L-rhamnosidase (r-Rha1), an enzyme previously identified from Aspergillus niger JMU-TS528, multiple arginine (Arg) residues were introduced into the r-Rha1 sequence to replace several lysine (Lys) residues that located on the surface of the folded r-Rha1. Hinted by in silico analysis, five surface Lys residues (K134, K228, K406, K440, K573) were targeted to produce a list of 5 single-residue mutants and 4 multiple-residue mutants using site-directed mutagenesis. Among these mutants, a double Lys to Arg mutant, i.e. K406R/K573R, showed the best thermostability improvement. The half-life of this mutant's enzyme activity increased 3 h at 60 °C, 23 min at 65 °C, and 3.5 min at 70 °C, when compared with the wild type. The simulated protein structure based interaction analysis and molecular dynamics calculation indicate that the thermostability improvement of the mutant K406R-K573R was possibly due to the extra hydrogen bonds, the additional cation-π interactions, and the relatively compact conformation. With the enhanced thermostability, the α-L-rhamnosidase mutant, K406R-K573R, has potentially broadened the r-Rha1 applications in food processing industry. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system.

    Science.gov (United States)

    Katano, Yuta; Li, Tongyang; Baba, Misato; Nakamura, Miyo; Ito, Masaaki; Kojima, Kenji; Takita, Teisuke; Yasukawa, Kiyoshi

    2017-12-01

    We attempted to increase the thermostability of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). The eight-site saturation mutagenesis libraries corresponding to Ala70-Arg469 in the whole MMLV RT (Thr24-Leu671), in each of which 1 out of 50 amino acid residues was replaced with other amino acid residue, were constructed. Seven-hundred and sixty eight MMLV RT clones were expressed using a cell-free protein expression system, and their thermostabilities were assessed by the temperature of thermal treatment at which they retained cDNA synthesis activity. One clone D200C was selected as the most thermostable variant. The highest temperature of thermal treatment at which D200C exhibited cDNA synthesis activity was 57ºC, which was higher than for WT (53ºC). Our results suggest that a combination of site saturation mutagenesis library and cell-free protein expression system might be useful for generation of thermostable MMLV RT in a short period of time for expression and selection.

  2. Characterization of a cold-active esterase from Serratia sp. and improvement of thermostability by directed evolution.

    Science.gov (United States)

    Jiang, Huang; Zhang, Shaowei; Gao, Haofeng; Hu, Nan

    2016-01-22

    In recent years, cold-active esterases have received increased attention due to their attractive properties for some industrial applications such as high catalytic activity at low temperatures. An esterase-encoding gene (estS, 909 bp) from Serratia sp. was identified, cloned and expressed in Escherichia coli DE3 (BL21). The estS encoded a protein (EstS) of 302 amino acids with a predicted molecular weight of 32.5 kDa. It showed the highest activity at 10 °C and pH 8.5. EstS was cold active and retained ~92 % of its original activity at 0 °C. Thermal inactivation analysis showed that the T1/2 value of EstS was 50 min at 50 °C (residual activity 41.23 %) after 1 h incubation. EstS is also quite stable in high salt conditions and displayed better catalytic activity in the presence of 4 M NaCl. To improve the thermo-stability of EstS, variants of estS gene were created by error-prone PCR. A mutant 1-D5 (A43V, R116W, D147N) that showed higher thermo-stability than its wild type predecessor was selected. 1-D5 showed enhanced T1/2 of 70 min at 50 °C and retained 63.29 % of activity after incubation at 50 °C for 60 min, which were about 22 % higher than the wild type (WT). CD spectrum showed that the secondary structure of WT and 1-D5 are more or less similar, but an increase in β-sheets was recorded, which enhanced the thermostability of mutant protein. EstS was a novel cold-active and salt-tolerant esterase and half-life of mutant 1-D5 was enhanced by 1.4 times compared with WT. The features of EstS are interesting and can be exploited for commercial applications. The results have also provided useful information about the structure and function of Est protein.

  3. Continuous D-tagatose production by immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor.

    Science.gov (United States)

    Ryu, Se-Ah; Kim, Chang Sup; Kim, Hye-Jung; Baek, Dae Heoun; Oh, Deok-Kun

    2003-01-01

    D-Tagatose was continuously produced using thermostable L-arabinose isomerase immobilized in alginate with D-galactose solution in a packed-bed bioreactor. Bead size, L/D (length/diameter) of reactor, dilution rate, total loaded enzyme amount, and substrate concentration were found to be optimal at 0.8 mm, 520/7 mm, 0.375 h(-1), 5.65 units, and 300 g/L, respectively. Under these conditions, the bioreactor produced about 145 g/L tagatose with an average productivity of 54 g tagatose/L x h and an average conversion yield of 48% (w/w). Operational stability of the immobilized enzyme was demonstrated, with a tagatose production half-life of 24 days.

  4. Discovery, cloning and characterisation of proline specific prolyl endopeptidase, a gluten degrading thermo-stable enzyme from Sphaerobacter thermophiles

    DEFF Research Database (Denmark)

    Shetty, Radhakrishna; Vestergaard, Mike; Jessen, Flemming

    2017-01-01

    processes occur at elevated temperature. We present in this paper, the discovery, cloning and characterisation of a novel recombinant thermostable gluten degrading enzyme, a proline specific prolyl endoprotease (PEP) from Sphaerobacter thermophiles. The molecular mass of the prolyl endopeptidase......Gluten free products have emerged during the last decades, as a result of a growing public concern and technological advancements allowing gluten reduction in food products. One approach is to use gluten degrading enzymes, typically at low or ambient temperatures, whereas many food production...... was estimated to be 77 kDa by using SDS-PAGE. Enzyme activity assays with a synthetic dipeptide Z-Gly-Pro-p-nitroanilide as the substrate revealed that the enzyme had optimal activity at pH 6.6 and was most active from pH 5.0-8.0. The optimum temperature was 63 °C and residual activity after one hour incubation...

  5. Preparation of lactose-free pasteurized milk with a recombinant thermostable β-glucosidase from Pyrococcus furiosus

    Science.gov (United States)

    2013-01-01

    Background Lactose intolerance is a common health concern causing gastrointestinal symptoms and avoidance of dairy products by afflicted individuals. Since milk is a primary source of calcium and vitamin D, lactose intolerant individuals often obtain insufficient amounts of these nutrients which may lead to adverse health outcomes. Production of lactose-free milk can provide a solution to this problem, although it requires use of lactase from microbial sources and increases potential for contamination. Use of thermostable lactase enzymes can overcome this issue by functioning under pasteurization conditions. Results A thermostable β-glucosidase gene from Pyrococcus furiosus was cloned in frame with the Saccharomyces cerecisiae a-factor secretory signal and expressed in Pichia pastoris strain X-33. The recombinant enzyme was purified by a one-step method of weak anion exchange chromatography. The optimum temperature and pH for this β-glucosidase activity was 100°C and pH 6.0, respectively. The enzyme activity was not significantly inhibited by Ca2+. We tested the additive amount, hydrolysis time, and the influence of glucose on the enzyme during pasteurization and found that the enzyme possessed a high level of lactose hydrolysis in milk that was not obviously influenced by glucose. Conclusions The thermostablity of this recombinant β-glucosidase, combined with its neutral pH activity and favorable temperature activity optima, suggest that this enzyme is an ideal candidate for the hydrolysis of lactose in milk, and it would be suitable for application in low-lactose milk production during pasteurization. PMID:24053641

  6. Effect of thermostable α-amylase injection on mechanical and physiochemical properties for saccharification of extruded corn starch.

    Science.gov (United States)

    Myat, Lin; Ryu, Gi-Hyung

    2014-01-30

    In industry, a jet cooker is used to gelatinize starch by mixing the starch slurry with steam under pressure at 100-175 °C. A higher degree of starch hydrolysis in an extruder is possible with glucoamylase. Unfortunately, it is difficult to carry out liquefaction and saccharification in parallel, because the temperature of gelatinization will be too high and will inactivate glucoamylase. Since the temperature for liquefaction and saccharification is different, it is hard to change the temperature from high (required for liquefaction) to low (required for saccharification). The industrial gelatinization process is usually carried out with 30-35% (w/w) dry solids starch slurry. Conventional jet cookers cannot be used any more at high substrate concentrations owing to higher viscosity. In this study, therefore, corn starch was extruded at different melt temperatures to overcome these limitations and to produce the highest enzyme-accessible starch extrudates. Significant effects on physical properties (water solubility index, water absorption index and color) and chemical properties (reducing sugar and % increase in reducing sugar after saccharification) were achieved by addition of thermostable α-amylase at melt temperatures of 115 and 135 °C. However, there was no significant effect on % increase in reducing sugar of extruded corn starch at 95 °C. The results show the great potential of extrusion with thermostable α-amylase injection at 115 and 135 °C as an effective pretreatment for breaking down starch granules, because of the significant increase (P < 0.05) in % reducing sugar and enzyme-accessible extrudates for saccharification yield. © 2013 Society of Chemical Industry.

  7. Optimization of physical factors affecting the production of thermo-stable organic solvent-tolerant protease from a newly isolated halo tolerant Bacillus subtilis strain Rand

    Directory of Open Access Journals (Sweden)

    Salleh Abu

    2009-04-01

    Full Text Available Abstract Background Many researchers have reported on the optimization of protease production; nevertheless, only a few have reported on the optimization of the production of organic solvent-tolerant proteases. Ironically, none has reported on thermostable organic solvent-tolerant protease to date. The aim of this study was to isolate the thermostable organic solvent-tolerant protease and identify the culture conditions which support its production. The bacteria of genus Bacillus are active producers of extra-cellular proteases, and the thermostability of enzyme production by Bacillus species has been well-studied by a number of researchers. In the present study, the Bacillus subtilis strain Rand was isolated from the contaminated soil found in Port Dickson, Malaysia. Results A thermostable organic solvent-tolerant protease producer had been identified as Bacillus subtilis strain Rand, based on the 16S rRNA analysis conducted, as well as the morphological characteristics and biochemical properties. The production of the thermostable organic solvent-tolerant protease was optimized by varying various physical culture conditions. Inoculation with 5.0% (v/v of (AB600 = 0.5 inoculum size, in a culture medium (pH 7.0 and incubated for 24 h at 37°C with 200 rpm shaking, was the best culture condition which resulted in the maximum growth and production of protease (444.7 U/ml; 4042.4 U/mg. The Rand protease was not only stable in the presence of organic solvents, but it also exhibited a higher activity than in the absence of organic solvent, except for pyridine which inhibited the protease activity. The enzyme retained 100, 99 and 80% of its initial activity, after the heat treatment for 30 min at 50, 55, and 60°C, respectively. Conclusion Strain Rand has been found to be able to secrete extra-cellular thermostable organic solvent-tolerant protease into the culture medium. The protease exhibited a remarkable stability towards temperature and organic

  8. Improved thermostability and enzyme activity of a recombinant phyA mutant phytase from Aspergillus niger N25 by directed evolution and site-directed mutagenesis.

    Science.gov (United States)

    Tang, Zizhong; Jin, Weiqiong; Sun, Rong; Liao, Yan; Zhen, Tianrun; Chen, Hui; Wu, Qi; Gou, Lin; Li, Chenlei

    2018-01-01

    We previously constructed three recombinant phyA mutant strains (PP-NP m -8, PP-NP ep -6A and I44E/T252R-PhyA), showing improved catalytic efficiency or thermostability of Aspergillus niger N25 phytase, by error-prone PCR or site-directed mutagenesis. In this study, directed evolution and site-directed mutagenesis were further applied to improve the modified phytase properties. After one-round error-prone PCR for phytase gene of PP-NP ep -6A, a single transformant, T195L/Q368E/F376Y, was obtained with the significant improvements in catalytic efficiency and thermostability. The phytase gene of T195L/Q368E/F376Y, combined with the previous mutant phytase genes of PP-NP ep -6A, PP-NP m -8 and I44E/T252R-PhyA, was then sequentially modified by DNA shuffling. Three genetically engineered strains with desirable properties were then obtained, namedQ172R, Q172R/K432R andQ368E/K432R. Among them, Q172R/K432R showed the highest thermostability with the longest half-life and the greatest remaining phytase activity after heat treatment, while Q368E/K432R showed the highest catalytic activity. Five substitutions (Q172R, T195L, Q368E, F376Y, K432R) identified from random mutagenesis were added sequentially to the phytase gene of PP-NP ep -6A to investigate how the mutant sites influence the properties of phytase. Characterization and structural analysis demonstrated that these mutations could produce cumulative or synergistic improvements in thermostability or catalytic efficiency of phytase. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Resolution Mechanism and Characterization of an Ammonium Chloride-Tolerant, High-Thermostable, and Salt-Tolerant Phenylalanine Dehydrogenase from Bacillus halodurans.

    Science.gov (United States)

    Jiang, Wei; Wang, Ya-Li; Fang, Bai-Shan

    2018-05-09

    As phenylalanine dehydrogenase (PheDH) plays an important role in the synthesis of chiral drug intermediates and detection of phenylketonuria, it is significant to obtain a PheDH with specific and high activity. Here, a PheDH gene, pdh, encoding a novel BhPheDH with 61.0% similarity to the known PheDH from Microbacterium sp., was obtained. The BhPheDH showed optimal activity at 60 °C and pH 7.0, and it showed better stability in hot environment (40-70 °C) than the PheDH from Nocardia sp. And its activity and thermostability could be significantly increased by sodium salt. After incubation for 2 h in 3 M NaCl at 60 °C, the residual activity of the BhPheDH was found to be 1.8-fold higher than that of the control group (without NaCl). The BhPheDH could tolerate high concentration of ammonium chloride and its activity could be also enhanced by the high concentration of ammonium salts. These characteristics indicate that the BhPheDH possesses better thermostability, ammonium chloride tolerance, halophilic mechanism, and high salt activation. The mechanism of thermostability and high salt tolerance of the BhPheDH was analyzed by molecular dynamics simulation. These results provide useful information about the enzyme with high-temperature activity, thermostability, halophilic mechanism, tolerance to high concentration of ammonium chloride, higher salt activation and enantio-selectivity, and the application of molecular dynamics simulation in analyzing the mechanism of these distinctive characteristics.

  10. Development of soluble and immobilized biocatalysts based on a recombinant thermostable ß-fructosidase enabling complete sucrose inversion at pasteurization temperatures

    OpenAIRE

    Menéndez, Carmen; Martínez, Duniesky; Trujillo, Luis E; Ramírez, Ricardo; Sobrino, Alina; Cutiño-Ávila, Bessy V; Basabe, Liliana; del Monte-Martínez, Alberto; Pérez, Enrique R; Hernández, Lázaro

    2014-01-01

    Biocatalysts for the industrial production of invert sugar are preferred to stably operate at high sucrose concentrations and pasteurization temperatures. Thermotoga maritima ß-fructosidase (BfrA) is more thermostable and less susceptible to substrate inhibition than the current commercial invertase from Saccharomyces cerevisiae. In this research, the non-saccharolytic host Pichia pastoris was engineered for BfrA production. Fed-batch fermentation of the recombinant yeast for 72 h using cane ...

  11. Thermo-stable carbon nanotube-TiO_2 nanocompsite as electron highways in dye-sensitized solar cell produced by bio-nano-process

    International Nuclear Information System (INIS)

    Inoue, Ippei; Yasueda, Hisashi; Yamauchi, Hirofumi; Okamoto, Naofumi; Toyoda, Kenichi; Horita, Masahiro; Ishikawa, Yasuaki; Uraoka, Yukiharu; Yamashita, Ichiro

    2015-01-01

    We produced a thermostable TiO_2-(anatase)-coated multi-walled-carbon-nanotube (MWNT) nanocomposite for use in dye-sensitized solar cells (DSSCs) using biological supuramolecules as catalysts. We synthesized two different sizes of iron oxide nanoparticles (NPs) and arrayed the NPs on a silicon substrate utilizing two kinds of genetically modified cage-shaped proteins with silicon-binding peptide aptamers on their outer surfaces. Chemical vapor deposition (CVD) with the vapor–liquid-solid phase (VLS) method was applied to the substrate, and thermostable MWNTs with a diameter of 6 ± 1 nm were produced. Using a genetically modified cage-shaped protein with carbon-nanomaterials binding and Ti-mineralizing peptides as a catalyst, we were able to mineralize a titanium compound around the surface of the MWNT. The products were sintered, and thin TiO_2-layer-coated MWNTs nanocomoposites were successfully produced. Addition of a 0.2 wt% TiO_2-coated MWNT nanocomposite to a DSSC photoelectrode improved current density by 11% and decreased electric resistance by 20% compared to MWNT-free reference DSSCs. These results indicate that a nanoscale TiO_2-layer-coated thermostable MWNT structure produced by our mutant proteins works as a superior electron transfer highway within TiO_2 photoelectrodes. (paper)

  12. Enzymatic liquefaction of agarose above the sol-gel transition temperature using a thermostable endo-type β-agarase, Aga16B.

    Science.gov (United States)

    Kim, Jung Hyun; Yun, Eun Ju; Seo, Nari; Yu, Sora; Kim, Dong Hyun; Cho, Kyung Mun; An, Hyun Joo; Kim, Jae-Han; Choi, In-Geol; Kim, Kyoung Heon

    2017-02-01

    The main carbohydrate of red macroalgae is agarose, a heterogeneous polysaccharide composed of D-galactose and 3,6-anhydro-L-galactose. When saccharifying agarose by enzymes, the unique physical properties of agarose, namely the sol-gel transition and the near-insolubility of agarose in water, limit the accessibility of agarose to the enzymes. Due to the lower accessibility of agarose to enzymes in the gel state than to the sol state, it is important to prevent the sol-gel transition by performing the enzymatic liquefaction of agarose at a temperature higher than the sol-gel transition temperature of agarose. In this study, a thermostable endo-type β-agarase, Aga16B, originating from Saccharophagus degradans 2-40 T , was characterized and introduced in the liquefaction process. Aga16B was thermostable up to 50 °C and depolymerized agarose mainly into neoagarooligosaccharides with degrees of polymerization 4 and 6. Aga16B was applied to enzymatic liquefaction of agarose at 45 °C, which was above the sol-gel transition temperature of 1 % (w/v) agarose (∼35 °C) when cooling agarose. This is the first systematic demonstration of enzymatic liquefaction of agarose, enabled by determining the sol-gel temperature of agarose under specific conditions and by characterizing the thermostability of an endo-type β-agarase.

  13. Improving the Efficiency of New Automatic Dishwashing Detergent Formulation by Addition of Thermostable Lipase, Protease and Amylase

    Directory of Open Access Journals (Sweden)

    Ashwini Naganthran

    2017-09-01

    Full Text Available The use of T1 lipase in automatic dishwashing detergent (ADD is well established, but efficiency in hard water is very low. A new enzymatic environmentally-friendly dishwashing was formulated to be efficient in both soft and hard water. Thermostable enzymes such as T1 lipase from Geobacillus strain T1, Rand protease from Bacillus subtilis strain Rand, and Maltogenic amylase from Geobacillus sp. SK70 were produced and evaluated for an automatic dishwashing detergent formulation. The components of the new ADD were optimized for compatibility with these three enzymes. In compatibility tests of the enzymes with different components, several criteria were considered. The enzymes were mostly stable in non-ionic surfactants, especially polyhydric alcohols, Glucopon UP 600, and in a mixture of sodium carbonate and glycine (30:70 buffer at a pH of 9.25. Sodium polyacrylate and sodium citrate were used in the ADD formulation as a dispersing agent and a builder, respectively. Dishwashing performance of the formulated ADDs was evaluated in terms of percent of soil removed using the Leenert‘s Improved Detergency Tester. The results showed that the combination of different hydrolysis enzymes could improve the washing efficiency of formulated ADD compared to the commercial ADD “Finish” at 40 and 50 C.

  14. Thermostable trypsin conjugates immobilized to biogenic magnetite show a high operational stability and remarkable reusability for protein digestion

    Science.gov (United States)

    Pečová, M.; Šebela, M.; Marková, Z.; Poláková, K.; Čuda, J.; Šafářová, K.; Zbořil, R.

    2013-03-01

    In this work, magnetosomes produced by microorganisms were chosen as a suitable magnetic carrier for covalent immobilization of thermostable trypsin conjugates with an expected applicability for efficient and rapid digestion of proteins at elevated temperatures. First, a biogenic magnetite was isolated from Magnetospirillum gryphiswaldense and its free surface was coated with the natural polysaccharide chitosan containing free amino and hydroxy groups. Prior to covalent immobilization, bovine trypsin was modified by conjugating with α-, β- and γ-cyclodextrin. Modified trypsin was bound to the magnetic carriers via amino groups using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide as coupling reagents. The magnetic biomaterial was characterized by magnetometric analysis and electron microscopy. With regard to their biochemical properties, the immobilized trypsin conjugates showed an increased resistance to elevated temperatures, eliminated autolysis, had an unchanged pH optimum and a significant storage stability and reusability. Considering these parameters, the presented enzymatic system exhibits properties that are superior to those of trypsin forms obtained by other frequently used approaches. The proteolytic performance was demonstrated during in-solution digestion of model proteins (horseradish peroxidase, bovine serum albumin and hen egg white lysozyme) followed by mass spectrometry. It is shown that both magnetic immobilization and chemical modification enhance the characteristics of trypsin making it a promising tool for protein digestion.

  15. Thermostable L-arabinose isomerase from Bacillus stearothermophilus IAM 11001 for D-tagatose production: gene cloning, purification and characterisation.

    Science.gov (United States)

    Cheng, Lifang; Mu, Wanmeng; Jiang, Bo

    2010-06-01

    D-Tagatose, as one of the rare sugars, has been found to be a natural and safe low-calorie sweetener in food products and is classified as a GRAS substance. L-Arabinose isomerase (L-AI, EC 5.3.1.4), catalysing the isomerisations of L-arabinose and D-galactose to L-ribulose and D-tagatose respectively, is considered to be the most promising enzyme for the production of D-tagatose. The araA gene encoding an L-AI from Bacillus stearothermophilus IAM 11001 was cloned, sequenced and overexpressed in Escherichia coli. The gene is composed of 1491 bp nucleotides and codes for a protein of 496 amino acid residues. The recombinant L-AI was purified to electrophoretical homogeneity by affinity chromatography. The purified enzyme was optimally active at 65 degrees C and pH 7.5 and had an absolute requirement for the divalent metal ion Mn(2+) for both catalytic activity and thermostability. The enzyme was relatively active and stable at acidic pH of 6. The bioconversion yield of D-galactose to D-tagatose by the purified L-AI after 12 h at 65 degrees C reached 36%. The purified L-AI from B. stearothermophilus IAM 11001 was characterised and shown to be a good candidate for potential application in D-tagatose production. Copyright (c) 2010 Society of Chemical Industry.

  16. Directed divergent evolution of a thermostable D-tagatose epimerase towards improved activity for two hexose substrates.

    Science.gov (United States)

    Bosshart, Andreas; Hee, Chee Seng; Bechtold, Matthias; Schirmer, Tilman; Panke, Sven

    2015-03-02

    Functional promiscuity of enzymes can often be harnessed as the starting point for the directed evolution of novel biocatalysts. Here we describe the divergent morphing of an engineered thermostable variant (Var8) of a promiscuous D-tagatose epimerase (DTE) into two efficient catalysts for the C3 epimerization of D-fructose to D-psicose and of L-sorbose to L-tagatose. Iterative single-site randomization and screening of 48 residues in the first and second shells around the substrate-binding site of Var8 yielded the eight-site mutant IDF8 (ninefold improved kcat for the epimerization of D-fructose) and the six-site mutant ILS6 (14-fold improved epimerization of L-sorbose), compared to Var8. Structure analysis of IDF8 revealed a charged patch at the entrance of its active site; this presumably facilitates entry of the polar substrate. The improvement in catalytic activity of variant ILS6 is thought to relate to subtle changes in the hydration of the bound substrate. The structures can now be used to select additional sites for further directed evolution of the ketohexose epimerase. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Thermostable gel polymer electrolyte based on succinonitrile and ionic liquid for high-performance solid-state supercapacitors

    Science.gov (United States)

    Pandey, Gaind P.; Liu, Tao; Hancock, Cody; Li, Yonghui; Sun, Xiuzhi Susan; Li, Jun

    2016-10-01

    A flexible, free-standing, thermostable gel polymer electrolyte based on plastic crystalline succinonitrile (SN) and ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate (BMImBF4) entrapped in copolymer poly(vinylidene fluoride-co-hexafluoropropylene) (PVdF-HFP) is prepared and optimized for application in solvent-free solid-state supercapacitors. The synthesized gel polymer electrolyte exhibits a high ionic conductivity over a wide temperature range (from ∼5 × 10-4 S cm-1 at -30 °C up to ∼1.5 × 10-2 S cm-1 at 80 °C) with good electrochemical stability window (-2.9 to 2.5 V). Thermal studies confirm that the SN containing gel polymer electrolyte remains stable in the same gel phase over a wide temperature range from -30 to 90 °C. The electric double layer capacitors (EDLCs) have been fabricated using activated carbon as active materials and new gel polymer electrolytes. Electrochemical performance of the EDLCs is assessed through cyclic voltammetry, galvanostatic charge-discharge cycling and impedance spectroscopy. The EDLC cells with the proper SN-containing gel polymer electrolyte has been found to give high specific capacitance 176 F g-1 at 0.18 A g-1 and 138 F g-1 at 8 A g-1. These solid-state EDLC cells show good cycling stability and the capability to retain ∼80% of the initial capacitance after 10,000 cycles.

  18. Thermostable hemicellulases of a bacterium, Geobacillus sp. DC3, isolated from the former Homestake gold mine in Lead, South Dakota.

    Science.gov (United States)

    Bergdale, Terran E; Hughes, Stephen R; Bang, Sookie S

    2014-04-01

    A thermophilic strain, Geobacillus sp. DC3, capable of producing hemicellulolytic enzymes was isolated from the 1.5-km depth of the former Homestake gold mine in Lead, South Dakota. The DC3 strain expressed a high level of extracellular endoxylanase at 39.5 U/mg protein with additional hemicellulases including β-xylosidase (0.209 U/mg) and arabinofuranosidase (0.230 U/mg), after the bacterium was grown in xylan for 24 h. Partially purified DC3 endoxylanase exhibited a molecular mass of approximately 43 kDa according to zymography with an optimal pH of 7 and optimal temperature of 70 °C. The kinetic constants, K m and V max, were 13.8 mg/mL and 77.5 μmol xylose/min·mg xylan, respectively. The endoxylanase was highly stable and maintained 70 % of its original activity after 16 h incubation at 70 °C. The thermostable properties and presence of three different hemicellulases of Geobacillus sp. DC3 strain support its potential application for industrial hydrolysis of renewable biomass such as lignocelluloses.

  19. CNA web server: rigidity theory-based thermal unfolding simulations of proteins for linking structure, (thermo-)stability, and function.

    Science.gov (United States)

    Krüger, Dennis M; Rathi, Prakash Chandra; Pfleger, Christopher; Gohlke, Holger

    2013-07-01

    The Constraint Network Analysis (CNA) web server provides a user-friendly interface to the CNA approach developed in our laboratory for linking results from rigidity analyses to biologically relevant characteristics of a biomolecular structure. The CNA web server provides a refined modeling of thermal unfolding simulations that considers the temperature dependence of hydrophobic tethers and computes a set of global and local indices for quantifying biomacromolecular stability. From the global indices, phase transition points are identified where the structure switches from a rigid to a floppy state; these phase transition points can be related to a protein's (thermo-)stability. Structural weak spots (unfolding nuclei) are automatically identified, too; this knowledge can be exploited in data-driven protein engineering. The local indices are useful in linking flexibility and function and to understand the impact of ligand binding on protein flexibility. The CNA web server robustly handles small-molecule ligands in general. To overcome issues of sensitivity with respect to the input structure, the CNA web server allows performing two ensemble-based variants of thermal unfolding simulations. The web server output is provided as raw data, plots and/or Jmol representations. The CNA web server, accessible at http://cpclab.uni-duesseldorf.de/cna or http://www.cnanalysis.de, is free and open to all users with no login requirement.

  20. Thermostability and surface morphology of nano- and micro-filled NBR/CSM and CR/CSM rubber blends

    Directory of Open Access Journals (Sweden)

    M. MARINOVIC-CINCOVIC

    2004-02-01

    Full Text Available Acrylonitrile-butadiene rubber (NBR, polychloroprene rubber (CR, chlorosulphonated polyethylene rubber (CSM and their blends were cross-linked with sulphur, ethylene-thiourea, magnesium oxide or their combination. The effect of nano- and micro- particle sized of 35 pphr SiO2 on the thermostability and surface morphology of all the crosslinked systems was investigated. Identification of the structure of nano- and micro- particle sized SiO2 filled NBR/CSM and CR/CSM crosslinked systems was carried out by Fourier transform infrared spectroscopy (FTIR with an attenuated total reflectance (ATR extension. The thermal stability of the nano- and micro- particle sized SiO2 filled NBR/CSM and CR/CSM crosslinked systems were carried out by thermogravimetric analysis (TGA. The glass transition temperature (Tg of the samples was determined by differential scanning calorimetry (DSC. The morphology of the fracture surface of the crosslinked systems was carried out by scanning electron microscope (SEM. The results show when filled with nano-particle sized of SiO2 NBR/CSM and CR/CSM polymer matrix have a strong peak from SiO–C at 1079 cm-1. This suggests the an interaction between the SiO2, which should lead to an increased thermal stability, higher values of Tg, better dispersion the nano-SiO2 and more polish, without cracks than micro-filled NBR/CSM and CR/CSM crosslinked systems.

  1. Production of an acidic and thermostable lipase of the mesophilic fungus Penicillium simplicissimum by solid-state fermentation.

    Science.gov (United States)

    Gutarra, Melissa L E; Godoy, Mateus G; Maugeri, Francisco; Rodrigues, Maria Isabel; Freire, Denise M G; Castilho, Leda R

    2009-11-01

    The production of a lipase by a wild-type Brazilian strain of Penicillium simplicissimum in solid-state fermentation of babassu cake, an abundant residue of the oil industry, was studied. The enzyme production reached about 90 U/g in 72 h, with a specific activity of 4.5 U/mg of total proteins. The crude lipase showed high activities at 35-60 degrees C and pH 4.0-6.0, with a maximum activity at 50 degrees C and pH 4.0-5.0. Enzyme stability was enhanced at pH 5.0 and 6.0, with a maximum half-life of 5.02 h at 50 degrees C and pH 5.0. Thus, this lipase shows a thermophilic and thermostable behavior, what is not common among lipases from mesophilic filamentous fungi. The crude enzyme catalysed the hydrolysis of triglycerides and p-nitrophenyl esters (C4:0-C18:0), preferably acting on substrates with medium-chain fatty acids. This non-purified lipase in addition to interesting properties showed a reduced production cost making feasible its applicability in many fields.

  2. Effect of chemical stabilizers on the thermostability and infectivity of a representative panel of freeze dried viruses.

    Directory of Open Access Journals (Sweden)

    Boris Pastorino

    Full Text Available As a partner of the European Virus Archive (EVA FP7 project, our laboratory maintains a large collection of freeze-dried viruses. The distribution of these viruses to academic researchers, public health organizations and industry is one major aim of the EVA consortium. It is known that lyophilization requires appropriate stabilizers to prevent inactivation of the virus. However, few studies have investigated the influence of different stabilizers and lyophilization protocols on the thermostability of different viruses. In order to identify optimal lyophilization conditions that will deliver maximum retention of viral infectivity titre, different stabilizer formulations containing trehalose, sorbitol, sucrose or foetal bovine serum were evaluated for their efficacy in stabilizing a representative panel of freeze dried viruses at different storage temperatures (-20°C, +4°C and +20°C for one week, the two latter mimicking suboptimal shipping conditions. The Tissue Culture Infectious Dose 50% (TCID50 assay was used to compare the titres of infectious virus. The results obtained using four relevant and model viruses (enveloped/non enveloped RNA/DNA viruses still serve to improve the freeze drying conditions needed for the development and the distribution of a large virus collection.

  3. Novel thermostable clostridial strains through protoplast fusion for enhanced biobutanol production at higher temperature—preliminary study

    Directory of Open Access Journals (Sweden)

    Muhammad Ferhan

    2016-01-01

    Full Text Available The objective of this study is to improve the thermal stability of clostridium strains for enhanced biobutanol production. Thermostable clostridia species were developed through protoplast fusion between mesophilic clostridial species (i.e., Clostridium beijerinckii and Clostridium acetobutylicum and thermophilic clostridial species (i.e., Clostridium thermocellum. Production of biobutanol was examined in the present preliminary study using the clostridium strains and their protoplast fusants using sugar mixture with composition identical to that of wheat straw acid hydrolysate. Maximum biobutanol production of 9.4 g/L was achieved by a fused strain at 45 °C with total sugar consumption of 66% compared to that at 35 °C (i.e., 8.4 g/L production and 64% total sugar consumption. Glucose and xylose uptake rates were generally higher compared to all other individual sugars in the feedstock. In general, average cell concentrations were in close proximity for all parenting and fused strains at 35 °C; i.e., in the range of 5.12 × 107 to 5.49 × 107 cells/mL. Average cell concentration of fusants between the mesophilic clostridial species and the thermophilic clostridial species slightly increased to ~ 5.62 × 107 cells/mL at a higher temperature of 45 °C. These results, in addition to the ones obtained for the butanol production, demonstrate enhanced thermal stability of both fusants at a higher temperature (45 °C.

  4. Immobilization of anaerobic thermophilic bacteria for the production of cell-free thermostable. alpha. -amylases and pullulanases

    Energy Technology Data Exchange (ETDEWEB)

    Klingeberg, M [Goettingen Univ. (Germany, F.R.). Inst. fuer Mikrobiologie; Vorlop, K D [Technische Univ. Braunschweig (Germany, F.R.). Inst. fuer Technische Chemie; Antranikian, G [Technische Univ. Hamburg-Harburg, Hamburg (Germany, F. R.). Arbeitsbereich Biotechnologie 1

    1990-08-01

    For the production of cell-free thermostable {alpha}-amylases and pullulanases various anaerobic thermophilic bacteria that belong to the genera Clostridium and Thermoanaerobacter were immobilized in calcium alginate gel beads. The entrapment of bacteria was performed in full was well as in hollow spheres. An optimal limited medium, which avoided bacterial outgrowth, was developed for the cultivation of immobilized organisms at 60deg C using 0.4% starch as substrate. Compared to non-immobilized cells these techniques allowed a significant increase (up to 5.6-fold) in the specific activities of the extracellular enzymes formed. An increase in the productivity of extracellular enzymes was observed after immobilization of bacteria in full spheres. In the case of C. thermosaccharolyticum, for instance, the productivity was raised from 90 units (U)/10{sup 12} cells up to 700 U/10{sup 12} cells. Electrophoretic analysis of the secreted proteins showed that in all cases most of the amylolytic enzymes formed were released into the culture medium. Proteins that had a molecular mass of less than 450 000 daltons could easily diffuse through the gel matrix. Cultivation of immobilized bacteria in semi-continuous and fed-batch cultures was also accompanied by an elevation in the concentration of cell-free enzymes. (orig.).

  5. Purification, characterization and gene cloning of thermostable O-acetyl-L-serine sulfhydrylase forming β-cyano-L-alanine

    International Nuclear Information System (INIS)

    Omura, Hironori; Yoshida, Toyokazu; Nagasawa, Toru; Kobayashi, Michihiko; Shimizu, Sakayu

    2003-01-01

    A thermophilic and cyanide ion-tolerant bacterium, Bacillus stearothermophilus CN3 isolated from a hot spring in Japan, was found to produce thermostable β-cyano-L-alanine synthase. The enzyme catalyzes the synthesis of β-cyano-L-alanine from O-acetyl-L-serine and cyanide ions. The purified enzyme has a molecular mass of approximately 70 kDa and consists of two identical sub-units. It was stable in the pH range of 6.0 to 10.0 and up to 70degC. The enzyme also catalyzes the synthesis of various β-substituted-L-alanine derivatives from O-acetyl-L-serine and nucleophilic reagents. The gene encoding the β-cyano-L-alanine synthase was isolated from B. stearothermophilus CN3. Sequence homology analysis revealed that the β-cyano-L-alanine synthase of the bacterium is O-acetyl-L-serine sulfhydrylase. A recombinant plasmid, constructed by ligation of the cloned gene and an expression vector, pKK223-3, was introduced into E. coli JM109. The transformed E. coli cells overexpressed β-cyano-L-alanine synthase. Heat stable β-cyano-L-alanine synthase can be applied to the synthesis of [4- 11 C]L-2,4-diaminobutyric acid as a tracer for positron emission tomography. (author)

  6. Antimicrobial activity and high thermostability of a novel BLIS secreted by Enterococcus Mundtii isolated from Lebanese cow’s milk

    Directory of Open Access Journals (Sweden)

    Imad AL Kassaa

    2016-12-01

    Full Text Available AL Kassaa, I., Safourim, N., Mostafa, N. and Hamze, M. Antimicrobial activity and high thermostability of a novel BLIS secreted by Enterococcus Mundtii isolated from lebanese cow’s milk. 2016. Lebanese Science Journal, 17(2: 166-176. Lactic acid bacteria (LAB are used in many fields such as fermentation agents, increasing nutritional value and improving organoleptic quality of food. Also they are used as probiotics and preservatives against pathogens and spoilage microbes by producing antimicrobial substances such as bacteriocins. Fifty cow’s milk samples were collected and 175 LAB isolates were isolated and identified by using biochemical method. Fifteen isolates showed an antimicrobial activity against Listeria monocytogenes ATCC® 19115™. One strain, BL4 which showed the strongest activity, was chosen to extract and characterize its antimicrobial substance in order to evaluate its potential use as a new food protective agent. This strain was identified as Enterococcus mundtii by pyrosequencing method. The active substance was extracted using solvent method. This Bacteriocin like Inhibitory Substances “BLIS” can support a high temperature (121 ˚C for a long time and resist pH variation. The BLIS BL4 can be considered as a peptide active against many food pathogen and food-spoilage microbes, such as Listeria monocytogenes and Penicillium spp. BLIS BL4 can be used in food application as bio-preservative to reduce food-spoilage and food-borne diseases in food products.

  7. Dynamic fluorescence studies of beta-glycosidase mutants from Sulfolobus solfataricus: effects of single mutations on protein thermostability.

    Science.gov (United States)

    Bismuto, Ettore; Febbraio, Ferdinando; Limongelli, Simona; Briante, Raffaella; Nucci, Roberto

    2003-04-01

    Multiple sequence alignment on 73 proteins belonging to glycosyl hydrolase family 1 reveals the occurrence of a segment (83-124) in the enzyme sequences from hyperthermophilic archaea bacteria, which is absent in all the mesophilic members of the family. The alignment of the known three-dimensional structures of hyperthermophilic glycosidases with the known ones from mesophilic organisms shows a similar spatial organizations of beta-glycosidases except for this sequence segment whose structure is located on the external surface of each of four identical subunits, where it overlaps two alpha-helices. Site-directed mutagenesis substituting N97 or S101 with a cysteine residue in the sequence of beta-glycosidase from hyperthermophilic archaeon Sulfolobus solfataricus caused some changes in the structural and dynamic properties as observed by circular dichroism in far- and near-UV light, as well as by frequency domain fluorometry, with a simultaneous loss of thermostability. The results led us to hypothesize an important role of the sequence segment present only in hyperthermophilic beta-glycosidases, in the thermal adaptation of archaea beta-glycosidases. The thermostabilization mechanism could occur as a consequence of numerous favorable ionic interactions of the 83-124 sequence with the other part of protein matrix that becomes more rigid and less accessible to the insult of thermal-activated solvent molecules. Copyright 2003 Wiley-Liss, Inc.

  8. Thermostable, salt tolerant, wide pH range novel chitobiase from Vibrio parahemolyticus: isolation, characterization, molecular cloning, and expression.

    Science.gov (United States)

    Zhu, B C; Lo, J Y; Li, Y T; Li, S C; Jaynes, J M; Gildemeister, O S; Laine, R A; Ou, C Y

    1992-07-01

    A chitobiase gene from Vibrio parahemolyticus was cloned into plasmid pUC18 in Escherichia coli strain DH5 alpha. The plasmid construct, pC120, contained a 6.4 kb Vibrio DNA insert. The recombinant gene expressed chitobiase [EC 3.2.1.30] activity similar to that found in the native Vibrio. The enzyme was purified by ion exchange, hydroxylapatite and gel permeation chromatographies, and exhibited an apparent molecular weight of 80 kDa on SDS-polyacrylamide gel electrophoresis. Chitobiose and 6 more substrates, including beta-N-acetyl galactosamine glycosides, were hydrolyzed by the recombinant chitobiase, indicating its putative classification as an hexosaminidase [EC 3.2.1.52]. The enzyme was resistant to denaturation by 2 M NaCl, thermostable at 45 degrees C and active over a very unusual (for glycosyl hydrolases) pH range, from 4 to 10. The purified cloned chitobiase gave 4 closely focussed bands on an isoelectric focusing gel, at pH 4 to 6.5. The N-terminal 43 amino acid sequence shows no homology with other proteins in commercial databanks or in the literature, and from its N-terminal sequence, appears to be a novel protein, unrelated in sequence to chitobiases from other Vibrios reported and unrelated to hexosaminidases from other organisms.

  9. Production and Characterization of Thermostable Bioplastic (Poly-beta-Hydroxybutyrate) from Bacillus Cereus NRRL-B-3711

    International Nuclear Information System (INIS)

    Asad-ur-Rehman, M.; Aslam, A.; Masood, R.; Aftab, M. N.; Ajmal, R.; Haq, I. U.

    2016-01-01

    The poly-beta-hydroxybutyrate (PHB) is a thermostable and biocompatible polyester produced by several bacteria under unbalanced nutritional conditions. Among Gram-positive bacteria, which are not well exploited for PHB production on industrial scale, Bacillus appears to be a prospective candidate due to its excellent biopolymer yield and less rigorous fermentation conditions. Batch culture fermentation was carried out for PHB production. The Bacillus cereus NRRL-B-3711 was the most efficient producer of PHB out of five Bacillus species. The optimized production was achieved with 2% glucose, 37 degree C, pH value of 8 and ammonium sulfate (2.5 g/L) as a nitrogen source. Carbon to nitrogen ratio of 10 significantly affects the PHB accumulation. The selected specie was able to accumulate PHB up to 56% (14.2) 0.07 g/L) on dry cell weight basis after optimization. This corresponds to a 1.87 folds increase in the production. The optical microscopy showed extremely flat surface of bioplastic thin film indicating the brittle failure of PHB under tensile loading. The FTIR analysis revealed C=O and -CH groups, with thermal properties e.g. Tg; 2 degree C, Tc; 54 degree C, Tm;162 degree C and percent crystallinity of 51.3 by differential scanning calorimeter (DSC), thus confirming the presence of PHB bioplastic and enhancing its industrial applications.The results surpassed those reported in the literature for PHB production. (author)

  10. Enhanced thermostability of silica-immobilized lipase from Bacillus coagulans BTS-3 and synthesis of ethyl propionate.

    Science.gov (United States)

    Kumar, Satyendra; Pahujani, Shweta; Ola, R P; Kanwar, S S; Gupta, Reena

    2006-06-01

    A lipase from the thermophilic isolate Bacillus coagulans BTS-3 was produced and purified. The enzyme was purified 40-fold to homogeneity by ammonium sulfate precipitation and DEAE-Sepharose column chromatography. Its molecular weight was 31 kDa on SDS-PAGE. The purified lipase was immobilized on silica and its binding efficiency was found to be 60%. The enzyme took 60 min to bind maximally onto the support. The pH and temperature optima of immobilized lipase were same as those of the free enzyme, i.e. 8.5 and 55 degrees C, respectively. The immobilized enzyme had shown marked thermostability on the elevated temperatures of 55, 60, 65 and 70 degrees C. The immobilized enzyme was reused for eigth cycles as it retained almost 80% of its activity. The catalytic activity of immobilized enzyme was enhanced in n-hexane and ethanol. The immobilized enzyme when used for esterification of ethanol and propionic acid showed 96% conversion in n-hexane in 12 h at 55 degrees C.

  11. A Phytase Characterized by Relatively High pH Tolerance and Thermostability from the Shiitake Mushroom Lentinus edodes

    Directory of Open Access Journals (Sweden)

    Guo-Qing Zhang

    2013-01-01

    Full Text Available A monomeric phytase with a molecular mass of 14 kDa was acquired from fresh fruiting bodies of the shiitake mushroom Lentinus edodes. The isolation procedure involved chromatography on DEAE-cellulose, CM-cellulose, Q-Sepharose, Affi-gel blue gel, and a final fast protein liquid chromatography-gel filtration on Superdex 75. The purified phytase demonstrated the unique N-terminal amino acid sequence DPKRTDQVN, which exhibited no sequence similarity with those of other phytases previously reported. It expressed its maximal activity at pH 5.0 and 37°C. Phytase activity manifested less than 20% change in activity over the pH range of 3.0–9.0, considerable thermostability with more than 60% residual activity at 70°C, and about 40% residual activity at 95°C. It displayed a wide substrate specificity on a variety of phosphorylated compounds with the following ranking: ATP > fructose-6-phosphate > AMP > glucose-6-phosphate > ADP > sodium phytate > β-glycerophosphate. The phytase activity was moderately stimulated by Ca2+, but inhibited by Al3+, Mn2+, Zn2+, and Cu2+ at a tested concentration of 5 mM.

  12. Purification, characterization and gene cloning of thermostable O-acetyl-L-serine sulfhydrylase forming {beta}-cyano-L-alanine

    Energy Technology Data Exchange (ETDEWEB)

    Omura, Hironori; Yoshida, Toyokazu; Nagasawa, Toru [Gifu Univ. (Japan). Dept. of Biomolecular Science; Kuroda, Masako [Ikeda Food Research Co., Ltd., Fukuyama, Hiroshima (Japan); Kobayashi, Michihiko; Shimizu, Sakayu [Kyoto Univ. (Japan). Agricultural Sciences

    2003-10-01

    A thermophilic and cyanide ion-tolerant bacterium, Bacillus stearothermophilus CN3 isolated from a hot spring in Japan, was found to produce thermostable {beta}-cyano-L-alanine synthase. The enzyme catalyzes the synthesis of {beta}-cyano-L-alanine from O-acetyl-L-serine and cyanide ions. The purified enzyme has a molecular mass of approximately 70 kDa and consists of two identical sub-units. It was stable in the pH range of 6.0 to 10.0 and up to 70degC. The enzyme also catalyzes the synthesis of various {beta}-substituted-L-alanine derivatives from O-acetyl-L-serine and nucleophilic reagents. The gene encoding the {beta}-cyano-L-alanine synthase was isolated from B. stearothermophilus CN3. Sequence homology analysis revealed that the {beta}-cyano-L-alanine synthase of the bacterium is O-acetyl-L-serine sulfhydrylase. A recombinant plasmid, constructed by ligation of the cloned gene and an expression vector, pKK223-3, was introduced into E. coli JM109. The transformed E. coli cells overexpressed {beta}-cyano-L-alanine synthase. Heat stable {beta}-cyano-L-alanine synthase can be applied to the synthesis of [4-{sup 11}C]L-2,4-diaminobutyric acid as a tracer for positron emission tomography. (author)

  13. A Phytase Characterized by Relatively High pH Tolerance and Thermostability from the Shiitake Mushroom Lentinus edodes

    Science.gov (United States)

    Zhang, Guo-Qing; Wu, Ying-Ying; Ng, Tzi-Bun; Chen, Qing-Jun; Wang, He-Xiang

    2013-01-01

    A monomeric phytase with a molecular mass of 14 kDa was acquired from fresh fruiting bodies of the shiitake mushroom Lentinus edodes. The isolation procedure involved chromatography on DEAE-cellulose, CM-cellulose, Q-Sepharose, Affi-gel blue gel, and a final fast protein liquid chromatography-gel filtration on Superdex 75. The purified phytase demonstrated the unique N-terminal amino acid sequence DPKRTDQVN, which exhibited no sequence similarity with those of other phytases previously reported. It expressed its maximal activity at pH 5.0 and 37°C. Phytase activity manifested less than 20% change in activity over the pH range of 3.0–9.0, considerable thermostability with more than 60% residual activity at 70°C, and about 40% residual activity at 95°C. It displayed a wide substrate specificity on a variety of phosphorylated compounds with the following ranking: ATP > fructose-6-phosphate > AMP > glucose-6-phosphate > ADP > sodium phytate > β-glycerophosphate. The phytase activity was moderately stimulated by Ca2+, but inhibited by Al3+, Mn2+, Zn2+, and Cu2+ at a tested concentration of 5 mM. PMID:23586045

  14. Production and characterization of thermostable alkaline protease of Bacillus subtilis (ATCC 6633) from optimized solid-state fermentation.

    Science.gov (United States)

    Chatterjee, Joyee; Giri, Sudipta; Maity, Sujan; Sinha, Ankan; Ranjan, Ashish; Rajshekhar; Gupta, Suvroma

    2015-01-01

    Proteases are the most important group of enzymes utilized commercially in various arenas of industries, such as food, detergent, leather, dairy, pharmaceutical, diagnostics, and waste management, accounting for nearly 20% of the world enzyme market. Microorganisms of specially Bacillus genera serve as a vast repository of diverse set of industrially important enzymes and utilized for the large-scale enzyme production using a fermentation technology. Approximately 30%-40% of the cost of industrial enzymes originates from the cost of the growth medium. This study is attempted to produce protease from Bacillus subtilis (ATCC 6633) after optimization of various process parameters with the aid of solid-state fermentation using a cheap nutrient source such as wheat bran. B. subtilis (ATCC 6633) produces proteases of molecular weight 36 and 20 kDa, respectively, in the fermented medium as evident from SDS zymogram. Alkaline protease activity has been detected with optimum temperature at 50 °C and is insensitive to ethylenediaminetetraacetic acid. This thermostable alkaline protease exhibits dual pH optimum at 7 and 10 with moderate pH stability at alkaline pH range. It preserves its activity in the presence of detergent such as SDS, Tween 20, and Triton X-100 and may be considered as an effective additive to detergent formulation with some industrial importance. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

  15. Engineering a disulfide bond in the lid hinge region of Rhizopus chinensis lipase: increased thermostability and altered acyl chain length specificity.

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    Xiao-Wei Yu

    Full Text Available The key to enzyme function is the maintenance of an appropriate balance between molecular stability and structural flexibility. The lid domain which is very important for "interfacial activation" is the most flexible part in the lipase structure. In this work, rational design was applied to explore the relationship between lid rigidity and lipase activity by introducing a disulfide bond in the hinge region of the lid, in the hope of improving the thermostability of R. chinensis lipase through stabilization of the lid domain without interfering with its catalytic performance. A disulfide bridge between F95C and F214C was introduced into the lipase from R. chinensis in the hinge region of the lid according to the prediction of the "Disulfide by Design" algorithm. The disulfide variant showed substantially improved thermostability with an eleven-fold increase in the t(1/2 value at 60°C and a 7°C increase of T(m compared with the parent enzyme, probably contributed by the stabilization of the geometric structure of the lid region. The additional disulfide bond did not interfere with the catalytic rate (k(cat and the catalytic efficiency towards the short-chain fatty acid substrate, however, the catalytic efficiency of the disulfide variant towards pNPP decreased by 1.5-fold probably due to the block of the hydrophobic substrate channel by the disulfide bond. Furthermore, in the synthesis of fatty acid methyl esters, the maximum conversion rate by RCLCYS reached 95% which was 9% higher than that by RCL. This is the first report on improving the thermostability of the lipase from R. chinensis by introduction of a disulfide bond in the lid hinge region without compromising the catalytic rate.

  16. Effect of pulsed electric field treatment on enzyme kinetics and thermostability of endogenous ascorbic acid oxidase in carrots (Daucus carota cv. Nantes).

    Science.gov (United States)

    Leong, Sze Ying; Oey, Indrawati

    2014-03-01

    The objective of this research was to study the enzyme kinetics and thermostability of endogenous ascorbic acid oxidase (AAO) in carrot purée (Daucus carota cv. Nantes) after being treated with pulsed electric field (PEF) processing. Various PEF treatments using electric field strength between 0.2 and 1.2kV/cm and pulsed electrical energy between 1 and 520kJ/kg were conducted. The enzyme kinetics and the kinetics of AAO thermal inactivation (55-70°C) were described using Michaelis-Menten model and first order reaction model, respectively. Overall, the estimated Vmax and KM values were situated in the same order of magnitude as the untreated carrot purée after being exposed to pulsed electrical energy between 1 and 400kJ/kg, but slightly changed at pulsed electrical energy above 500kJ/kg. However, AAO presented different thermostability depending on the electric field strength applied. After PEF treatment at the electric field strength between 0.2 and 0.5kV/cm, AAO became thermolabile (i.e. increase in inactivation rate (k value) at reference temperature) but the temperature dependence of k value (Ea value) for AAO inactivation in carrot purée decreased, indicating that the changes in k values were less temperature dependent. It is obvious that PEF treatment affects the temperature stability of endogenous AAO. The changes in enzyme kinetics and thermostability of AAO in carrot purée could be related to the resulting carrot purée composition, alteration in intracellular environment and the effective concentration of AAO released after being subjected to PEF treatment. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Bovine pancreatic trypsin inhibitor immobilized onto sepharose as a new strategy to purify a thermostable alkaline peptidase from cobia (Rachycentron canadum) processing waste.

    Science.gov (United States)

    França, Renata Cristina da Penha; Assis, Caio Rodrigo Dias; Santos, Juliana Ferreira; Torquato, Ricardo José Soares; Tanaka, Aparecida Sadae; Hirata, Izaura Yoshico; Assis, Diego Magno; Juliano, Maria Aparecida; Cavalli, Ronaldo Olivera; Carvalho, Luiz Bezerra de; Bezerra, Ranilson Souza

    2016-10-15

    A thermostable alkaline peptidase was purified from the processing waste of cobia (Rachycentron canadum) using bovine pancreatic trypsin inhibitor (BPTI) immobilized onto Sepharose. The purified enzyme had an apparent molecular mass of 24kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. Its optimal temperature and pH were 50°C and 8.5, respectively. The enzyme was thermostable until 55°C and its activity was strongly inhibited by the classic trypsin inhibitors N-ρ-tosyl-l-lysine chloromethyl ketone (TLCK) and benzamidine. BPTI column allowed at least 15 assays without loss of efficacy. The purified enzyme was identified as a trypsin and the N-terminal amino acid sequence of this trypsin was IVGGYECTPHSQAHQVSLNSGYHFC, which was highly homologous to trypsin from cold water fish species. Using Nα-benzoyl-dl-arginine ρ-nitroanilide hydrochloride (BApNA) as substrate, the apparent km value of the purified trypsin was 0.38mM, kcat value was 3.14s(-1), and kcat/km was 8.26s(-1)mM(-1). The catalytic proficiency of the purified enzyme was 2.75×10(12)M(-1) showing higher affinity for the substrate at the transition state than other fish trypsin. The activation energy (AE) of the BApNA hydrolysis catalyzed by this enzyme was estimated to be 11.93kcalmol(-1) while the resulting rate enhancement of this reaction was found to be approximately in a range from 10(9) to 10(10)-fold evidencing its efficiency in comparison to other trypsin. This new purification strategy showed to be appropriate to obtain an alkaline peptidase from cobia processing waste with high purification degree. According with N-terminal homology and kinetic parameters, R. canadum trypsin may gathers desirable properties of psychrophilic and thermostable enzymes. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Constraint Network Analysis (CNA): a Python software package for efficiently linking biomacromolecular structure, flexibility, (thermo-)stability, and function.

    Science.gov (United States)

    Pfleger, Christopher; Rathi, Prakash Chandra; Klein, Doris L; Radestock, Sebastian; Gohlke, Holger

    2013-04-22

    For deriving maximal advantage from information on biomacromolecular flexibility and rigidity, results from rigidity analyses must be linked to biologically relevant characteristics of a structure. Here, we describe the Python-based software package Constraint Network Analysis (CNA) developed for this task. CNA functions as a front- and backend to the graph-based rigidity analysis software FIRST. CNA goes beyond the mere identification of flexible and rigid regions in a biomacromolecule in that it (I) provides a refined modeling of thermal unfolding simulations that also considers the temperature-dependence of hydrophobic tethers, (II) allows performing rigidity analyses on ensembles of network topologies, either generated from structural ensembles or by using the concept of fuzzy noncovalent constraints, and (III) computes a set of global and local indices for quantifying biomacromolecular stability. This leads to more robust results from rigidity analyses and extends the application domain of rigidity analyses in that phase transition points ("melting points") and unfolding nuclei ("structural weak spots") are determined automatically. Furthermore, CNA robustly handles small-molecule ligands in general. Such advancements are important for applying rigidity analysis to data-driven protein engineering and for estimating the influence of ligand molecules on biomacromolecular stability. CNA maintains the efficiency of FIRST such that the analysis of a single protein structure takes a few seconds for systems of several hundred residues on a single core. These features make CNA an interesting tool for linking biomacromolecular structure, flexibility, (thermo-)stability, and function. CNA is available from http://cpclab.uni-duesseldorf.de/software for nonprofit organizations.

  19. Optimization of Thermostable Alpha-Amylase Production Via Mix Agricultural-Residues and Bacillus amyloliquefaciens

    Directory of Open Access Journals (Sweden)

    Shalini RAI

    2014-03-01

    Full Text Available This study reports utilization of mixture of wheat and barley bran (1:1 for the production of thermostable alpha-amylase enzyme through a spore former, heat tolerant strain of Bacillus amyloliquefaciens in solid state fermentation. Maximum yield of alpha-amylase (252.77 U mL-1 was obtained in following optimized conditions, inoculums size 2 mL (2 × 106 CFU/mL, moisture 80%, pH 7±0.02, NaCl (3%, temperature 38±1°C, incubation for 72 h, maltose (1% and tryptone (1%. After SSF crude enzyme was purified via ammonium sulfate precipitation, ion exchange and column chromatography by DEAE Cellulose. Purified protein showed a molecular weight of 42 kDa by SDS-PAGE electrophoresis. After purification, purified enzyme was characterized against several enzymes inhibitors such as temperature, NaCl, pH, metal and surfactants. Pure enzyme was highly active over broad temperature (50-70°C, NaCl concentration (0.5-4 M, and pH (6-10 ranges, indicating it’s a thermoactive and alkali-stable nature. Moreover, CaCl2, MnCl2, =-mercaptoethanol were found to stimulate the amylase activity, whereas FeCl3, sodium dodecyl sulfate (SDS, CuCl3 and ethylenediaminetetraacetic acid (EDTA strongly inhibited the enzyme. Moreover, enzyme specificity and thermal stability conformed by degradation of different soluble starch up to 55°C. Therefore, the present study proved that the extracellular alpha-amylase extracted through wheat flour residues by organism B. amyloliquefaciens MCCB0075, both have considerable potential for industrial application owing to its properties.

  20. Process optimization for production and purification of a thermostable, organic solvent tolerant lipase from Acinetobacter sp. AU07

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    P. Gururaj

    Full Text Available ABSTRACT The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5 U/mL was observed at 30 ºC and pH 7, using a 0.5% (v/v inoculum, 2% (v/v castor oil (inducer, and agitation 150 rpm. The optimized conditions from the shake flask experiments were validated in a 3 L lab scale bioreactor, and the lipase production increased to 48 U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45 kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50 ºC and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98 U/mg, 0.51 mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.

  1. Alkaline thermostable pectinase enzyme from Aspergillus niger strain MCAS2 isolated from Manaslu Conservation Area, Gorkha, Nepal.

    Science.gov (United States)

    Khatri, Bhim Prakash; Bhattarai, Tribikram; Shrestha, Sangita; Maharjan, Jyoti

    2015-01-01

    Pectinase enzymes are one of the commercially important enzymes having great potential in various industries especially in food industry. Pectinases accounts for 25 % of global food enzymes produced and their market is increasing day by day. Therefore, the exploration of microorganism with novel characteristics has always been the focus of the research. Microorganism dwelling in unique habitat may possess unique characteristics. As such, a pectinase producing fungus Aspergillus niger strain MCAS2 was isolated from soil of Manaslu Conservation Area (MCA), Gorkha, Nepal. The optimum production of pectinase enzyme was observed at 48 h of fermentation. The pectinase enzyme was partially purified by cold acetone treatment followed by Sephadex G-75 gel filtration chromatography. The partially purified enzyme exhibited maximum activity 60 U/mg which was almost 8.5-fold higher than the crude pectinase. The approximate molecular weight of the enzyme was found to be 66 kDa as observed from SDS-PAGE. The pectinase enzyme was active at broad range of temperature (30-70 °C) and pH (6.2-9.2). Optimum temperature and pH of the pectinase enzyme were 50 °C and 8.2 respectively. The enzyme was stable up to 70 °C and about 82 % of pectinase activity was still observed at 100 °C. The thermostable and alkaline nature of this pectinase can meet the demand of various industrial processes like paper and pulp industry, in textile industry, fruit juice industry, plant tissue maceration and wastewater treatment. In addition, the effect of different metal ions on pectinase activity was also studied.

  2. A decoy set for the thermostable subdomain from chicken villin headpiece, comparison of different free energy estimators

    Directory of Open Access Journals (Sweden)

    Tosatto Silvio CE

    2005-12-01

    Full Text Available Abstract Background Estimators of free energies are routinely used to judge the quality of protein structural models. As these estimators still present inaccuracies, they are frequently evaluated by discriminating native or native-like conformations from large ensembles of so-called decoy structures. Results A decoy set is obtained from snapshots taken from 5 long (100 ns molecular dynamics (MD simulations of the thermostable subdomain from chicken villin headpiece. An evaluation of the energy of the decoys is given using: i a residue based contact potential supplemented by a term for the quality of dihedral angles; ii a recently introduced combination of four statistical scoring functions for model quality estimation (FRST; iii molecular mechanics with solvation energy estimated either according to the generalized Born surface area (GBSA or iv the Poisson-Boltzmann surface area (PBSA method. Conclusion The decoy set presented here has the following features which make it attractive for testing energy scoring functions: 1 it covers a broad range of RMSD values (from less than 2.0 Å to more than 12 Å; 2 it has been obtained from molecular dynamics trajectories, starting from different non-native-like conformations which have diverse behaviour, with secondary structure elements correctly or incorrectly formed, and in one case folding to a native-like structure. This allows not only for scoring of static structures, but also for studying, using free energy estimators, the kinetics of folding; 3 all structures have been obtained from accurate MD simulations in explicit solvent and after molecular mechanics (MM energy minimization using an implicit solvent method. The quality of the covalent structure therefore does not suffer from steric or covalent problems. The statistical and physical effective energy functions tested on the set behave differently when native simulation snapshots are included or not in the set and when averaging over the

  3. Removal of distal protein-water hydrogen bonds in a plant epoxide hydrolase increases catalytic turnover but decreases thermostability.

    Science.gov (United States)

    Thomaeus, Ann; Naworyta, Agata; Mowbray, Sherry L; Widersten, Mikael

    2008-07-01

    A putative proton wire in potato soluble epoxide hydrolase 1, StEH1, was identified and investigated by means of site-directed mutagenesis, steady-state kinetic measurements, temperature inactivation studies, and X-ray crystallography. The chain of hydrogen bonds includes five water molecules coordinated through backbone carbonyl oxygens of Pro(186), Leu(266), His(269), and the His(153) imidazole. The hydroxyl of Tyr(149) is also an integrated component of the chain, which leads to the hydroxyl of Tyr(154). Available data suggest that Tyr(154) functions as a final proton donor to the anionic alkylenzyme intermediate formed during catalysis. To investigate the role of the putative proton wire, mutants Y149F, H153F, and Y149F/H153F were constructed and purified. The structure of the Y149F mutant was solved by molecular replacement and refined to 2.0 A resolution. Comparison with the structure of wild-type StEH1 revealed only subtle structural differences. The hydroxyl group lost as a result of the mutation was replaced by a water molecule, thus maintaining a functioning hydrogen bond network in the proton wire. All mutants showed decreased catalytic efficiencies with the R,R-enantiomer of trans-stilbene oxide, whereas with the S,S-enantiomer, k (cat)/K (M) was similar or slightly increased compared with the wild-type reactions. k (cat) for the Y149F mutant with either TSO enantiomer was increased; thus the lowered enzyme efficiencies were due to increases in K (M). Thermal inactivation studies revealed that the mutated enzymes were more sensitive to elevated temperatures than the wild-type enzyme. Hence, structural alterations affecting the hydrogen bond chain caused increases in k (cat) but lowered thermostability.

  4. Purification and Characterization of Thermostable and Detergent-Stable α-Amylase from Anoxybacillus sp. AH1

    Directory of Open Access Journals (Sweden)

    Fatma Matpan Bekler

    2016-01-01

    Full Text Available A thermostable and detergent-stable α-amylase from a newly isolated Anoxybacillus sp. AH1 was purifi ed and characterized. Maximum enzyme production (1874.8 U/mL was obtained at 24 h of incubation. The amylase was purified by using Sephadex G-75 gel filtration, after which an 18-fold increase in specific activity and a yield of 9 % were achieved. The molecular mass of the purified enzyme was estimated at 85 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE. The optimum pH and temperature values of the enzyme were 7.0 and 60 °C, respectively. The enzyme was highly stable in the presence of 30 % glycerol, retaining 85 % of its original activity at 60 °C within 120 min. Km and vmax values were 0.102 μmol and 0.929 μmol/min, espectively, using Lineweaver-Burk plot. The enzyme activity was increased by various detergents, but it was significantly inhibited in the presence of urea. Mg2+ and Ca2+ also significantly activated α-amylase, while Zn2+, Cu2+ and metal ion chelators ethylenediaminetetraacetic acid (EDTA and 1,10-phenanthroline (phen greatly inhibited the enzyme activity. α-Amylase activity was enhanced by β-mercaptoethanol (β-ME and dithiothreitol (DTT to a great extent, but inhibited by p-chloromercuribenzoic acid (PCMB. Iodoacetamide (IAA and N-ethylmaleimide (NEM had a slight, whereas phenylmethylsulfonyl fluoride (PMSF had a strong inhibitory effect on the amylase activity.

  5. Enzymatic conversion of D-galactose to D-tagatose: heterologous expression and characterisation of a thermostable L-arabinose isomerase from Thermoanaerobacter mathranii.

    Science.gov (United States)

    Jørgensen, F; Hansen, O C; Stougaard, P

    2004-06-01

    The ability to convert D-galactose into D-tagatose was compared among a number of bacterial L-arabinose isomerases ( araA). One of the most efficient enzymes, from the anaerobic thermophilic bacterium Thermoanaerobacter mathranii, was produced heterologously in Escherichia coli and characterised. Amino acid sequence comparisons indicated that this enzyme is only distantly related to the group of previously known araA sequences in which the sequence similarity is evident. The substrate specificity and the Michaelis-Menten constants of the enzyme determined with L-arabinose, D-galactose and D-fucose also indicated that this enzyme is an unusual, versatile L-arabinose isomerase which is able to isomerise structurally related sugars. The enzyme was immobilised and used for production of D-tagatose at 65 degrees C. Starting from a 30% solution of D-galactose, the yield of D-tagatose was 42% and no sugars other than D-tagatose and D-galactose were detected. Direct conversion of lactose to D-tagatose in a single reactor was demonstrated using a thermostable beta-galactosidase together with the thermostable L-arabinose isomerase. The two enzymes were also successfully combined with a commercially available glucose isomerase for conversion of lactose into a sweetening mixture comprising lactose, glucose, galactose, fructose and tagatose.

  6. Effects of Calcium Ions on the Thermostability and Spectroscopic Properties of the LH1-RC Complex from a New Thermophilic Purple Bacterium Allochromatium tepidum.

    Science.gov (United States)

    Kimura, Yukihiro; Lyu, Shuwen; Okoshi, Akira; Okazaki, Koudai; Nakamura, Natsuki; Ohashi, Akira; Ohno, Takashi; Kobayashi, Manami; Imanishi, Michie; Takaichi, Shinichi; Madigan, Michael T; Wang-Otomo, Zheng-Yu

    2017-05-18

    The light harvesting-reaction center (LH1-RC) complex from a new thermophilic purple sulfur bacterium Allochromatium (Alc.) tepidum was isolated and characterized by spectroscopic and thermodynamic analyses. The purified Alc. tepidum LH1-RC complex showed a high thermostability comparable to that of another thermophilic purple sulfur bacterium Thermochromatium tepidum, and spectroscopic characteristics similar to those of a mesophilic bacterium Alc. vinosum. Approximately 4-5 Ca 2+ per LH1-RC were detected by inductively coupled plasma atomic emission spectroscopy and isothermal titration calorimetry. Upon removal of Ca 2+ , the denaturing temperature of the Alc. tepidum LH1-RC complex dropped accompanied by a blue-shift of the LH1 Q y absorption band. The effect of Ca 2+ was also observed in the resonance Raman shift of the C3-acetyl νC═O band of bacteriochlorophyll-a, indicating changes in the hydrogen-bonding interactions between the pigment and LH1 polypeptides. Thermodynamic parameters for the Ca 2+ -binding to the Alc. tepidum LH1-RC complex indicated that this reaction is predominantly driven by the largely favorable electrostatic interactions that counteract the unfavorable negative entropy change. Our data support a hypothesis that Alc. tepidum may be a transitional organism between mesophilic and thermophilic purple bacteria and that Ca 2+ is one of the major keys to the thermostability of LH1-RC complexes in purple bacteria.

  7. Crystallization and preliminary X-ray study of a family 10 alkali-thermostable xylanase from alkalophilic Bacillus sp. strain NG-27

    International Nuclear Information System (INIS)

    Manikandan, K.; Bhardwaj, Amit; Ghosh, Amit; Reddy, V. S.; Ramakumar, S.

    2005-01-01

    A family 10 alkali-thermostable xylanase from Bacillus sp. NG-27 has been crystallized. A diffraction data set has been collected to 2.2 Å resolution. Xylanases (EC 3.2.1.8) catalyze the hydrolysis of β-1,4-glycosidic linkages within xylan, a major hemicellulose component in the biosphere. The extracellular endoxylanase (XylnA) from the alkalophilic Bacillus sp. strain NG-27 belongs to family 10 of the glycoside hydrolases. It is active at 343 K and pH 8.4. Moreover, it has attractive features from the point of view of utilization in the paper pulp, animal feed and baking industries since it is an alkali-thermostable protein. In this study, XylnA was purified from the native host source and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 174.5, b = 54.7, c = 131.5 Å, β = 131.2°, and diffract to better than 2.2 Å resolution

  8. Crystallization and preliminary X-ray study of a family 10 alkali-thermostable xylanase from alkalophilic Bacillus sp. strain NG-27

    Energy Technology Data Exchange (ETDEWEB)

    Manikandan, K. [Department of Physics, Indian Institute of Science, Bangalore 560 012 (India); Bhardwaj, Amit [International Centre for Genetic Engineering and Biotechnology, New Delhi 110 067 (India); Ghosh, Amit [Institute of Microbial Technology, Sector 39-A, Chandigarh 160 036 (India); Reddy, V. S. [International Centre for Genetic Engineering and Biotechnology, New Delhi 110 067 (India); Ramakumar, S., E-mail: ramak@physics.iisc.ernet.in [Department of Physics, Indian Institute of Science, Bangalore 560 012 (India); Bioinformatics Centre, Indian Institute of Science, Bangalore 560 012 (India)

    2005-08-01

    A family 10 alkali-thermostable xylanase from Bacillus sp. NG-27 has been crystallized. A diffraction data set has been collected to 2.2 Å resolution. Xylanases (EC 3.2.1.8) catalyze the hydrolysis of β-1,4-glycosidic linkages within xylan, a major hemicellulose component in the biosphere. The extracellular endoxylanase (XylnA) from the alkalophilic Bacillus sp. strain NG-27 belongs to family 10 of the glycoside hydrolases. It is active at 343 K and pH 8.4. Moreover, it has attractive features from the point of view of utilization in the paper pulp, animal feed and baking industries since it is an alkali-thermostable protein. In this study, XylnA was purified from the native host source and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 174.5, b = 54.7, c = 131.5 Å, β = 131.2°, and diffract to better than 2.2 Å resolution.

  9. Study of the leaching of heavy metals from waste water sludge and incinerator's ash, using coupled thermostated columns and DTPA as complex agent

    International Nuclear Information System (INIS)

    Vite T, J.; Vite T, M.; Guerrero D, J.; Carreno de Leon, M.C.

    2000-01-01

    We studied the metallic composition from waste water sludge and incinerators ashes of an incinerator located in Toluca, Mexico, the qualitative studies were made using the Activation Analysis technique, and fluorescence X-ray techniques. The quantitative analysis of heavy metals in the wastes were made using Inductively coupled plasma atomic emission spectrometry (Icp-Aes). For leaching the samples, we used four coupled thermostated columns, each one had a p H of 2,5, 7 and 10. The flux of the air was of 1600 cc/min. The temperature was maintain constant in 60 Centigrade using a thermostated system. For this study we used 100 g of wastes mixed with mineral acid or sodium hydroxide to reach p H 2,5,7 and 10. We added a reducing and tensoactive agents and finally DTPA as complex agent. With this method, we obtain a better leaching efficiency using a complex agent. However the high DTPA cost, make this process expansive that is why we recommend to work with another classes of complex agents, that be cheaper to leach metals of different chemistry matrix. (Author)

  10. Construction of a highly thermostable 1,3-1,4-β-glucanase by combinational mutagenesis and its potential application in the brewing industry.

    Science.gov (United States)

    Niu, Chengtuo; Zhu, Linjiang; Hill, Annie; Alex Speers, R; Li, Qi

    2017-01-01

    To improve the thermostability and catalytic property of a mesophilic 1,3-1,4-β-glucanase by combinational mutagenesis and to test its effect in congress mashing. A mutant β-glucanase (rE-BglTO) constructed by combinational mutagenesis showed a 25 °C increase in optimal temperature (to 70 °C) a 19.5 °C rise in T 50 value and a 15.6 °C increase in melting temperature compared to wild-type enzyme. Its half-life values at 60 and 70 °C were 152 and 99 min, which were 370 and 800 % higher than those of wild-type enzyme. Besides, its specific activity and k cat value were 42,734 U mg -1 and 189 s -1 while its stability under acidic conditions was also improved. In flask fermentation, the catalytic activity of rE-BglTO reached 2381 U ml -1 , which was 63 % higher than that of wild-type enzyme. The addition of rE-BglTO in congress mashing decreased the filtration time and viscosity by 21.3 and 9.6 %, respectively. The mutant β-glucanase showed high catalytic activity and thermostability which indicated that rE-BglTO is a good candidate for application in the brewing industry.

  11. Purification and properties of a thermostable pullulanase from Clostridium thermosulfurgenes EM1 which hydrolyses both. alpha. -1,6 and. alpha. -1,4-glycosidic linkages

    Energy Technology Data Exchange (ETDEWEB)

    Spreinat, A [Goettingen Univ. (Germany, F.R.). Inst. fuer Mikrobiologie; Antranikian, G [Technische Univ. Hamburg-Harburg, Hamburg (Germany, F. R.). Arbeitsbereich Biotechnologie 1

    1990-08-01

    A novel thermostable pullulanase, secreted by the thermophilic anaerobic bacterium Clostridium thermosulfurogenes EM1, was purified and characterized. Applying anion exchange chromatography and gel filtration the enzyme was purified 47-fold and had a specific activity of 200 units/mg. The molecular mass of this thermostable enzyme was determined to be 102 000 daltons and consisted of a single subunit. The enzyme was able to attack specifically the {alpha}-1,6-glycosidic linkages in pullulan and caused its complete hydrolysis to maltotriose. Surprisingly and unlike the enzyme from Klebsiella pneumoniae, the purified enzyme from this anaerobic thermophile exhibited, in addition to its debranching and pullulanase activity, an {alpha}-1,4 hydrolysing activity as well. By the action of this single polypeptide chain various branched and linear polysaccharides were completely converted to two major products, namely maltose and maltotriose. The K{sub m} values of this enzyme for pullulan and amylose were determined to be 1.33 mg/ml and 0.38 mg/ml, respectively. This debranching enzyme displays a temperature optimum at 60deg-65deg C and a pH optimum at 5.5-6.0. The application of this new class of pullulanase (pullulanase type II) in industry will significantly enhance the starch saccharification process. (orig.).

  12. Crystallization and preliminary X-ray crystallographic analysis of highly thermostable L2 lipase from the newly isolated Bacillus sp. L2

    International Nuclear Information System (INIS)

    Shariff, Fairolniza Mohd; Rahman, Raja Noor Zaliha Raja Abd.; Ali, Mohd Shukuri Mohamad; Chor, Adam Leow Thean; Basri, Mahiran; Salleh, Abu Bakar

    2010-01-01

    Thermostable recombinant L2 lipase from thermophilic Bacillus sp. L2 has been crystallized by using counter-diffusion method and diffracted to 2.7 Å resolution. The crystal belongs to the primitive orthorhombic space group P2 1 2 1 2 1 with unit-cell parameters a = 87.44, b = 94.90, c = 126.46 Å. Purified thermostable recombinant L2 lipase from Bacillus sp. L2 was crystallized by the counter-diffusion method using 20% PEG 6000, 50 mM MES pH 6.5 and 50 mM NaCl as precipitant. X-ray diffraction data were collected to 2.7 Å resolution using an in-house Bruker X8 PROTEUM single-crystal diffractometer system. The crystal belonged to the primitive orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 87.44, b = 94.90, c = 126.46 Å. The asymmetric unit contained one single molecule of protein, with a Matthews coefficient (V M ) of 2.85 Å 3 Da −1 and a solvent content of 57%

  13. Properties of a novel thermostable glucose isomerase mined from Thermus oshimai and its application to preparation of high fructose corn syrup.

    Science.gov (United States)

    Jia, Dong-Xu; Zhou, Lin; Zheng, Yu-Guo

    2017-04-01

    Glucose isomerase (GI) is used in vitro to convert d-glucose to d-fructose, which is capable of commercial producing high fructose corn syrup (HFCS). To manufacture HFCS at elevated temperature and reduce the cost of enriching syrups, novel refractory GIs from Thermoanaerobacterium xylanolyticum (TxGI), Thermus oshimai (ToGI), Geobacillus thermocatenulatus (GtGI) and Thermoanaerobacter siderophilus (TsGI) were screened via genome mining approach. The enzymatic characteristics research showed that ToGI had higher catalytic efficiency and superior thermostability toward d-glucose among the screened GIs. Its optimum temperature reached 95°C and could retain more than 80% of initial activity in the presence of 20mM Mn 2+ at 85°C for 48h. The K m and k cat /K m values for ToGI were 81.46mM and 21.77min -1 mM -1 , respectively. Furthermore, the maximum conversion yield of 400g/L d-glucose to d-fructose at 85°C was 52.16%. Considering its excellent high thermostability and ameliorable application performance, ToGI might be promising for realization of future industrial production of HFCS at elevated temperature. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Production of extremely alkaliphilic, halotolerent, detergent, and thermostable mannanase by the free and immobilized cells of Bacillus halodurans PPKS-2. Purification and characterization.

    Science.gov (United States)

    Vijayalaxmi, S; Prakash, P; Jayalakshmi, S K; Mulimani, V H; Sreeramulu, K

    2013-09-01

    The alkaliphilic Bacillus halodurans strain PPKS-2 was shown to produce extracellular extreme alkaliphilic, halotolerent, detergent, and thermostable mannanase activity. The cultural conditions for the maximum enzyme production were optimized with respect to pH, temperature, NaCl, and inexpensive agro wastes as substrates. Mannanase production was enhanced more than 4-fold in the presence of 1 % defatted copra meal and 0.5 % peptone or feather hydrolysate at pH 11 and 40 °C. Mannanase was purified to 10.3-fold with 34.6 % yield by ion exchange and gel filtration chromatography methods. Its molecular mass was estimated to be 22 kDa by SDS-PAGE. The mannanase had maximal activity at pH 11 and 70 °C. This enzyme was active over a broad range of NaCl (0-16 %) and thermostable retaining 100 % of the original activity at 70 °C for 3 h. Immobilization of whole cells proved to be effective for continuous production of mannanase. Since the strain PPKS-2 grows on cheaper agro wastes such as defatted copra meal, corn husk, jowar bagasse, and wheat bran, these can be exploited for mannanase production on an industrial scale.

  15. Isolation, production, purification and characterization of an organic-solvent-thermostable alkalophilic cellulase from Bacillus vallismortis RG-07.

    Science.gov (United States)

    Gaur, Rajeeva; Tiwari, Soni

    2015-03-19

    The rising concerns about the scarcity of fossil fuels, the emission of green house gasses and air pollution by incomplete combustion of fossil fuel have also resulted in an increasing focus on the use of cellulases to perform enzymatic hydrolysis of the lignocellulosic materials for the generation of bioethanol. The aim of this study was to isolate a potential thermo-solvent tolerant cellulase producing bacterium from natural resources, and then applied for purification and characterization. The purified enzyme was to be accessible for the bioethanol production as well as industrial exploitation (discuss in our next study). It is the first instance when thermo-solvent tolerant cellulase producing bacterium was isolated from soil sample. The culture was identified as Bacillus vallismortis RG-07 by 16S rDNA sequence analysis. Bacillus vallismortis RG-07 reported maximum cellulase production from sugarcane baggase (4105 U ml(-1)) used as agro-waste carbon source. The cellulase enzyme produced by the Bacillus sp. was purified by (NH4)2SO4 precipitation, ion exchange and gel filtration chromatography, with overall recovery of 28.8%. The molecular weight of purified cellulase was 80 kDa as revealed by SDS-PAGE and activity gel analysis. The optimum temperature and pH for enzyme activity was determined as 65°C and 7.0 and it retained 95 and 75% of activity even at 95°C, and 9.0 respectively. The enzyme activity was enhanced in the presence of organic solvents (30%) n-dodecane, iso-octane, n-decane, xylene, toluene, n-haxane, n-butanol, and cyclohexane, after prolonged incubation (7 days). The enzyme activity was also stimulated by Ca(2+), mercaptoethanol, Tween-60, and Sodium hypochloride whereas strongly inhibited by Hg. Kinetic analysis of purified enzyme showed the Km and Vmax to be 1.923 mg ml(-1) and 769.230 μg ml(-1) min(-1), respectively. The unique property of solvent-thermostable-alkalophilic, nature proves the potential candidature of this isolate for

  16. phenylalanine and l-tyrosine as chiral micellar media for the cat

    African Journals Online (AJOL)

    potential chiral micellar catalysts for the Diels-Alder reaction between methyl acrylate and .... hydrophobic effects, electrostatic interactions and the accompanying medium effects. In the case .... liquids as effective organocatalysts for Diels-Alder reaction. Green Chem. 2014, 16 .... Colloids and Surfaces A: Physicochem.

  17. phenylalanine and l-tyrosine as chiral micellar media for the cat

    African Journals Online (AJOL)

    polar solvents [15-18]. Hence, surfactants offer the possibility for organic reactions to occur in aqueous media, and from the viewpoint of green chemistry, water is safer, harmless and environmentally benign [19]. However, there has been limited work on the use of chiral micellar media to catalyze Diels-. Alder reactions.

  18. Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity.

    Science.gov (United States)

    Yang, Guang; Yao, Hua; Mozzicafreddo, Matteo; Ballarini, Patrizia; Pucciarelli, Sandra; Miceli, Cristina

    2017-07-01

    The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii ( Ef Amy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, Ef Amy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active Ef Amy with improved thermostability and catalytic efficiency at low temperatures. We engineered two Ef Amy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of Ef Amy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model. IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the

  19. Characterization of mutants expressing thermostable D1 and D2 polypeptides of photosystem II in the cyanobacterium Synechococcus elongatus PCC 7942.

    Science.gov (United States)

    Haraguchi, Norihisa; Kaseda, Jun; Nakayama, Yasumune; Nagahama, Kazuhiro; Ogawa, Takahira; Matsuoka, Masayoshi

    2018-06-08

    Photosystem II complex embedded in thylakoid membrane performs oxygenic photosynthesis where the reaction center D1/D2 heterodimer accommodates all components of the electron transport chain. To express thermostable D1/D2 heterodimer in a cyanobacterium Synechococcus elongatus PCC 7942, we constructed a series of mutant strains whose psbA1 and psbD1 genes encoding, respectively, the most highly expressed D1 and D2 polypeptides were replaced with those of a thermophilic strain, Thermosynechococcus vulcanus. Because the C-terminal 16 amino acid sequences of D1 polypeptides should be processed prior to maturation but diverge from each other, we also constructed the psbA1ΔC-replaced strain expressing a thermostable D1 polypeptide devoid of the C-terminal extension. The psbA1/psbD1-replaced strain showed decreased growth rate and oxygen evolution rate, suggesting inefficient photosystem II. Immunoblot analyses for thermostable D1, D2 polypeptides revealed that the heterologous D1 protein was absent in thylakoid membrane from any mutant strains with psbA1, psbA1ΔC, and psbA1/psbD1-replacements, whereas the heterologous D2 protein was present in thylakoid membrane as well as purified photosystem II complex from the psbA1/psbD1-replaced strain. In the latter strain, the compensatory expression of psbA3 and psbD2 genes was elevated. These data suggest that heterologous D2 polypeptide could be combined with the host D1 polypeptide to form chimeric D1/D2 heterodimer, whereas heterologous D1 polypeptide even without the C-terminal extension was unable to make complex with the host D2 polypeptide. Since the heterologous D1 could not be detected even in the whole cells of psbA1/psbD1-replaced strain, the rapid degradation of unprocessed or unassembled heterologous D1 was implicated. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  20. Thermostable Shelf Life Study

    Science.gov (United States)

    Perchonok, M. H.; Antonini, D. K.

    2008-01-01

    The objective of this project is to determine the shelf life end-point of various food items by means of actual measurement or mathematical projection. The primary goal of the Advanced Food Technology Project in these long duration exploratory missions is to provide the crew with a palatable, nutritious and safe food system while minimizing volume, mass, and waste. The Mars missions could be as long as 2.5 years with the potential of the food being positioned prior to the crew arrival. Therefore, it is anticipated that foods that are used during the Mars missions will require a 5 year shelf life. Shelf life criteria are safety, nutrition, and acceptability. Any of these criteria can be the limiting factor in determining the food's shelf life. Due to the heat sterilization process used for the thermostabilized food items, safety will be preserved as long as the integrity of the package is maintained. Nutrition and acceptability will change over time. Since the food can be the sole source of nutrition to the crew, a significant loss in nutrition may determine when the shelf life endpoint has occurred. Shelf life can be defined when the food item is no longer acceptable. Acceptability can be defined in terms of appearance, flavor, texture, or aroma. Results from shelf life studies of the thermostabilized food items suggest that the shelf life of the foods range from 0 months to 8 years, depending on formulation.

  1. Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-β-mannosidase from Aspergillus niger BK01

    Directory of Open Access Journals (Sweden)

    Sigoillot Jean-Claude

    2009-11-01

    Full Text Available Abstract Background Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-β-mannosidases (1,4-β-D-mannanases catalyze the random hydrolysis of β-1,4-mannosidic linkages in the main chain of β-mannans. Biodegradation of β-mannans by the action of thermostable mannan endo-1,4-β-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS. Results A gene encoding mannan endo-1,4-β-mannosidase or 1,4-β-D-mannan mannanohydrolase (E.C. 3.2.1.78, commonly termed β-mannanase, from Aspergillus niger BK01, which belongs to glycosyl hydrolase family 5 (GH5, was cloned and successfully expressed heterologously (up to 243 μg of active recombinant protein per mL in Pichia pastoris. The enzyme was secreted by P. pastoris and could be collected from the culture supernatant. The purified enzyme appeared glycosylated as a single band on SDS-PAGE with a molecular mass of approximately 53 kDa. The recombinant β-mannanase is highly thermostable with a half-life time of approximately 56 h at 70°C and pH 4.0. The optimal temperature (10-min assay and pH value for activity are 80°C and pH 4.5, respectively. The enzyme is not only active towards structurally different mannans but also exhibits low activity towards birchwood xylan. Apparent Km values of the enzyme for konjac glucomannan (low viscosity, locust bean gum galactomannan, carob galactomannan (low viscosity, and 1,4-β-D-mannan (from carob are 0.6 mg mL-1, 2.0 mg mL-1, 2.2 mg mL-1 and 1.5 mg mL-1, respectively, while the kcat values for these

  2. Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-beta-mannosidase from Aspergillus niger BK01.

    Science.gov (United States)

    Do, Bien-Cuong; Dang, Thi-Thu; Berrin, Jean-Guy; Haltrich, Dietmar; To, Kim-Anh; Sigoillot, Jean-Claude; Yamabhai, Montarop

    2009-11-13

    Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-beta-mannosidases (1,4-beta-D-mannanases) catalyze the random hydrolysis of beta-1,4-mannosidic linkages in the main chain of beta-mannans. Biodegradation of beta-mannans by the action of thermostable mannan endo-1,4-beta-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS). A gene encoding mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannan mannanohydrolase (E.C. 3.2.1.78), commonly termed beta-mannanase, from Aspergillus niger BK01, which belongs to glycosyl hydrolase family 5 (GH5), was cloned and successfully expressed heterologously (up to 243 microg of active recombinant protein per mL) in Pichia pastoris. The enzyme was secreted by P. pastoris and could be collected from the culture supernatant. The purified enzyme appeared glycosylated as a single band on SDS-PAGE with a molecular mass of approximately 53 kDa. The recombinant beta-mannanase is highly thermostable with a half-life time of approximately 56 h at 70 degrees C and pH 4.0. The optimal temperature (10-min assay) and pH value for activity are 80 degrees C and pH 4.5, respectively. The enzyme is not only active towards structurally different mannans but also exhibits low activity towards birchwood xylan. Apparent Km values of the enzyme for konjac glucomannan (low viscosity), locust bean gum galactomannan, carob galactomannan (low viscosity), and 1,4-beta-D-mannan (from carob) are 0.6 mg mL-1, 2.0 mg mL-1, 2.2 mg mL-1 and 1.5 mg mL-1, respectively, while the kcat values for these

  3. Isolation and Characterization of Thermophilic Bacteria from Jordanian Hot Springs: Bacillus licheniformis and Thermomonas hydrothermalis Isolates as Potential Producers of Thermostable Enzymes.

    Science.gov (United States)

    Mohammad, Balsam T; Al Daghistani, Hala I; Jaouani, Atef; Abdel-Latif, Saleh; Kennes, Christian

    2017-01-01

    The aim of this study was the isolation and characterization of thermophilic bacteria from hot springs in Jordan. Ten isolates were characterized by morphological, microscopic, biochemical, molecular, and physiological characteristics. Sequencing of the 16S rDNA of the isolates followed by BLAST search revealed that nine strains could be identified as Bacillus licheniformis and one isolate as Thermomonas hydrothermalis . This is the first report on the isolation of Thermomonas species from Jordanian hot springs. The isolates showed an ability to produce some thermostable enzymes such as amylase, protease, cellulose, gelatins, and lecithin. Moreover, the UPGMA dendrogram of the enzymatic characteristics of the ten isolates was constructed; results indicated a high phenotypic diversity, which encourages future studies to explore further industrial and environmental applications.

  4. Isolation and Characterization of Thermophilic Bacteria from Jordanian Hot Springs: Bacillus licheniformis and Thermomonas hydrothermalis Isolates as Potential Producers of Thermostable Enzymes

    Directory of Open Access Journals (Sweden)

    Balsam T. Mohammad

    2017-01-01

    Full Text Available The aim of this study was the isolation and characterization of thermophilic bacteria from hot springs in Jordan. Ten isolates were characterized by morphological, microscopic, biochemical, molecular, and physiological characteristics. Sequencing of the 16S rDNA of the isolates followed by BLAST search revealed that nine strains could be identified as Bacillus licheniformis and one isolate as Thermomonas hydrothermalis. This is the first report on the isolation of Thermomonas species from Jordanian hot springs. The isolates showed an ability to produce some thermostable enzymes such as amylase, protease, cellulose, gelatins, and lecithin. Moreover, the UPGMA dendrogram of the enzymatic characteristics of the ten isolates was constructed; results indicated a high phenotypic diversity, which encourages future studies to explore further industrial and environmental applications.

  5. A Thermostable Salmonella Phage Endolysin, Lys68, with Broad Bactericidal Properties against Gram-Negative Pathogens in Presence of Weak Acids

    DEFF Research Database (Denmark)

    Oliveira, Hugo; Thiagarajan, Viruthachalam; Walmagh, Maarten

    2014-01-01

    Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that has encouraged the development of new antimicrobial agents. This paper characterizes a Salmonella phage endolysin (Lys68) and demonstrates its potential antimicrobial effectiveness when combined...... with organic acids towards Gram-negative pathogens. Biochemical characterization reveals that Lys68 is more active at pH 7.0, maintaining 76.7% of its activity when stored at 4°C for two months. Thermostability tests showed that Lys68 is only completely inactivated upon exposure to 100°C for 30 min......, and circular dichroism analysis demonstrated the ability to refold into its original conformation upon thermal denaturation. It was shown that Lys68 is able to lyse a wide panel of Gram-negative bacteria (13 different species) in combination with the outer membrane permeabilizers EDTA, citric and malic acid...

  6. Pigeon pea waste as a novel, inexpensive, substrate for production of a thermostable alkaline protease from thermoalkalophilic Bacillus sp. JB-99.

    Science.gov (United States)

    Johnvesly, B; Manjunath, B R; Naik, G R

    2002-03-01

    Thermoalkaliphilic Bacillus sp. JB-99 was grown in a 250 ml Erlenmeyer flask containing 50 ml medium containing (g/l) Pigeon pea waste 10; NaNO3, 5.0; K2HPO4, 5.0; MgSO4 x 2H2O, 0.2 and Na2CO3, 10.0. Incubations were carried out at 50 degrees C on a rotary incubator shaker for 15 h. A high level of extra cellular thermostable protease activity was observed after 24 h incubation. The optimum temperature and pH for activity were 70 degrees C and 11, respectively, so this enzyme showed stable activity at high temperature and under alkaline conditions.

  7. A thermostable Salmonella phage endolysin, Lys68, with broad bactericidal properties against gram-negative pathogens in presence of weak acids.

    Directory of Open Access Journals (Sweden)

    Hugo Oliveira

    Full Text Available Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that has encouraged the development of new antimicrobial agents. This paper characterizes a Salmonella phage endolysin (Lys68 and demonstrates its potential antimicrobial effectiveness when combined with organic acids towards Gram-negative pathogens. Biochemical characterization reveals that Lys68 is more active at pH 7.0, maintaining 76.7% of its activity when stored at 4°C for two months. Thermostability tests showed that Lys68 is only completely inactivated upon exposure to 100°C for 30 min, and circular dichroism analysis demonstrated the ability to refold into its original conformation upon thermal denaturation. It was shown that Lys68 is able to lyse a wide panel of Gram-negative bacteria (13 different species in combination with the outer membrane permeabilizers EDTA, citric and malic acid. While the EDTA/Lys68 combination only inactivated Pseudomonas strains, the use of citric or malic acid broadened Lys68 antibacterial effect to other Gram-negative pathogens (lytic activity against 9 and 11 species, respectively. Particularly against Salmonella Typhimurium LT2, the combinatory effect of malic or citric acid with Lys68 led to approximately 3 to 5 log reductions in bacterial load/CFUs after 2 hours, respectively, and was also able to reduce stationary-phase cells and bacterial biofilms by approximately 1 log. The broad killing capacity of malic/citric acid-Lys68 is explained by the destabilization and major disruptions of the cell outer membrane integrity due to the acidity caused by the organic acids and a relatively high muralytic activity of Lys68 at low pH. Lys68 demonstrates good (thermostability properties that combined with different outer membrane permeabilizers, could become useful to combat Gram-negative pathogens in agricultural, food and medical industry.

  8. Characterization of a thermostable recombinant l-rhamnose isomerase from Caldicellulosiruptor obsidiansis OB47 and its application for the production of l-fructose and l-rhamnulose.

    Science.gov (United States)

    Chen, Ziwei; Xu, Wei; Zhang, Wenli; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

    2018-04-01

    l-Hexoses are rare sugars that are important components and precursors in the synthesis of biological compounds and pharmaceutical drugs. l-Rhamnose isomerase (L-RI, EC 5.3.1.14) is an aldose-ketose isomerase that plays a significant role in the production of l-sugars. In this study, a thermostable, l-sugar-producing L-RI from the hyperthermophile Caldicellulosiruptor obsidiansis OB47 was characterized. The recombinant L-RI displayed maximal activity at pH 8.0 and 85 °C and was significantly activated by Co 2+ . It exhibited a relatively high thermostability, with measured half-lives of 24.75, 11.55, 4.15 and 3.30 h in the presence of Co 2+ at 70, 75, 80 and 85 °C, respectively. Specific activities of 277.6, 57.9, 13.7 and 9.6 U mg -1 were measured when l-rhamnose, l-mannose, d-allose and l-fructose were used as substrates, respectively. l-Rhamnulose was produced with conversion ratios of 44.0% and 38.6% from 25 and 50 g L -1 l-rhamnose, respectively. l-Fructose was also efficiently produced by the L-RI, with conversion ratios of 67.0% and 58.4% from 25 and 50 g L -1 l-mannose, respectively. The recombinant L-RI could effectively catalyze the formation of l-rhamnulose and l-fructose, suggesting that it was a promising candidate for industrial production of l-rhamnulose and l-fructose. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  9. Allergy to fish collagen: Thermostability of collagen and IgE reactivity of patients' sera with extracts of 11 species of bony and cartilaginous fish.

    Science.gov (United States)

    Kobayashi, Yukihiro; Kuriyama, Takuma; Nakagawara, Ryoko; Aihara, Michiko; Hamada-Sato, Naoko

    2016-10-01

    Parvalbumin was identified as a major fish allergen, and has been well investigated. Collagen was identified as a second allergen; however, its allergenic properties remain uncharacterized. Although fish is an important staple in coastal countries, its thermostability is unknown. Therefore, we aimed to determine the thermostability of fish collagen as an allergen. Meat of seven bony and four cartilaginous fishes was heated at various temperatures and times, and extracts were analyzed using SDS-PAGE, IgE-ELISA, and SPTs. Collagen was dissolved from heated meat of Pacific mackerel into a crude extract. Collagen in the extracts was degraded at a high heating load-140 °C (10 min) or 100 °C (320 min). However, ELISA revealed the IgE reactivities of patients' sera with the extracts were unchanged even after heating the samples. Patients strongly reacted to extract proteins of other bony fish, which were detected by patients' IgE even after heating at 100 °C (320 min). In contrast, reactivities of the extracts of cartilaginous fish were lower than those of bony fish. SPTs in one patient revealed that all bony and cartilaginous fish extracts prepared from heated meat elicited allergic reactions. The IgE reactivity of patients' sera to fish collagen in extracts was retained even when fish meat was treated by a high heating load. As for the fish collagen, the IgE reactivities to cartilaginous fish were lower than that to bony fish. Reducing IgE reactivity to fish meat using heat is difficult, and other modalities will be required to produce hypoallergenic fish meat. Copyright © 2016 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.

  10. Crystallization and preliminary X-ray crystallographic analysis of EstE1, a new and thermostable esterase cloned from a metagenomic library

    Energy Technology Data Exchange (ETDEWEB)

    Byun, Jung-Sue [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Protein Network Research Center, Yonsei University, Seoul 120-749 (Korea, Republic of); Rhee, Jin-Kyu [Department of Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Dong-Uk [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Oh, Jong-Won [Department of Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Cho, Hyun-Soo, E-mail: hscho8@yonsei.ac.kr [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Protein Network Research Center, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2006-02-01

    Recombinant EstE1 protein with a histidine tag at the C-terminus was overexpressed in Escherichia coli strain BL21(DE3) and then purified by affinity chromatography. The protein was then crystallized at 290 K by the hanging-drop vapour-diffusion method. EstE1, a new thermostable esterase, was isolated by functional screening of a metagenomic DNA library from thermal environment samples. This enzyme showed activity towards short-chain acyl derivatives of length C4–C6 at a temperature of 303–363 K and displayed a high thermostability above 353 K. EstE1 has 64 and 57% amino-acid sequence similarity to est{sub pc}-encoded carboxylesterase from Pyrobaculum calidifontis and AFEST from Archaeoglobus fulgidus, respectively. The recombinant protein with a histidine tag at the C-terminus was overexpressed in Escherichia coli strain BL21(DE3) and then purified by affinity chromatography. The protein was crystallized at 290 K by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 2.3 Å resolution from an EstE1 crystal; the crystal belongs to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 73.71, c = 234.23 Å. Assuming the presence of four molecules in the asymmetric unit, the Matthews coefficient V{sub M} is calculated to be 2.2 Å{sup 3} Da{sup −1} and the solvent content is 44.1%.

  11. Eukaryote-Made Thermostable DNA Polymerase Enables Rapid PCR-Based Detection of Mycoplasma, Ureaplasma and Other Bacteria in the Amniotic Fluid of Preterm Labor Cases.

    Science.gov (United States)

    Ueno, Tomohiro; Niimi, Hideki; Yoneda, Noriko; Yoneda, Satoshi; Mori, Masashi; Tabata, Homare; Minami, Hiroshi; Saito, Shigeru; Kitajima, Isao

    2015-01-01

    Intra-amniotic infection has long been recognized as the leading cause of preterm delivery. Microbial culture is the gold standard for the detection of intra-amniotic infection, but several days are required, and many bacterial species in the amniotic fluid are difficult to cultivate. We developed a novel nested-PCR-based assay for detecting Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples within three hours of sample collection. To detect prokaryotes, eukaryote-made thermostable DNA polymerase, which is free from bacterial DNA contamination, is used in combination with bacterial universal primers. In contrast, to detect eukaryotes, conventional bacterially-made thermostable DNA polymerase is used in combination with fungal universal primers. To assess the validity of the PCR assay, we compared the PCR and conventional culture results using 300 amniotic fluid samples. Based on the detection level (positive and negative), 93.3% (280/300) of Mycoplasma, 94.3% (283/300) of Ureaplasma, 89.3% (268/300) of other bacteria and 99.7% (299/300) of fungi matched the culture results. Meanwhile, concerning the detection of bacteria other than Mycoplasma and Ureaplasma, 228 samples were negative according to the PCR method, 98.2% (224/228) of which were also negative based on the culture method. Employing the devised primer sets, mixed amniotic fluid infections of Mycoplasma, Ureaplasma and/or other bacteria could be clearly distinguished. In addition, we also attempted to compare the relative abundance in 28 amniotic fluid samples with mixed infection, and judged dominance by comparing the Ct values of quantitative real-time PCR. We developed a novel PCR assay for the rapid detection of Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples. This assay can also be applied to accurately diagnose the absence of bacteria in samples. We believe that this assay will positively contribute to the treatment of intra-amniotic infection and

  12. 包覆赤磷制备工艺及其热安定性研究%Preparation technique of microcapsulated red phosphorusand its thermostability

    Institute of Scientific and Technical Information of China (English)

    刘杰; 关华; 宋东明

    2017-01-01

    为了降低赤磷的吸湿性,提高其热安定性,该文采用氢氧化铝-密胺树脂包覆赤磷(MRP).通过单因素实验优化了制备包覆赤磷的工艺,采用傅里叶变换红外(FTIR)光谱仪和扫描电子显微镜(SEM)对包覆层进行了表征,用热重-差热分析法(TG-DTA)分析了包覆前后赤磷样品的热安定性.结果表明,制备包覆赤磷的最佳工艺参数为,九水合硝酸铝的质量为0.10 g,甲醛和三聚氰胺总质量为0.40 g、摩尔比为3∶1,反应温度为80 ℃,反应时间为1 h.在此工艺条件下,包覆赤磷(MRP)的吸湿率降幅最大,达到96.88%,且包覆物质量分数小于5%,包覆材料在赤磷表面的包覆状况良好.相比于未包覆赤磷,包覆后赤磷的外推起始温度、峰值温度和外推终点温度分别提高42.8 ℃、51.7 ℃和48.4 ℃;自发火温度提高58.8 ℃,表明包覆赤磷的热安定性大幅提高.%In order to reduce the hygroscopicity of red phosphorus and improve its thermostability,the aluminium hydroxide/melamine-formaldehyde resin microcapsulated red phosphorus(MRP)is successfully prepared.The process of preparing the coated red phosphorus is optimized by the single factor test.The coating layer of the red phosphorus is characterized by the Fourier transform infrared(FTIR)spectrometer and the scanning electron microscopy(SEM).The thermostability of the red phosphorus(RP)and the MRP is investigated by the thermogravimetric analysis-differential thermal analysis(TG-DTA)in air atmosphere.Results show that the optimal process parameters for preparing the coated red phosphorus are,the mass of the aluminum nitrate nonahydrate 0.10 g,the total mass of the formaldehyde and melamine 0.40 g,the molar ratio of the formaldehyde and the melamine 3∶1,the reaction temperature 80 ℃,the reaction time 1 h.Under these optimal process parameters,the moisture absorption ratio of MRP reduces 96.88%(the greatest reduction),the mass fraction of coating materials is below 5

  13. Enhancement of the thermostability of Hydrogenobacter thermophilus cytochrome c(552) through introduction of an extra methylene group into its hydrophobic protein interior.

    Science.gov (United States)

    Tai, Hulin; Irie, Kiyofumi; Mikami, Shin-ichi; Yamamoto, Yasuhiko

    2011-04-19

    Careful scrutiny of the protein interior of Hydrogenobacter thermophilus cytochrome c(552) (HT) on the basis of its X-ray structure [Travaglini-Allocatelli, C., Gianni, S., Dubey, V. K., Borgia, A., Di Matteo, A., Bonivento, D., Cutruzzola, F., Bren, K. L., and Brunori, M. (2005) J. Biol. Chem. 280, 25729-25734] indicated that a void space, which is large enough to accommodate a methyl group, exists in the hydrophobic protein interior near the heme. We tried to reduce the void space through the replacement of a Val by Ile or Leu (Val/Ile or Val/Leu mutation), and then the structural and functional consequences of these two mutations were characterized in order to elucidate the relationship between the nature of the packing of hydrophobic residues and the functional properties of the protein. The study demonstrated striking differences in the structural and functional consequences between the two mutations. The Val/Ile mutation was found to cause further enhancement of the thermostability of the oxidized HT, as reflected in the increase of the denaturation temperature (T(m)) value by ∼ 3 deg, whereas the thermostability of the reduced form was essentially unaffected. As a result, the redox potential (E(m)) of the Val/Ile mutant exhibited a negative shift of ∼ 50 mV relative to that of the wild-type protein in an enthalpic manner, this being consistent with our previous finding that a protein with higher stability in its oxidized form exhibits a lower E(m) value [Terui, N., Tachiiri, N., Matsuo, H., Hasegawa, J., Uchiyama, S., Kobayashi, Y., Igarashi, Y., Sambongi, Y., and Yamamoto, Y. (2003) J. Am. Chem. Soc. 125, 13650-13651]. In contrast, the Val/Leu mutation led to a decrease in thermostability of both the redox forms of the protein, as reflected in the decreases of the T(m) values of the oxidized and reduced proteins by ∼ 3 and ∼ 5 deg, respectively, and the E(m) value of the Val/Leu mutant happened to be similar to that of the Val/Ile one. The E

  14. Cloning, Expression and Characterization of a Thermostable Esterase HydS14 from Actinomadura sp. Strain S14 in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Pichapak Sriyapai

    2015-06-01

    Full Text Available A thermostable esterase gene (hydS14 was cloned from an Actinomadura sp. S14 gene library. The gene is 777 bp in length and encodes a polypeptide of 258 amino acid residues with no signal peptide, no N-glycosylation site and a predicted molecular mass of 26,604 Da. The encoded protein contains the pentapeptide motif (GYSLG and catalytic triad (Ser88-Asp208-His235 of the esterase/lipase superfamily. The HydS14 sequence shows 46%–64% identity to 23 sequences from actinomycetes (23 α/β-hydrolases, has three conserved regions, and contains the novel motif (GY(FSLG, which distinguishes it from other clusters in the α/β-hydrolase structural superfamily. A plasmid containing the coding region (pPICZαA-hydS14 was used to express HydS14 in Pichia pastoris under the control of the AOXI promoter. The recombinant HydS14 collected from the supernatant had a molecular mass of ~30 kDa, which agrees with its predicted molecular mass without N-glycosylation. HydS14 had an optimum temperature of approximately 70 °C and an optimum pH of 8.0. HydS14 was stable at 50 and 60 °C for 120 min, with residual activities of above 80% and above 90%, respectively, as well as 50% activity at pH 6.0–8.0 and pH 9.0, respectively. The enzyme showed higher activity with p-nitrophenyl-C2 and C4. The Km and Vmax values for p-nitrophenyl-C4 were 0.21 ± 0.02 mM and 37.07 ± 1.04 μmol/min/mg, respectively. The enzyme was active toward short-chain p-nitrophenyl ester (C2–C6, displaying optimal activity with p-nitrophenyl-C4 (Kcat/Km = 11.74 mM−1·S−1. In summary, HydS14 is a thermostable esterase from Actinomadura sp. S14 that has been cloned and expressed for the first time in Pichia pastoris.

  15. A feasible enzymatic process for D-tagatose production by an immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor.

    Science.gov (United States)

    Kim, Hye-Jung; Ryu, Se-Ah; Kim, Pil; Oh, Deok-Kun

    2003-01-01

    To develop a feasible enzymatic process for d-tagatose production, a thermostable l-arabinose isomerase, Gali152, was immobilized in alginate, and the galactose isomerization reaction conditions were optimized. The pH and temperature for the maximal galactose isomerization reaction were pH 8.0 and 65 degrees C in the immobilized enzyme system and pH 7.5 and 60 degrees C in the free enzyme system. The presence of manganese ion enhanced galactose isomerization to tagatose in both the free and immobilized enzyme systems. The immobilized enzyme was more stable than the free enzyme at the same pH and temperature. Under stable conditions of pH 8.0 and 60 degrees C, the immobilized enzyme produced 58 g/L of tagatose from 100 g/L galactose in 90 h by batch reaction, whereas the free enzyme produced 37 g/L tagatose due to its lower stability. A packed-bed bioreactor with immobilized Gali152 in alginate beads produced 50 g/L tagatose from 100 g/L galactose in 168 h, with a productivity of 13.3 (g of tagatose)/(L-reactor.h) in continuous mode. The bioreactor produced 230 g/L tagatose from 500 g/L galactose in continuous recycling mode, with a productivity of 9.6 g/(L.h) and a conversion yield of 46%.

  16. Protein Thermostability Is Owing to Their Preferences to Non-Polar Smaller Volume Amino Acids, Variations in Residual Physico-Chemical Properties and More Salt-Bridges.

    Science.gov (United States)

    Panja, Anindya Sundar; Bandopadhyay, Bidyut; Maiti, Smarajit

    2015-01-01

    Protein thermostability is an important field for its evolutionary perspective of mesophilic versus thermophilic relationship and for its industrial/ therapeutic applications. Presently, a total 400 (200 thermophilic and 200 mesophilic homologue) proteins were studied utilizing several software/databases to evaluate their amino acid preferences. Randomly selected 50 homologous proteins with available PDB-structure of each group were explored for the understanding of the protein charges, isoelectric-points, hydrophilicity, hydrophobicity, tyrosine phosphorylation and salt-bridge occurrences. These 100 proteins were further probed to generate Ramachandran plot/data for the gross secondary structure prediction in and comparison between the thermophilic and mesophilic proteins. Present results strongly suggest that nonpolar smaller volume amino acids Ala (χ2 = 238.54, psalt bridges in this study. The average percentage of salt-bridge of thermophiles is found to be higher by 20% than their mesophilic homologue. The GLU-HIS and GLU-LYS salt-bridge dyads are calculated to be significantly higher (psalt-bridges and smaller volume nonpolar residues (Gly, Ala and Val) and lesser occurrence of bulky polar residues in the thermophilic proteins. A more stoichiometric relationship amongst these factors minimized the hindrance due to side chain burial and increased compactness and secondary structural stability in thermophilic proteins.

  17. Characterization of an organic solvent-tolerant thermostable glucoamylase from a halophilic isolate, Halolactibacillus sp. SK71 and its application in raw starch hydrolysis for bioethanol production.

    Science.gov (United States)

    Yu, Hui-Ying; Li, Xin

    2014-01-01

    A halophilic bacterium Halolactibacillus sp. SK71 producing extracellular glucoamylase was isolated from saline soil of Yuncheng Salt Lake, China. Enzyme production was strongly influenced by the salinity of growth medium with maximum in the presence of 5% NaCl. The glucoamylase was purified to homogeneity with a molecular mass of 78.5 kDa. It showed broad substrate specificity and raw starch hydrolyzing activity. Analysis of hydrolysis products from soluble starch by thin-layer chromatography revealed that glucose was the sole end-product, indicating the enzyme was a true glucoamylase. Optimal enzyme activity was found to be at 70°C, pH 8.0, and 7.5% NaCl. In addition, it was highly active and stable over broad ranges of temperature (0-100°C), pH (7.0-12.0), and NaCl concentration (0-20%), showing excellent thermostable, alkali stable, and halotolerant properties. Furthermore, it displayed high stability in the presence of hydrophobic organic solvents. The purified glucoamylase was applied for raw corn starch hydrolysis and subsequent bioethanol production using Saccharomyces cerevisiae. The yield in terms of grams of ethanol produced per gram of sugar consumed was 0.365 g/g, with 71.6% of theoretical yield from raw corn starch. This study demonstrated the feasibility of using enzymes from halophiles for further application in bioenergy production. © 2014 American Institute of Chemical Engineers.

  18. Development of a D-amino acids electrochemical sensor based on immobilization of thermostable D-Proline dehydrogenase within agar gel membrane

    International Nuclear Information System (INIS)

    Tani, Yuji; Tanaka, Katsuhito; Yabutani, Tomoki; Mishima, Yuji; Sakuraba, Haruhiko; Ohshima, Toshihisa; Motonaka, Junko

    2008-01-01

    A novel biosensor for determination of D-amino acids (DAAs) in biological samples by using an electrode based on immobilization of a thermostable D-Proline dehydrogenase (D-Pro DH) within an agar gel membrane was developed. The electrode was simply prepared by spin-coating the agar solution with the D-Pro DH on a glassy carbon (GC) electrode. An electrocatalytic oxidation current of 2,6-dichloroindophenol (DCIP) was observed at -100 mV vs. Ag/AgCl with the addition of 5 and 20 mmol L -1 D-proline. The current response and its relative standard deviation were 0.15 μA and 7.6% (n = 3), respectively, when it was measured in a pH 8.0 phosphate buffer solution containing 10 mmol L -1 D-proline and 0.5 mmol L -1 DCIP at 50 deg. C. The current response of D-proline increased with increase of the temperature of the sample solution up to 70 deg. C. The electrocatalytic response at the D-Pro DH/agar immobilized electrode subsequently maintained for 80 days. Finally, the D-Pro DH/agar immobilized electrode was applied to determination of DAAs in a human urine sample. The determined value of DAAs in the human urine and its R.S.D. were 1.39 ± 0.12 mmol L -1 and 8.9% (n = 3), respectively

  19. Engineering of Yersinia Phytases to Improve Pepsin and Trypsin Resistance and Thermostability and Application Potential in the Food and Feed Industry.

    Science.gov (United States)

    Niu, Canfang; Yang, Peilong; Luo, Huiying; Huang, Huoqing; Wang, Yaru; Yao, Bin

    2017-08-30

    Susceptibility to proteases usually limits the application of phytase. We sought to improve the pepsin and trypsin resistance of YeAPPA from Yersinia enterocolitica and YkAPPA from Y. kristensenii by optimizing amino acid polarity and charge. The predicted pepsin/trypsin cleavage sites F89/K226 in pepsin/trypsin-sensitive YeAPPA and the corresponding sites (F89/E226) in pepsin-sensitive but trypsin-resistant YkAPPA were substituted with S and H, respectively. Six variants were produced in Pichia pastoris for catalytic and biochemical characterization. F89S, E226H, and F89S/E226H elevated pepsin resistance and thermostability and K226H and F89S/K226H improved pepsin and trypsin resistance and stability at 60 °C and low pH. All the variants increased the ability of the proteins to hydrolyze phytate in corn meal by 2.6-14.9-fold in the presence of pepsin at 37 °C and low pH. This study developed a genetic manipulation strategy specific for pepsin/trypsin-sensitive phytases that can improve enzyme tolerance against proteases and heat and benefit the food and feed industry in a cost-effective way.

  20. Identification of a recombinant inulin fructotransferase (difructose dianhydride III forming) from Arthrobacter sp. 161MFSha2.1 with high specific activity and remarkable thermostability.

    Science.gov (United States)

    Wang, Xiao; Yu, Shuhuai; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

    2015-04-08

    Difructose dianhydride III (DFA III) is a functional carbohydrate produced from inulin by inulin fructotransferase (IFTase, EC 4.2.2.18). In this work, an IFTase gene from Arthrobacter sp. 161MFSha2.1 was cloned and expressed in Escherachia coli. The recombinant enzyme was purified by metal affinity chromatography. It showed significant inulin hydrolysis activity, and the produced main product from inulin was determined as DFA III by nuclear magnetic resonance analysis. The molecular mass of the purified protein was calculated to be 43 and 125 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively, suggesting the native enzyme might be a homotrimer. The recombinant enzyme showed maximal activity as 2391 units/mg at pH 6.5 and 55 °C. It displayed the highest thermostability among previously reported IFTases (DFA III forming) and was stable up to 80 °C for 4 h of incubation. The smallest substrate was determined as nystose. The conversion ratio of inulin to DFA III reached 81% when 100 g/L inulin was catalyzed by 80 nM recombinant enzyme for 20 min at pH 6.5 and 55 °C. All of these data indicated that the IFTase (DFA III forming) from Arthrobacter sp. 161MFSha2.1 had great potential for industrial DFA III production.

  1. Bioconversion of D-galactose to D-tagatose: continuous packed bed reaction with an immobilized thermostable L-arabinose isomerase and efficient purification by selective microbial degradation.

    Science.gov (United States)

    Liang, Min; Chen, Min; Liu, Xinying; Zhai, Yafei; Liu, Xian-wei; Zhang, Houcheng; Xiao, Min; Wang, Peng

    2012-02-01

    The continuous enzymatic conversion of D-galactose to D-tagatose with an immobilized thermostable L-arabinose isomerase in packed-bed reactor and a novel method for D-tagatose purification were studied. L-arabinose isomerase from Thermoanaerobacter mathranii (TMAI) was recombinantly overexpressed and immobilized in calcium alginate. The effects of pH and temperature on D-tagatose production reaction catalyzed by free and immobilized TMAI were investigated. The optimal condition for free enzyme was pH 8.0, 60°C, 5 mM MnCl(2). However, that for immobilized enzyme was pH 7.5, 75°C, 5 mM MnCl(2). In addition, the catalytic activity of immobilized enzyme at high temperature and low pH was significantly improved compared with free enzyme. The optimum reaction yield with immobilized TMAI increased by four percentage points to 43.9% compared with that of free TMAI. The highest productivity of 10 g/L h was achieved with the yield of 23.3%. Continuous production was performed at 70°C; after 168 h, the reaction yield was still above 30%. The resultant syrup was then incubated with Saccharomyces cerevisiae L1 cells. The selective degradation of D-galactose was achieved, obtaining D-tagatose with the purity above 95%. The established production and separation methods further potentiate the industrial production of D-tagatose via bioconversion and biopurification processes.

  2. Crystallization and preliminary X-ray crystallographic analysis of l-rhamnose isomerase with a novel high thermostability from Bacillus halodurans

    International Nuclear Information System (INIS)

    Doan, Thi-Ngoc-Thanh; Prabhu, Ponnandy; Kim, Jin-Kwang; Ahn, Yeh-Jin; Natarajan, Sampath; Kang, Lin-Woo; Park, Geon Tae; Lim, Sang-Boem; Lee, Jung-Kul

    2010-01-01

    l-Rhamnose isomerase (l-RhI) from B. halodurans has been purified and crystallized. The crystals of l-RhI belonged to the monoclinic space group P2 1 , with unit-cell parameters a = 83.2, b = 164.9, c = 92.0 Å, β = 116.0°, and diffracted to 2.5 Å resolution. l-Rhamnose isomerases catalyze isomerization between l-rhamnose (6-deoxy-l-mannose) and l-rhamnulose (6-deoxy-l-fructose), which is the first step in rhamnose catabolism. l-Rhamnose isomerase from Bacillus halodurans ATCC BAA-125 (BHRI) exhibits interesting characteristics such as high thermostability and selective substrate specificity. BHRI fused with an HHHHHH sequence was purified and crystallized in order to elucidate the molecular basis of its unique enzymatic properties. The crystals were grown by the hanging-drop vapour-diffusion method and belonged to the monoclinic space group P2 1 , with unit-cell parameters a = 83.2, b = 164.9, c = 92.0 Å, β = 116.0°. Diffraction data were collected to 2.5 Å resolution. According to a Matthews coefficient calculation, there are four monomers in the asymmetric unit with a V M of 3.0 Å 3 Da −1 and a solvent content of 59.3%. The initial structure of BHRI has been determined by the molecular-replacement method

  3. Purification and Characterization of a New Thermostable, Haloalkaline, Solvent Stable, and Detergent Compatible Serine Protease from Geobacillus toebii Strain LBT 77

    Directory of Open Access Journals (Sweden)

    Wajdi Thebti

    2016-01-01

    Full Text Available A new thermostable, haloalkaline, solvent stable SDS-induced serine protease was purified and characterized from a thermophilic Geobacillus toebii LBT 77 newly isolated from a Tunisian hot spring. This study reveals the potential of the protease from Geobacillus toebii LBT 77 as an additive to detergent with spectacular proprieties described for the first time. The protease was purified to homogeneity by ammonium sulfate precipitation followed by Sephadex G-75 and DEAE-Cellulose chromatography. It was a monomeric enzyme with molecular weight of 30 kDa. The optimum pH, temperature, and NaCl for maximum protease activity were 13.0, 95°C, and 30%, respectively. Activity was stimulated by Ca2+, Mg2+, DTNB, β-mercaptoethanol, and SDS. The protease was extremely stable even at pH 13.25, 90°C, and 30% NaCl and in the presence of hydrophilic, hydrophobic solvents at high concentrations. The high compatibility with ionic, nonionic, and commercial detergents confirms the utility as an additive to cleaning products. Kinetic and thermodynamic characterization of protease revealed Km=1 mg mL−1,  Vmax=217.5 U mL−1, Kcat/Km=99 mg mL−1 S−1, Ea=51.5 kJ mol−1, and ΔG⁎=56.5 kJ mol−1.

  4. Molecular Dynamic Simulation of Space and Earth-Grown Crystal Structures of Thermostable T1 Lipase Geobacillus zalihae Revealed a Better Structure.

    Science.gov (United States)

    Ishak, Siti Nor Hasmah; Aris, Sayangku Nor Ariati Mohamad; Halim, Khairul Bariyyah Abd; Ali, Mohd Shukuri Mohamad; Leow, Thean Chor; Kamarudin, Nor Hafizah Ahmad; Masomian, Malihe; Rahman, Raja Noor Zaliha Raja Abd

    2017-09-25

    Less sedimentation and convection in a microgravity environment has become a well-suited condition for growing high quality protein crystals. Thermostable T1 lipase derived from bacterium Geobacillus zalihae has been crystallized using the counter diffusion method under space and earth conditions. Preliminary study using YASARA molecular modeling structure program for both structures showed differences in number of hydrogen bond, ionic interaction, and conformation. The space-grown crystal structure contains more hydrogen bonds as compared with the earth-grown crystal structure. A molecular dynamics simulation study was used to provide insight on the fluctuations and conformational changes of both T1 lipase structures. The analysis of root mean square deviation (RMSD), radius of gyration, and root mean square fluctuation (RMSF) showed that space-grown structure is more stable than the earth-grown structure. Space-structure also showed more hydrogen bonds and ion interactions compared to the earth-grown structure. Further analysis also revealed that the space-grown structure has long-lived interactions, hence it is considered as the more stable structure. This study provides the conformational dynamics of T1 lipase crystal structure grown in space and earth condition.

  5. thermo-stable alpha-amylase(S) from irradiated microbial origin utilizing agricultural and environmental wastes under solid state fermentation conditions

    International Nuclear Information System (INIS)

    Moustafa, O.E.A.

    2002-01-01

    an investigation concerning the production of thermo-stable α-amylases by thermophilic bacterial and fungal isolates has been undertaken. nine thermophilic bacteria and five teen fungi were isolated from different localities viz. phyllosphere of water hyacinth, different desert plants leaves, fermented dough, oven dust, garbage , and soil. their amylolytic activities were tested by dinitrosalicylic acid color reagent (Dns) method when grown on some environmental pollutants (garbage and water hyacinth) as well as industrial wastes (Bagasse, biscuit, corn flex and dough residues ) as the sole carbon source at 65 o C for bacterial and at 50 o C for fungal isolates . isolates No. B 1 ,B 2 ,B 5 ,B 6 ,B 7 ,B 8 ,B 9 , and F 4 ,F 6 ,F 8 ,F 1 2,F 1 3 and F 1 5, exhibited the highest α -amylase production when grown on water hyacinth, while B 4 ,F 3 ,F 1 1 and F 1 3, on dough ; (B 3 ,F 9 and F 1 0 ) on bagasse and ( F 1 ,F 2 ,F 5 ,F 7 ,F 1 1 and F 1 4) on garbage. Out of the nine identified bacterial isolated, only two isolates viz; actinobacillus actinomycetemcomitans, B1 and strepto bacillus moniliformis, B 7 , exhibited the ability to produce high percentages of α amylases at 55 o C (while still able to produce the enzyme within 45-70 o C)

  6. Purification and biochemical characterization of a novel thermostable serine alkaline protease from Aeribacillus pallidus C10: a potential additive for detergents.

    Science.gov (United States)

    Yildirim, Vildan; Baltaci, Mustafa Ozkan; Ozgencli, Ilknur; Sisecioglu, Melda; Adiguzel, Ahmet; Adiguzel, Gulsah

    2017-12-01

    An extracellular thermostable alkaline serine protease enzyme from Aeribacillus pallidus C10 (GenBank No: KC333049), was purified 4.85 and 17. 32-fold with a yield of 26.9 and 19.56%, respectively, through DE52 anion exchange and Probond affinity chromatography. The molecular mass of the enzyme was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with approximately 38.35 kDa. The enzyme exhibited optimum activity at pH 9 and at temperature 60 °C. It was determined that the enzyme had remained stable at the range of pH 7.0-10.0, and that it had preserved more than 80% of its activity at a broad temperature range (20-80 °C). The enzyme activity was found to retain more than 70% and 55% in the presence of organic solvents and commercial detergents, respectively. In addition, it was observed that the enzyme activity had increased in the presence of 5% SDS. K M and V max values were calculated as 0.197 mg/mL and 7.29 μmol.mL - 1 .min - 1 , respectively.

  7. Characterisation of the thermostable protease AprX in strains of Pseudomonas fluorescens and impact on the shelf-life of dairy products: preliminary results

    Directory of Open Access Journals (Sweden)

    Nadia Andrea Andreani

    2016-12-01

    Full Text Available Bacterial proteases are involved in food spoilage and shelf-life reduction. Among the bacterial proteases, a predominant role in spoilage of dairy products seems to be played by the thermostable metallo-protease AprX, which is produced by various strains of Pseudomonas fluorescens. Differences in AprX enzyme activity among different strains were highlighted, but the most proteolytic strains were not identified. In this study, the presence of the aprX gene was evaluated in 69 strains isolated from food matrices and 18 reference strains belonging to the P. fluorescens group, which had been previously typed by the multi locus sequence typing method. Subsequently, a subset of reference strains was inoculated in ultra-high temperature milk, and the expression of the aprX gene was evaluated at 22 and 6°C. On the same milk samples, the proteolytic activity was then evaluated through Azocasein and trinitrobenzenesulfonic acid solution assays. Finally, to assess the applicability of the former assay directly on dairy products the proteolityc activity was tested on industrial ricotta samples using the Azocasein assay. These results demonstrate the spread of aprX gene in most strains tested and the applicability of Azocasein assay to monitor the proteolytic activity in dairy products.

  8. A metagenome-derived thermostable β-glucanase with an unusual module architecture which defines the new glycoside hydrolase family GH148.

    Science.gov (United States)

    Angelov, Angel; Pham, Vu Thuy Trang; Übelacker, Maria; Brady, Silja; Leis, Benedikt; Pill, Nicole; Brolle, Judith; Mechelke, Matthias; Moerch, Matthias; Henrissat, Bernard; Liebl, Wolfgang

    2017-12-11

    The discovery of novel and robust enzymes for the breakdown of plant biomass bears tremendous potential for the development of sustainable production processes in the rapidly evolving new bioeconomy. By functional screening of a metagenomic library from a volcano soil sample a novel thermostable endo-β-glucanase (EngU) which is unusual with regard to its module architecture and cleavage specificity was identified. Various recombinant EngU variants were characterized. Assignment of EngU to an existing glycoside hydrolase (GH) family was not possible. Two regions of EngU showed weak sequence similarity to proteins of the GH clan GH-A, and acidic residues crucial for catalytic activity of EngU were identified by mutation. Unusual, a carbohydrate-binding module (CBM4) which displayed binding affinity for β-glucan, lichenin and carboxymethyl-cellulose was found as an insertion between these two regions. EngU hydrolyzed β-1,4 linkages in carboxymethyl-cellulose, but displayed its highest activity with mixed linkage (β-1,3-/β-1,4-) glucans such as barley β-glucan and lichenin, where in contrast to characterized lichenases cleavage occurred predominantly at the β-1,3 linkages of C4-substituted glucose residues. EngU and numerous related enzymes with previously unknown function represent a new GH family of biomass-degrading enzymes within the GH-A clan. The name assigned to the new GH family is GH148.

  9. Evaluating the effects of buffer conditions and extremolytes on thermostability of granulocyte colony-stimulating factor using high-throughput screening combined with design of experiments.

    Science.gov (United States)

    Ablinger, Elisabeth; Hellweger, Monika; Leitgeb, Stefan; Zimmer, Andreas

    2012-10-15

    In this study, we combined a high-throughput screening method, differential scanning fluorimetry (DSF), with design of experiments (DoE) methodology to evaluate the effects of several formulation components on the thermostability of granulocyte colony stimulating factor (G-CSF). First we performed a primary buffer screening where we tested thermal stability of G-CSF in different buffers, pH values and buffer concentrations. The significance of each factor and the two-way interactions between them were studied by multivariable regression analysis. pH was identified as most critical factor regarding thermal stability. The most stabilizing buffer, sodium glutamate, and sodium acetate were determined for further investigations. Second we tested the effect of 6 naturally occurring extremolytes (trehalose, sucrose, ectoine, hydroxyectoine, sorbitol, mannitol) on the thermal stability of G-CSF, using a central composite circumscribed design. At low pH (3.8) and low buffer concentration (5 mM) all extremolytes led to a significant increase in thermal stability except the addition of ectoine which resulted in a strong destabilization of G-CSF. Increasing pH and buffer concentration led to an increase in thermal stability with all investigated extremolytes. The described systematic approach allowed to create a ranking of stabilizing extremolytes at different buffer conditions. Copyright © 2012. Published by Elsevier B.V.

  10. A Thermophilic Phage Endolysin Fusion to a Clostridium perfringens-Specific Cell Wall Binding Domain Creates an Anti-Clostridium Antimicrobial with Improved Thermostability

    Directory of Open Access Journals (Sweden)

    Steven M. Swift

    2015-06-01

    Full Text Available Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Many enzymes are added to animal feed during production and are subjected to high-heat stress during feed processing. To produce a thermostabile endolysin for treating poultry, an E. coli codon-optimized gene was synthesized that fused the N-acetylmuramoyl-L-alanine amidase domain from the endolysin of the thermophilic bacteriophage ɸGVE2 to the cell-wall binding domain (CWB from the endolysin of the C. perfringens-specific bacteriophage ɸCP26F. The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures. PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl. The protein was able to withstand temperatures up to 50° C and still lyse C. perfringens. Herein, we report the construction and characterization of a thermostable chimeric endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production.

  11. Effect of enzymatic (thermostable α-amylase) treatment on the physicochemical and antioxidant properties of extruded rice incorporated with soybean flour.

    Science.gov (United States)

    Xu, Enbo; Wu, Zhengzong; Pan, Xiaowei; Long, Jie; Wang, Fang; Xu, Xueming; Jin, Zhengyu; Jiao, Aiquan

    2016-04-15

    In order to determine the effect of enzymatic extrusion on the physicochemical and antioxidant properties of rice/soybean mixture, different mass ratios (100/0, 95/5, 85/15, 70/30, 50/50 and 25/75%, w/w) were treated with thermostable α-amylase. The reduced special mechanical energy and the enhanced product temperature were closely and regularly linked with the increase of soybean content. The bulk density and water solubility index increased, and the water absorption index and viscosities decreased remarkably after enzymatic extrusion, however, the modification caused by α-amylase were dramatically eliminated with the increase of soybean content to ∼50%. Moreover, the addition of enzyme exhibited an improvement of the total phenolic/flavonoid content (TPC/TFC) and antioxidant capacities compared to traditional extrusion. The TPC/TFC retention of extrudate (ratios of 85/15 and 70/30%) attained over 90%, but dramatically decreased (72.91 and 67.81%, respectively) with soybean added to 75%, probably due to the great reduction of starch substrate for enzymatic hydrolysis. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  12. Enhanced production of alkaline thermostable keratinolytic protease from calcium alginate immobilized cells of thermoalkalophilic Bacillus halodurans JB 99 exhibiting dehairing activity.

    Science.gov (United States)

    Shrinivas, Dengeti; Kumar, Raghwendra; Naik, G R

    2012-01-01

    The thermoalkalophilic Bacillus halodurans JB 99 cells known for production of novel thermostable alkaline keratinolytic protease were immobilized in calcium alginate matrix. Batch and repeated batch cultivation using calcium alginate immobilized cells were studied for alkaline protease production in submerged fermentation. Immobilized cells with 2.5% alginate and 350 beads/flask of initial cell loading showed enhanced production of alkaline protease by 23.2% (5,275 ± 39.4 U/ml) as compared to free cells (4,280 ± 35.4 U/ml) after 24 h. In the semicontinuous mode of cultivation, immobilized cells under optimized conditions produced an appreciable level of alkaline protease in up to nine cycles and reached a maximal value of 5,975 U/ml after the seventh cycle. The enzyme produced from immobilized cells efficiently degraded chicken feathers in the presence of a reducing agent which can help the poultry industry in the management of keratin-rich waste and obtaining value-added products.

  13. A highly thermostable alkaline cellulase-free xylanase from thermoalkalophilic Bacillus sp. JB 99 suitable for paper and pulp industry: purification and characterization.

    Science.gov (United States)

    Shrinivas, Dengeti; Savitha, Gunashekaran; Raviranjan, Kumar; Naik, Gajanan Ramchandra

    2010-11-01

    A highly thermostable alkaline xylanase was purified to homogeneity from culture supernatant of Bacillus sp. JB 99 using DEAE-Sepharose and Sephadex G-100 gel filtration with 25.7-fold increase in activity and 43.5% recovery. The molecular weight of the purified xylanase was found to be 20 kDA by SDS-PAGE and zymogram analysis. The enzyme was optimally active at 70 °C, pH 8.0 and stable over pH range of 6.0-10.0.The relative activity at 9.0 and 10.0 were 90% and 85% of that of pH 8.0, respectively. The enzyme showed high thermal stability at 60 °C with 95% of its activity after 5 h. The K (m) and V (max) of enzyme for oat spelt xylan were 4.8 mg/ml and 218.6 µM min(-1) mg(-1), respectively. Analysis of N-terminal amino acid sequence revealed that the xylanase belongs to glycosyl hydrolase family 11 from thermoalkalophilic Bacillus sp. with basic pI. Substrate specificity showed a high activity on xylan-containing substrate and cellulase-free nature. The hydrolyzed product pattern of oat spelt xylan on thin-layer chromatography suggested xylanase as an endoxylanase. Due to these properties, xylanase from Bacillus sp. JB 99 was found to be highly compatible for paper and pulp industry.

  14. An organic solvent-, detergent-, and thermo-stable alkaline protease from the mesophilic, organic solvent-tolerant Bacillus licheniformis 3C5.

    Science.gov (United States)

    Rachadech, W; Navacharoen, A; Ruangsit, W; Pongtharangkul, T; Vangnai, A S

    2010-01-01

    Bacillus licheniformis 3C5, isolated as mesophilic bacterium, exhibited tolerance towards a wide range of non-polar and polar organic solvents at 45 degrees C. It produced an extracellular organic solvent-stable protease with an apparent molecular mass of approximately 32 kDa. The inhibitory effect of PMSF and EDTA suggested it is likely to be an alkaline serine protease. The protease was active over abroad range of temperatures (45-70 degrees C) and pH (8-10) range with an optimum activity at pH 10 and 65 degrees C. It was comparatively stable in the presence ofa relatively high concentration (35% (v/v)) of organic solvents and various types of detergents even at a relatively high temperature (45 degrees C). The protease production by B. licheniformis 3C5 was growth-dependent. The optimization of carbon and nitrogen sources for cell growth and protease production revealed that yeast extract was an important medium component to support both cell growth and the protease production. The overall properties of the protease produced by B. licheniformis 3C5 suggested that this thermo-stable, solvent-stable, detergent-stable alkaline protease is a promising potential biocatalyst for industrial and environmental applications.

  15. Lactate dehydrogenase of Mugil sp. (Mugilidae, Perciformes. Lack of electrokinetic, thermostability and kinetic differences among individuals with different number of scales

    Directory of Open Access Journals (Sweden)

    Marcelo dos Santos

    2000-03-01

    Full Text Available The scale number in lateral sets (SNS of Mugil sp. (Mugilidae, Perciformes collected in the lagoon-estuarine region of Cananéia, State of São Paulo ranges from 33 to 39. Electrokinetic, kinetic and thermostability properties of lactate dehydrogenase (LDH were tested to determine if individuals with different SNS correspond to different species or populations of mullet. As in many other teleosts, LDH-A*, LDH-B*, and LDH-C* loci were detected. Through a two-fold serial dilution method applied to 10 different tissues of Mugil sp., a bidirectionally divergent expression of these loci was suggested. No association among LDH electrophoretic pattern, thermal inactivation, kinetic responses and different SNS was observed. The apparent Km (pyr values obtained here were similar to Km values obtained by other authors for muscle and heart LDH or their purified isoforms. The effect of NaCl on Km and Vmax values of Mugil sp. (35 and 39 SNS individuals indicates that this salt behaves as a competitive inhibitor, since it decreases enzyme-substrate affinity. Thus, electrokinetic and thermostability behavior, Km and Vmax values and the effect of NaCl do not permit us to consider these mullets, with SNS ranging from 33 to 39, as belonging to different populations or species.O número de escamas em séries laterais (SNS de exemplares de Mugil sp. (Mugilidae, Perciformes coletados na região estuarino-lagunar de Cananéia, Estado de São Paulo, varia de 33 a 39. A fim de tentar determinar se exemplares com diferentes SNS corresponderiam a diferentes espécies ou populações de tainhas, foram analisadas as propriedades eletrocinéticas, cinéticas e de termoestabilidade da sua lactato desidrogenase (LDH. A exemplo de muitos teleósteos, a LDH de Mugil sp. mostrou-se codificada por 3 locos gênicos: LDH-A*, LDH-B* e LDH-C*. Método de diluições seriadas aplicado a 10 diferentes tecidos dessa espécie sugeriu um padrão bidirecionalmente divergente de express

  16. Establishment and application of a modified membrane-blot assay for Rhizomucor miehei lipases aimed at improving their methanol tolerance and thermostability.

    Science.gov (United States)

    He, Dong; Luo, Wen; Wang, Zhiyuan; Lv, Pengmei; Yuan, Zhenhong; Huang, Shaowei; Xv, Jingliang

    2017-07-01

    Directed evolution has been proved an effective way to improve the stability of proteins, but high throughput screening assays for directed evolution with simultaneous improvement of two or more properties are still rare. In this study, we aimed to establish a membrane-blot assay for use in the high-throughput screening of Rhizomucor miehei lipases (RMLs). With the assistance of the membrane-blot screening assay, a mutant E47K named G10 that showed improved thermal stability was detected in the first round of error-prone PCR. Using G10 as the parent, two variants G10-11 and G10-20 that showed improved thermal stability and methanol tolerance without loss of activity compared to the wild type RML were obtained. The T 50 60 -value of G10-11 and G10-20 increased by 12°C and 6.5°C, respectively. After incubation for 1h, the remaining residual activity of G10-11 and G10-20 was 63.45% and 74.33%, respectively, in 50% methanol, and 15.98% and 30.22%, respectively, in 80% methanol. Thus, we successfully developed a membrane-blot assay that could be used for the high-throughput screening of RMLs with improved thermostability and methanol tolerance. Based on our findings, we believe that our newly developed membrane-blot assay will have potential applications in directed evolution in the future. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Display of a thermostable lipase on the surface of a solvent-resistant bacterium, Pseudomonas putida GM730, and its applications in whole-cell biocatalysis

    Directory of Open Access Journals (Sweden)

    Kwon Seok-Joon

    2006-04-01

    Full Text Available Abstract Background Whole-cell biocatalysis in organic solvents has been widely applied to industrial bioprocesses. In two-phase water-solvent processes, substrate conversion yields and volumetric productivities can be limited by the toxicity of solvents to host cells and by the low mass transfer rates of the substrates from the solvent phase to the whole-cell biocatalysts in water. Results To solve the problem of solvent toxicity, we immobilized a thermostable lipase (TliA from Pseudomonas fluorescens on the cell surface of a solvent-resistant bacterium, Pseudomonas putida GM730. Surface immobilization of enzymes eliminates the mass-transfer limitation imposed by the cell wall and membranes. TliA was successfully immobilized on the surface of P. putida cells using the ice-nucleation protein (INP anchoring motif from Pseudomonas syrinage. The surface location was confirmed by flow cytometry, protease accessibility and whole-cell enzyme activity using a membrane-impermeable substrate. Three hundred and fifty units of whole-cell hydrolytic activity per gram dry cell mass were obtained when the enzyme was immobilized with a shorter INP anchoring motif (INPNC. The surface-immobilized TliA retained full enzyme activity in a two-phase water-isooctane reaction system after incubation at 37°C for 12 h, while the activity of the free form enzyme decreased to 65% of its initial value. Whole cells presenting immobilized TliA were shown to catalyze three representative lipase reactions: hydrolysis of olive oil, synthesis of triacylglycerol and chiral resolution. Conclusion In vivo surface immobilization of enzymes on solvent-resistant bacteria was demonstrated, and appears to be useful for a variety of whole-cell bioconversions in the presence of organic solvents.

  18. Application of a novel alkali-tolerant thermostable DyP-type peroxidase from Saccharomonospora viridis DSM 43017 in biobleaching of eucalyptus kraft pulp.

    Directory of Open Access Journals (Sweden)

    Wangning Yu

    Full Text Available Saccharomonospora viridis is a thermophilic actinomycete that may have biotechnological applications because of its dye decolorizing activity, though the enzymatic oxidative system responsible for this activity remains elusive. Bioinformatic analysis revealed a DyP-type peroxidase gene in the genome of S. viridis DSM 43017 with sequence similarity to peroxidase from dye-decolorizing microbes. This gene, svidyp, consists of 1,215 bp encoding a polypeptide of 404 amino acids. The gene encoding SviDyP was cloned, heterologously expressed in Escherichia coli, and then purified. The recombinant protein could efficiently decolorize several triarylmethane dyes, anthraquinonic and azo dyes under neutral to alkaline conditions. The optimum pH and temperature for SviDyP was pH 7.0 and 70°C, respectively. Compared with other DyP-type peroxidases, SviDyP was more active at high temperatures, retaining>63% of its maximum activity at 50-80°C. It also showed broad pH adaptability (>35% activity at pH 4.0-9.0 and alkali-tolerance (>80% activity after incubation at pH 5-10 for 1 h at 37°C, and was highly thermostable (>60% activity after incubation at 70°C for 2 h at pH 7.0. SviDyP had an accelerated action during the biobleaching of eucalyptus kraft pulp, resulting in a 21.8% reduction in kappa number and an increase of 2.98% (ISO in brightness. These favorable properties make SviDyP peroxidase a promising enzyme for use in the pulp and paper industries.

  19. Thermostable adenylate kinase technology: a new process indicator and its use as a validation tool for the reprocessing of surgical instruments.

    Science.gov (United States)

    Hesp, J R; Poolman, T M; Budge, C; Batten, L; Alexander, F; McLuckie, G; O'Brien, S; Wells, P; Raven, N D H; Sutton, J M

    2010-02-01

    Adenylate kinase (tAK), a thermostable enzyme, was assessed as a possible means of providing a quantitative measure of cleaning efficacy suitable for validating the performance of an automated washer disinfector (AWD) during routine use. Two indicator formulations were developed using either a commercially available washer disinfector soil or a protein-based soil. Each indicator consisted of 100 microg (in test soil) of tAK dried on to a steel or plastic surface. These indicators were placed in each basket of a washer disinfector and processed alongside soiled surgical instruments during a standard day's operation. After processing, remaining tAK activity was detected using a rapid enzyme assay (2 min detection time) in a handheld hygiene monitor. The amount of tAK remaining on each indictor after a full AWD cycle was found to range from 0.1 to 0.4 ng, which represented a mean log(10) removal of 5.8+/-0.3. There was no statistical difference in the residual tAK activity between individual runs or the position of the indicator in the machine. The tAK indicator was also used to analyse the protein removal within each component of the wash cycle. These results demonstrated that all phases of the wash process contributed to the removal of the protein load, with the main wash alone being responsible for 3.6-4.0 log(10) reductions in protein activity. We propose that a quantitative cleaning index using such rapid readout indicator devices would provide a valuable addition to the methodologies for validating cleaning processes.

  20. Characterization of a Highly Thermostable and Organic Solvent-Tolerant Copper-Containing Polyphenol Oxidase with Dye-Decolorizing Ability from Kurthia huakuii LAM0618T.

    Directory of Open Access Journals (Sweden)

    Xiang Guo

    Full Text Available Laccases are green biocatalysts that possess attractive advantages for the treatment of resistant environmental pollutants and dye effluents. A putative laccase-like gene, laclK, encoding a protein of 29.3 kDa and belonging to the Cu-oxidase_4 superfamily, was cloned and overexpressed in Escherichia coli. The purified recombinant protein LaclK (LaclK was able to oxidize typical laccase substrates such as 2,6-dimethoxyphenol and l-dopamine. The characteristic adsorption maximums of typical laccases at 330 nm and 610 nm were not detected for LaclK. Cu2+ was essential for substrate oxidation, but the ratio of copper atoms/molecule of LaclK was determined to only be 1:1. Notably, the optimal temperature of LaclK was 85°C with 2,6-dimethoxyphenol as substrates, and the half-life approximately 3 days at 80°C. Furthermore, 10% (v/v organic solvents (methanol, ethanol, isopropyl alcohol, butyl alcohol, Triton x-100 or dimethyl sulfoxide could promote enzymatic activity. LaclK exhibited wide-spectrum decolorization ability towards triphenylmethane dyes, azo dyes and aromatic dyes, decolorizing 92% and 94% of Victoria Blue B (25 μM and Ethyl Violet (25 μM, respectively, at a concentration of 60 U/L after 1 h of incubation at 60°C. Overall, we characterized a novel thermostable and organic solvent-tolerant copper-containing polyphenol oxidase possessing dye-decolorizing ability. These unusual properties make LaclK an alternative for industrial applications, particularly processes that require high-temperature conditions.

  1. Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-β-mannosidase from Aspergillus niger BK01

    OpenAIRE

    Sigoillot Jean-Claude; Kim-Anh To; Haltrich Dietmar; Berrin Jean-Guy; Thi-Thu Dang; Bien-Cuong Do; Yamabhai Montarop

    2009-01-01

    Abstract Background Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-β-mannosidases (1,4-β-D-mannanases) catalyze the random hydrolysis of β-1,4-mannosidic linkages in the main chain of β-mannans. Biodegradation of β-mannans by the action of thermostable mannan endo-1,4-β-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pret...

  2. Enzymatic syntheses of some 11C-labelled analogues of L-tyrosine and L-tryptophan

    International Nuclear Information System (INIS)

    Bjurling, P.; Malmborg, P.; Langstroem, B.

    1990-01-01

    In the elucidation of biochemical processes by use of positron emission tomography (PET), the multi-tracer approach can be valuable. In previous work, the authors have been studying the dopaminergic and serotonergic neurosystems by use of 11 C-labelled L-DOPA and 5-hydroxy-L-tryptophan, respectively. They have now developed the syntheses of several analogues of tyrosine and tryptophan, labelled with 11 C in the β-position, which are of interest for use in similar applications

  3. Singlet oxygen generation during the oxidation of L-tyrosine and L-dopa with mushroom tyrosinase

    Energy Technology Data Exchange (ETDEWEB)

    Miyaji, Akimitsu [Department of Environmental Chemistry and Engineering, Tokyo Institute of Technology, 4259-G1-14, Nagatsuta-cho, Midori-ku, Yokohama 226-8502 (Japan); Kohno, Masahiro [Department of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-G1-25 Nagatsuta-cho, Midori-ku, Yokohama 226-8502 (Japan); Inoue, Yoshihiro [Showa Pharmaceutical University, 3-3165 Higashi-tamagawagakuen, Machida, Tokyo 194-8543 (Japan); Baba, Toshihide, E-mail: tbaba@chemenv.titech.ac.jp [Department of Environmental Chemistry and Engineering, Tokyo Institute of Technology, 4259-G1-14, Nagatsuta-cho, Midori-ku, Yokohama 226-8502 (Japan)

    2016-03-18

    The generation of singlet oxygen during the oxidation of tyrosine and L-dopa using mushroom tyrosinase in a phosphate buffer (pH 7.4), the model of melanin synthesis in melanocytes, was examined. The reaction was performed in the presence of 2,2,6,6-tetramethyl-4-piperidone (4-oxo-TEMP), an acceptor of singlet oxygen and the electron spin resonance (ESR) of the spin adduct, 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMPO), was measured. An increase in the ESR signal attributable to 4-oxo-TEMPO was observed during the oxidation of tyrosine and L-dopa with tyrosinase, indicating the generation of singlet oxygen. The results suggest that {sup 1}O{sub 2} generation via tyrosinase-catalyzed melanin synthesis occurs in melanocyte. - Highlights: • Generation of singlet oxygen was observed during tyrosinase-catalyzed tyrosine oxidation. • The singlet oxygen generated when tyrosine was converted into dopachrome. • The amount of singlet oxygen is not sufficient for cell toxicity. • It decreased when the hydroxyl radicals and/or superoxide anions were trapped.

  4. Increasing the Thermostable Sugar-1-Phosphate Nucleotidylyltransferase Activities of the Archaeal ST0452 Protein through Site Saturation Mutagenesis of the 97th Amino Acid Position.

    Science.gov (United States)

    Honda, Yuki; Zang, Qian; Shimizu, Yasuhiro; Dadashipour, Mohammad; Zhang, Zilian; Kawarabayasi, Yutaka

    2017-02-01

    , a thermostable enzyme from the thermophilic archaeon Sulfolobus tokodaii, exhibited increased activity following single amino acid substitutions of Ala. In this study, ST0452 proteins exhibiting a further increase in activity were created using a site saturation mutagenesis strategy at the 97th position. Kinetic analyses showed that the increased activities of the mutant proteins were principally due to increased apparent k cat values. These mutant proteins might suggest clues regarding the mechanism underlying the reaction process and provide very important information for the design of synthetic improved enzymes, and they can be used as powerful biocatalysts for the production of sugar nucleotide molecules. Moreover, this work generated useful proteins for three-dimensional structural analysis clarifying the processes underlying the regulation and mechanism of enzymatic activity. Copyright © 2017 American Society for Microbiology.

  5. Overexpression of a novel thermostable and chloride-tolerant laccase from Thermus thermophilus SG0.5JP17-16 in Pichia pastoris and its application in synthetic dye decolorization.

    Directory of Open Access Journals (Sweden)

    Huiping Liu

    Full Text Available Laccases have been used for the decolorization and detoxification of synthetic dyes due to their ability to oxidize a wide variety of dyes with water as the sole byproduct. A putative laccase gene (LacTT from Thermus thermophilus SG0.5JP17-16 was screened using the genome mining approach, and it was highly expressed in Pichia pastoris, yielding a high laccase activity of 6130 U/L in a 10-L fermentor. The LacTT open reading frame encoded a protein of 466 amino acid residues with four putative Cu-binding regions. The optimal pH of the recombinant LacTT was 4.5, 6.0, 7.5 and 8.0 with 2,2'-azino-bis(3-ethylbenzothazoline-6-sulfonic acid (ABTS, syringaldazine (SGZ, guaiacol, and 2,6-dimethoxyphenol (2,6-DMP as the substrate, respectively. The optimal temperature of LacTT was 90°C with guaiacol as the substrate. LacTT was highly stable at pH 4.0-11.0 and thermostable at 40°C-90°C, confirming that it is a pH-stable and thermostable laccase. Furthermore, LacTT also exhibited high tolerance to halides such as NaCl, NaBr and NaF, and decolorized 100%, 94%, 94% and 73% of Congo Red, Reactive Black B and Reactive Black WNN, and Remazol Brilliant Blue R, respectively. Interestingly, addition of high concentration of NaCl increased the RBBR decolorization efficiency of LacTT. These results suggest that LacTT is a good candidate for industrial applications such as dyestuff processing and degradation of dyes in textile wastewaters.

  6. Application of alkaline thermo-stable lipase(s) enzyme produced from irradiated microbial isolate in the field of detergent technology

    International Nuclear Information System (INIS)

    Ahmed, O.E.A.M.S

    2010-01-01

    (s) from Bacillus brevis B 2 was studied. It was found that:1-The optimum incubation temperature for maximum lipase(s) formation is 70 degree C after 24h at ph 11.5.2- Maximum lipase(s) biosynthesis attained at concentration of 1% for each of fish-wastes and yeast extract with inoculum size equal 2 ml/100 ml culture medium incubating under shaking condition.3-Using fish-wastes-yeast extract medium fortified with disaccharide (sucrose or maltose or lactose) was the best nutritional medium for lipase formation. On contrary none of the tested nitrogen sources affect the induction of the enzyme.4-The addition of some surfactant to the nutritional medium revealed that Tween-20 slightly increase lipase(s) synthesis while the presence of zinc sulfate (100 ppm) and folic acid (500 ppm) enhanced the formation of lipase(s) by B. brevis B 2 . 5-Exposure of the experimental organism to 1 KGy gamma-irradiation resulted in slight increase in the level of enzyme. Thermoalkalo-stable lipase(s) from B. brevis B 2 was purified 21.8-fold using ammonium sulphate precipitation (100% saturation) followed by fractionation on Sephadex G-200 column chromatography. The properties of the enzyme were studied.Maximal enzyme activity occurred at 80 degree C. In addition, exposure of the enzyme to 70-80 degree C for 1h resulted remaining activities equal 98-90% respectively, indicating that it was thermo-stable. Optimum ph for maximal enzyme activity was ph 9.5 and exposure of the enzyme to alkaline condition (ph 9 - 10.5) for 1h resulted remaining activity from 80-90%, indicating the alkalo-stable nature of the enzyme. - The relationship between enzyme activity and enzyme concentration indicated that the extent of catalytic action was a function of the concentration of the enzyme. Maximal enzyme activity obtained after 60 minute and the reaction rate increased as a result of increasing substrate concentration, up to 1.2 mg of p-nitrophenyl palmitate, with some saturation at the highest concentration

  7. Effects of Mutations and Ligands on the Thermostability of the l-Arginine/Agmatine Antiporter AdiC and Deduced Insights into Ligand-Binding of Human l-Type Amino Acid Transporters

    Directory of Open Access Journals (Sweden)

    Hüseyin Ilgü

    2018-03-01

    Full Text Available The l-arginine/agmatine transporter AdiC is a prokaryotic member of the SLC7 family, which enables pathogenic enterobacteria to survive the extremely acidic gastric environment. Wild-type AdiC from Escherichia coli, as well as its previously reported point mutants N22A and S26A, were overexpressed homologously and purified to homogeneity. A size-exclusion chromatography-based thermostability assay was used to determine the melting temperatures (Tms of the purified AdiC variants in the absence and presence of the selected ligands l-arginine (Arg, agmatine, l-arginine methyl ester, and l-arginine amide. The resulting Tms indicated stabilization of AdiC variants upon ligand binding, in which Tms and ligand binding affinities correlated positively. Considering results from this and previous studies, we revisited the role of AdiC residue S26 in Arg binding and proposed interactions of the α-carboxylate group of Arg exclusively with amide groups of the AdiC backbone. In the context of substrate binding in the human SLC7 family member l-type amino acid transporter-1 (LAT1; SLC7A5, an analogous role of S66 in LAT1 to S26 in AdiC is discussed based on homology modeling and amino acid sequence analysis. Finally, we propose a binding mechanism for l-amino acid substrates to LATs from the SLC7 family.

  8. Effects of Mutations and Ligands on the Thermostability of the l-Arginine/Agmatine Antiporter AdiC and Deduced Insights into Ligand-Binding of Human l-Type Amino Acid Transporters.

    Science.gov (United States)

    Ilgü, Hüseyin; Jeckelmann, Jean-Marc; Colas, Claire; Ucurum, Zöhre; Schlessinger, Avner; Fotiadis, Dimitrios

    2018-03-20

    The l-arginine/agmatine transporter AdiC is a prokaryotic member of the SLC7 family, which enables pathogenic enterobacteria to survive the extremely acidic gastric environment. Wild-type AdiC from Escherichia coli, as well as its previously reported point mutants N22A and S26A, were overexpressed homologously and purified to homogeneity. A size-exclusion chromatography-based thermostability assay was used to determine the melting temperatures ( T m s) of the purified AdiC variants in the absence and presence of the selected ligands l-arginine (Arg), agmatine, l-arginine methyl ester, and l-arginine amide. The resulting T m s indicated stabilization of AdiC variants upon ligand binding, in which T m s and ligand binding affinities correlated positively. Considering results from this and previous studies, we revisited the role of AdiC residue S26 in Arg binding and proposed interactions of the α-carboxylate group of Arg exclusively with amide groups of the AdiC backbone. In the context of substrate binding in the human SLC7 family member l-type amino acid transporter-1 (LAT1; SLC7A5), an analogous role of S66 in LAT1 to S26 in AdiC is discussed based on homology modeling and amino acid sequence analysis. Finally, we propose a binding mechanism for l-amino acid substrates to LATs from the SLC7 family.

  9. Study of the leaching of heavy metals from waste water sludge and incinerator's ash, using coupled thermostated columns and DTPA as complex agent; Estudio de la extraccion de metales pesados de lodos y cenizas de aguas residuales usando columnas termostatizadas acopladas y DTPA como agente complejante

    Energy Technology Data Exchange (ETDEWEB)

    Vite T, J.; Vite T, M.; Guerrero D, J.; Carreno de Leon, M.C. [Departamento de Estudios del Ambiente, Instituto Nacional de Investigaciones Nucleares, A.P. 18-1027, 11801 Mexico D.F. (Mexico)

    2000-07-01

    We studied the metallic composition from waste water sludge and incinerators ashes of an incinerator located in Toluca, Mexico, the qualitative studies were made using the Activation Analysis technique, and fluorescence X-ray techniques. The quantitative analysis of heavy metals in the wastes were made using Inductively coupled plasma atomic emission spectrometry (Icp-Aes). For leaching the samples, we used four coupled thermostated columns, each one had a p H of 2,5, 7 and 10. The flux of the air was of 1600 cc/min. The temperature was maintain constant in 60 Centigrade using a thermostated system. For this study we used 100 g of wastes mixed with mineral acid or sodium hydroxide to reach p H 2,5,7 and 10. We added a reducing and tensoactive agents and finally DTPA as complex agent. With this method, we obtain a better leaching efficiency using a complex agent. However the high DTPA cost, make this process expansive that is why we recommend to work with another classes of complex agents, that be cheaper to leach metals of different chemistry matrix. (Author)

  10. Patterns of failure for patients with glioblastoma following O-(2-[18F]fluoroethyl)-L-tyrosine PET- and MRI-guided radiotherapy

    DEFF Research Database (Denmark)

    Lundemann, Michael; Costa, Junia Cardoso; Law, Ian

    2017-01-01

    evaluable, with median follow-up of 45months. Central, in-field, marginal and distant recurrences were observed for 82%, 10%, 2%, and 6% of the patients, respectively. We found a volumetric overlap of 26%, 31% and 39% of the RV with the contrast-enhancing MR volume, PETvol and the composite MRPETvol...

  11. Influence of preseasonal treatment with L-tyrosine-adsorbed allergoids on IgE-mediated histamine release from basophils of children suffering from allergic diseases.

    Science.gov (United States)

    Wegner, F; Fenkes, A; Stemmann, E A; Reinhardt, D

    1981-04-01

    In 10 children suffering from allergic pollinosis and/or asthma, a preseasonal hyposensitization scheme with 3 weekly injections of a glutaraldehyde-modified, tyrosine-adsorbed grass-pollen allergen reduced the histamine release from basophils in response to increasing concentrations of antigen. The decrease in histamine release which occurred 1 week after the injection course was even maintained during the pollen season. The inhibition was only obtained when basophils were incubated with the serum of patients, but not with the serum of normals, indicating that blocking antibodies may have occurred. In contrast to what has been observed in the treated patients' group, 5 patients, who were not included in the hyposensitization scheme, showed identical histamine release curves during the whole investigation period. Specific IgE did not increase after the treatment course and shows the same behaviour as the untreated patients. Thus, as treatment with glutaraldehyde modified, tyrosine-adsorbed allergoids is safe to administer, requires only 3 injections, reduces histamine release from basophils by production of "blocking" antibodies, it appears to be a useful tool in the hyposensitization treatment.

  12. RESOLUTE PET/MRI Attenuation Correction for O-(2-F-fluoroethyl)-L-tyrosine (FET) in Brain Tumor Patients with Metal Implants

    DEFF Research Database (Denmark)

    Ladefoged, Claes N; Andersen, Flemming L; Kjær, Andreas

    2017-01-01

    of agreement for TMAX/B was for RESOLUTE (-3%; 4%), Dixon (-9%; 16%), and UTE (-7%; 10%). The absolute error when measuring BTV was 0.7 ± 1.9 mL (N.S) with RESOLUTE, 5.3 ± 10 mL using Dixon, and 1.7 ± 3.7 mL using UTE. RESOLUTE performed best in the identification of the location of peak activity and in brain...... to be quantitatively correct in order to be used clinically, which require accurate attenuation correction (AC) in PET/MRI. The aim of this study was to evaluate the use of the subject-specific MR-derived AC method RESOLUTE in post-operative brain tumor patients.Methods:We analyzed 51 post-operative brain tumor...... patients (68 examinations, 200 MBq [18F]-FET) investigated in a PET/MRI scanner. MR-AC maps were acquired using: (1) the Dixon water fat separation sequence, (2) the ultra short echo time (UTE) sequences, (3) calculated using our new RESOLUTE methodology, and (4) a same day low-dose CT used as reference...

  13. The homogentisate pathway: a central catabolic pathway involved in the degradation of L-phenylalanine, L-tyrosine, and 3-hydroxyphenylacetate in Pseudomonas putida.

    Science.gov (United States)

    Arias-Barrau, Elsa; Olivera, Elías R; Luengo, José M; Fernández, Cristina; Galán, Beatriz; García, José L; Díaz, Eduardo; Miñambres, Baltasar

    2004-08-01

    Pseudomonas putida metabolizes Phe and Tyr through a peripheral pathway involving hydroxylation of Phe to Tyr (PhhAB), conversion of Tyr into 4-hydroxyphenylpyruvate (TyrB), and formation of homogentisate (Hpd) as the central intermediate. Homogentisate is then catabolized by a central catabolic pathway that involves three enzymes, homogentisate dioxygenase (HmgA), fumarylacetoacetate hydrolase (HmgB), and maleylacetoacetate isomerase (HmgC), finally yielding fumarate and acetoacetate. Whereas the phh, tyr, and hpd genes are not linked in the P. putida genome, the hmgABC genes appear to form a single transcriptional unit. Gel retardation assays and lacZ translational fusion experiments have shown that hmgR encodes a specific repressor that controls the inducible expression of the divergently transcribed hmgABC catabolic genes, and homogentisate is the inducer molecule. Footprinting analysis revealed that HmgR protects a region in the Phmg promoter that spans a 17-bp palindromic motif and an external direct repetition from position -16 to position 29 with respect to the transcription start site. The HmgR protein is thus the first IclR-type regulator that acts as a repressor of an aromatic catabolic pathway. We engineered a broad-host-range mobilizable catabolic cassette harboring the hmgABC, hpd, and tyrB genes that allows heterologous bacteria to use Tyr as a unique carbon and energy source. Remarkably, we show here that the catabolism of 3-hydroxyphenylacetate in P. putida U funnels also into the homogentisate central pathway, revealing that the hmg cluster is a key catabolic trait for biodegradation of a small number of aromatic compounds.

  14. Amino acid solutions for premature neonates during the first week of life: the role of N-acetyl-L-cysteine and N-acetyl-L-tyrosine

    NARCIS (Netherlands)

    van Goudoever, J. B.; Sulkers, E. J.; Timmerman, M.; Huijmans, J. G.; Langer, K.; Carnielli, V. P.; Sauer, P. J.

    1994-01-01

    Tyrosine and cyst(e)ine are amino acids that are thought to be essential for preterm neonates. These amino acids have low stability (cyst(e)ine) or low solubility (tyrosine) and are therefore usually present only in small amounts in amino acid solutions. Acetylation improves the stability and

  15. The synthesis of 18F-labelled amino acid O-(2-[18F]fluoroethyl)-L-tyrosine (FET) in NPI

    Czech Academy of Sciences Publication Activity Database

    Švecová, Helena; Procházka, Libor; Fedorova, S.; Kropáček, Martin; Melichar, František

    2007-01-01

    Roč. 2, č. 331 (2007), s. 34-34 ISSN 1619-7070 R&D Projects: GA MPO 2A-1TP1/055 Institutional research plan: CEZ:AV0Z10480505 Keywords : [18F]FET * PET * tumor imaging Subject RIV: FR - Pharmacology ; Medidal Chemistry

  16. The metabolism of L-phenylalanine and L-tyrosine by liver cells isolated from adrenalectomized rats and from streptozotocin-diabetic rats.

    OpenAIRE

    Stanley, J C; Fisher, M J; Pogson, C I

    1985-01-01

    Flux through, and maximal activities of, key enzymes of phenylalanine and tyrosine degradation were measured in liver cells prepared from adrenalectomized rats and from streptozotocin-diabetic rats. Adrenalectomy decreased the phenylalanine hydroxylase flux/activity ratio; this was restored by steroid treatment in vivo. Changes in the phosphorylation state of the hydroxylase may mediate these effects; there was no significant change in the maximal activity of the hydroxylase. Tyrosine metabol...

  17. A micro-PET/CT approach using O-(2-[{sup 18}F]fluoroethyl)-L-tyrosine in an experimental animal model of F98 glioma for BNCT

    Energy Technology Data Exchange (ETDEWEB)

    Menichetti, L., E-mail: luca.menichetti@ifc.cnr.it [CNR Institute of Clinical Physiology, Pisa (Italy); Petroni, D.; Panetta, D. [CNR Institute of Clinical Physiology, Pisa (Italy); Burchielli, S. [Fondazione CNR/Regione Toscana G. Monasterio, Pisa (Italy); Bortolussi, Silva [Dept. Theoretical and Nuclear Physics, University of Pavia, Pavia (Italy); Matteucci, M. [Scuola Superiore Sant' Anna, Pisa (Italy); Pascali, G.; Del Turco, S. [CNR Institute of Clinical Physiology, Pisa (Italy); Del Guerra, A. [Department of Physics, University of Pisa, Pisa (Italy); Altieri, S. [Dept. Theoretical and Nuclear Physics, University of Pavia, Pavia (Italy); Salvadori, P.A. [CNR Institute of Clinical Physiology, Pisa (Italy)

    2011-12-15

    The present study focuses on a micro-PET/CT application to be used for experimental Boron Neutron Capture Therapy (BNCT), which integrates, in the same frame, micro-CT derived anatomy and PET radiotracer distribution. Preliminary results have demonstrated that {sup 18}F-fluoroethyl-tyrosine (FET)/PET allows the identification of the extent of cerebral lesions in F98 tumor bearing rat. Neutron autoradiography and {alpha}-spectrometry on axial tissues slices confirmed the tumor localization and extraction, after the administration of fructose-boronophenylalanine (BPA). Therefore, FET-PET approach can be used to assess the transport, the net influx, and the accumulation of FET, as an aromatic amino acid analog of BPA, in experimental animal model. Coregistered micro-CT images allowed the accurate morphological localization of the radiotracer distribution and its potential use for experimental BNCT.

  18. Production and characterization of thermostable xylanase from ...

    African Journals Online (AJOL)

    ajl2

    2013-02-20

    Feb 20, 2013 ... Penicillium, .... production in liquid medium ranged from 0.11 to 0.21. U/ml (data not shown). ... Lane 2 represents the negative control (Bacillus sp.). Lane 3 is .... circulans D1 in submerged fermentation using response surface.

  19. Biochemical characterization of a thermoactive and thermostable ...

    African Journals Online (AJOL)

    faouzi

    h on a rotary shaker set at 200 rpm at a temperature of 30°C. Growth was followed ... The medium culture, obtained after 24 h with an inoculum size of 2. × 10⁷ cells/ml, was .... (1997) showed that SHL was not inactivated by anionic detergents ...

  20. Characterization and engineering of thermostable glycoside hydrolases

    NARCIS (Netherlands)

    Lieshout, van J.F.T.

    2007-01-01

    Glycosidehydrolasesform a class of enzymes that play an important role in sugar-converting processes. They are applied as biocatalyst in both the hydrolysis of natural polymers to mono- andoligo-saccharides, and the reverse hydrolysis or

  1. Optimization and Characterization of Thermostable Endo and ...

    African Journals Online (AJOL)

    http://dx.doi.org/10.4314/star.v3i4.16 · AJOL African Journals Online. HOW TO USE AJOL... for Researchers · for Librarians · for Authors · FAQ's · More about AJOL · AJOL's Partners · Terms and Conditions of Use · Contact AJOL · News. OTHER RESOURCES... for Researchers · for Journals · for Authors · for Policy Makers ...

  2. Production and characterization of thermostable xylanase from ...

    African Journals Online (AJOL)

    ajl2

    2013-02-20

    Feb 20, 2013 ... produced from Trichoderma (Huitron et al., 2008). Only a ... around their colonies against a red background, were selected and .... Resistance to antibiotic ..... Xylanases of marine fungi potential use for biobleaching of paper.

  3. Biochemical characterization of thermostable cellulase enzyme from ...

    African Journals Online (AJOL)

    user

    2012-05-29

    May 29, 2012 ... tested for their ability to produce cellulase complex enzyme by growing on a defined substrates as well ... In the current industrial processes, cellulolytic enzymes ... energy sources such as glucose, ethanol, hydrogen and.

  4. Purification and characterization of a thermostable glucoamylase ...

    African Journals Online (AJOL)

    Glucoamylase (GA) from Aspergillus flavus HBF34 strain was partially purified 120 folds using starch affinity chromatography. Two isoenzymes (GA1 and GA2) were identified by polyacrylamide gel electrophoresis (PAGE) zymography. Sodium dodecyl sulfate (SDS)-PAGE analysis revealed that one of the enzymes consist ...

  5. Purification and characterization of a thermostable glucoamylase ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-06-07

    Jun 7, 2010 ... was 6.0 and the optimum temperature was 60°C. GA was found to be stable at temperatures up to .... on starch plates were inoculated to test for amylase secretion, ..... starch-digesting glucoamylase production of Rhizopus sp.

  6. Engineering thermostable xylanase enzyme mutant from Bacillus ...

    African Journals Online (AJOL)

    user

    2010-11-22

    Nov 22, 2010 ... waste treatment, fuel and chemical production, paper and pulp industries; but these applications ... approaches have been taken: screening organisms from various ... and site-directed mutagenesis was applied on this.

  7. Effect of metal Ions (Ni2+, Cu2+ and Zn2+) and water coordination on the structure of L-phenylalanine, L-tyrosine, L-tryptophan and their zwitterionic forms

    NARCIS (Netherlands)

    Remko, Milan; Fitz, Daniel; Broer, Ria; Rode, Bernd Michael

    2011-01-01

    Methods of quantum chemistry have been applied to double-charged complexes involving the transition metals Ni2+, Cu2+ and Zn2+ with the aromatic amino acids (AAA) phenylalanine, tyrosine and tryptophan. The effect of hydration on the relative stability and geometry of the individual species studied

  8. Production of Thermostab Streptomyc ction of Thermostable Alkaline ...

    African Journals Online (AJOL)

    and temperature of 500C. Therefore ... Actinomycetes species and determining the cultural conditions se yields. ... ials for protease production on skimmed milk agar, only Norcar ... produced crude enzyme demonstrated optimal activity at pH 8.

  9. PCR performance of a thermostable heterodimeric archaeal DNA polymerase

    Science.gov (United States)

    Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

    2014-01-01

    DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications. PMID:24847315

  10. A thermostable Cas9 with increased lifetime in human plasma

    OpenAIRE

    Harrington, LB; Paez-Espino, D; Staahl, BT; Chen, JS; Ma, E; Kyrpides, NC; Doudna, JA

    2017-01-01

    © 2017 The Author(s). CRISPR-Cas9 is a powerful technology that has enabled genome editing in a wide range of species. However, the currently developed Cas9 homologs all originate from mesophilic bacteria, making them susceptible to degradation and unsuitable for applications requiring cleavage at elevated temperatures. Here, we show that the Cas9 protein from the thermophilic bacterium Geobacillus stearothermophilus (GeoCas9) catalyzes RNA-guided DNA cleavage at elevated temperatures. GeoCas...

  11. Thermostability of xylanolytic enzymes produced by Lentinula edodes UFV70

    Science.gov (United States)

    Ribeiro, Liliane Fraga Costa; Vaz, Marcelo Gomes Marçal Vieira; Chaves-Alves, Virgínia Maria; Vanetti, Maria Cristina Dantas; Vanetti, Maria Cristina Dantas; Kasuya, Maria Catarina Megumi; Passos, Flávia Maria Lopes; do Nascimento, Antônio Galvão

    2012-01-01

    Xylanolytic enzymes produced by Lentinula edodes UFV70, cultivated in eucalyptus sawdust/rice bran medium, were stable at 50, 60 and 65°C for 21 hours, losing only 15–25% activity. Fungus incubation at 50°C for 12 hours and at 65°C for 24 hours increased the amount of xylose produced. PMID:24031818

  12. Characterization of a thermostable Bacillus subtilis β-amylase

    African Journals Online (AJOL)

    ... 70 0C respectively, and the thermal stability curve gave a maximum activity of 9.75 U at 70oC for 60 min of incubation. Bacillus subtilis â-amylase is valuable for maltose production, which can be hydrolyzed further by other groups of amylase for the production of high cassava glucose syrup used as sweeteners in the food ...

  13. Thermostability of reconstituted newcastle disease virus strains at 36 ...

    African Journals Online (AJOL)

    Haemagglutination (HA) test was employed to determine the stability of HA titers of reconstituted form of Hitchner – B1 (B1), LaSota (L) and Komarov (K) strains of Newcastle Disease Vaccine (NDV) at 360c. The temperature treatment method was through incubation (in water bath) of the reconstituted vaccines at selected ...

  14. Characterization of two thermostable inulinases from Rhizopus oligosporus NRRL 2710

    Directory of Open Access Journals (Sweden)

    Saleh A. Mohamed

    2015-06-01

    Full Text Available Two inulinases (Inu2 and Inu3 were purified from Rhizopus oligosporus NRRL 2710 by chromatography on DEAE-Sepharose and Sephacryl S-200 columns. The molecular weight of Inu2 and Inu3 were determined to be 76 and 30 kDa, respectively. Inu2 and Inu3 had the same pH optimum at 5.0, temperature optimum at 50 and 60 °C, and thermal stability up to 60 and 70 °C for 1 h, respectively. Inu2 and Inu3 had low km values (0.93 and 0.70 mM, respectively indicating the high affinity toward inulin. Mg2+, Ca2+, Zn2+ and EDTA did not significantly influence the enzyme activity. Ni2+, Cu2+, Fe2+ and Co2+ showed a partial inhibitory effect, and Hg2+ had a strong inhibitory effect. p-Chloromercuribenzoate had a partial inhibitory effect on Inu2. From these findings, R. oligosporus inulinases can be beneficial enzymes for industrial enzymatic production of high fructose syrup.

  15. Purification and biochemical properties of a new thermostable ...

    African Journals Online (AJOL)

    The enzyme was stable for a long time-period up to 50°C and for 1 h at 60°C. Although the xylanase had a lower carboxymethylcellulase activity, it lacked activity towards substituted xylan, xylobiose, inulin, starch, polygalacturonic acid or pNP glycosides. Kinetic parameters indicated higher efficiency in the hydrolysis of ...

  16. PCR performance of a thermostable heterodimeric archaeal DNA polymerase

    Directory of Open Access Journals (Sweden)

    Tom eKillelea

    2014-05-01

    Full Text Available DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR, cDNA cloning, genome sequencing and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3’ primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications.

  17. Thermostability of sperm nuclei assessed by microinjection into hamster oocytes

    Science.gov (United States)

    Nuclei isolated from spermatozoa of various species (golden hamster, mouse, human, rooster, and the fish tilapia) were heated at 60 degrees-125 degrees C for 20-120 min and then microinjected into hamster oocytes to determine whether they could decondense and develop into pronucl...

  18. A thermostable messenger RNA based vaccine against rabies.

    Science.gov (United States)

    Stitz, Lothar; Vogel, Annette; Schnee, Margit; Voss, Daniel; Rauch, Susanne; Mutzke, Thorsten; Ketterer, Thomas; Kramps, Thomas; Petsch, Benjamin

    2017-12-01

    Although effective rabies virus vaccines have been existing for decades, each year, rabies virus infections still cause around 50.000 fatalities worldwide. Most of these cases occur in developing countries, where these vaccines are not available. The reasons for this are the prohibitive high costs of cell culture or egg grown rabies virus vaccines and the lack of a functional cold chain in many regions in which rabies virus is endemic. Here, we describe the excellent temperature resistance of a non-replicating mRNA based rabies virus vaccine encoding the rabies virus glycoprotein (RABV-G). Prolonged storage of the vaccine from -80°C to up to +70°C for several months did not impact the protective capacity of the mRNA vaccine. Efficacy after storage was demonstrated by the induction of rabies specific virus neutralizing antibodies and protection in mice against lethal rabies infection. Moreover, storing the vaccine at oscillating temperatures between +4° and +56°C for 20 cycles in order to simulate interruptions of the cold chain during vaccine transport, did not affect the vaccine's immunogenicity and protective characteristics, indicating that maintenance of a cold chain is not essential for this vaccine.

  19. Enhancing RGI lyase thermostability by targeted single point mutations

    DEFF Research Database (Denmark)

    Silva, Inês R.; Larsen, Dorte Møller; Jers, Carsten

    2013-01-01

    Rhamnogalacturonan I lyase (RGI lyase) (EC 4.2.2.-) catalyzes the cleavage of rhamnogalacturonan I in pectins by β-elimination. In this study the thermal stability of a RGI lyase (PL 11) originating from Bacillus licheniformis DSM 13/ATCC14580 was increased by a targeted protein engineering...

  20. Research of thermostability of phthalates of oxyalkylated alcohols

    Directory of Open Access Journals (Sweden)

    MAZITOVA Aliya Karamovna

    2018-04-01

    Full Text Available The production of polyvinyl chloride (PVC requires implication of a large number of plasticizers. Ester compounds are the most suitable for plasticization. Currently the existing range of industrial plasticizers does not satisfy a need of the polymer industry and it is of great importance to develop new plasticizers that meet modern requirements. Therefore, the elaboration of new ester compounds with effective plasticizing properties, is relevant and has not only scientific value, but also practical one. One of the basic requirements for ester plasticizers is their resistance to thermal oxidation. Accordingly, we considered the effect of oxygen at 150оC (the temperature of conversion of PVC-plastic compounds on a wide range of oxyalated alcohol phthalates developed earlier. The first group consisted of aromatic alcohols, the second group – phthalates of oxyalkylated aliphatic alcohols. The first group showed high thermal stability, the latter should be stabilized by the addition of antioxidants. It is known that aromatic hydrocarbon derivatives are widely used as tabilizers of thermooxidative disrupture. The main function of the mentioned compounds is to suppress the catalytic action of air oxygen in energy impacts on polymers and plasticizers. Consequently, we investigated the effect of Agidol-1 on increasing the stability of phthalate plasticizers to thermaloxidative degradation.

  1. Purification of a thermostable antinociceptive lectin isolated from Andira anthelmia.

    Science.gov (United States)

    Nascimento, Kyria Santiago; Nascimento, Francisco Lucas Faustino do; Silva, Mayara Torquato Lima; Nobre, Camila Bezerra; Moreira, Cleane Gomes; Brizeno, Luiz André Cavalcante; da Ponte, Edson Lopes; Assreuy, Ana Maria Sampaio; Cavada, Benildo Sousa

    2016-06-01

    Andira anthelmia (tribe Dalbergieae), a plant from Brazilian Amazon, possesses a seed lectin that was purified by affinity chromatography in sepharose-mannose. This novel Dalbergieae lectin, named AAL, agglutinated rabbit erythrocytes treated with trypsin. The hemagglutinating activity of AAL was maintained after incubation at a wide range of temperature (40 to 70 °C) and pH, was shown to be dependent on divalent cations, and was inhibited by d-mannose and d-sucrose. AAL showed an electrophoretic profile in sodium dodecyl sulfate-polyacrylamide gel electrophoresis similar to other lectins of the tribe Dalbergieae, presenting a double band of molecular weight with approximately 20 kDa and other minor bands of 17, 15, and 13 kDa, being the smaller fragment glycosylated. AAL injected by intravenous route in mice showed antinociceptive activity in two behavioral tests (writhing and formalin). In the writhing test induced by acetic acid, AAL showed inhibitory effect at 0.01 mg/kg (68%), 0.1 mg/kg (46%) and 1 mg/kg (74%). In the formalin test, AAL (0.1 mg/kg) inhibited by 48% the licking time in the inflammatory phase, an effect that was recovered by the lectin association with mannose. In conclusion, AAL presents analgesic effect involving the lectin domain via peripheral mechanisms of inflammatory nociception. This activity highlights the importance of lectins as tools to be used for understanding the interaction of protein-carbohydrate in processes associated to inflammatory pain. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  2. Directed evolution of a thermostable l-aminoacylase biocatalyst

    DEFF Research Database (Denmark)

    Parker, Brenda M.; Taylor, Ian N.; Woodley, John

    2011-01-01

    Enzymes from extreme environments possess highly desirable traits of activity and stability for application under process conditions. One such example is l-aminoacylase (E.C. 3.5.1.14) from Thermococcus litoralis (TliACY), which catalyzes the enantioselective amide hydrolysis of N-protected l......-amino acids, useful for resolving racemic mixtures in the preparation of chiral intermediates. Variants of this enzyme with improved activity and altered substrate preference are highly desirable. We have created a structural homology model of the enzyme and applied various two different directed evolution....... The substrate preference of wild type decreases with increasingly branched and sterically hindered substrates. However, the mutant S100T/M106K disrupted this simple trend by selectively improving the substrate preference for N-benzoyl valine, with a >30-fold shift in the ratio of kcat values for N...

  3. Characterization of Thermostable Cellulases Produced by Bacillus and Geobacillus Strains

    Science.gov (United States)

    Bacterial community composition of thermophilic (60 deg C) mixed cellulose-enrichment cultures was examined by constructing a 16S rDNA clone library which demonstrated major lineages affiliated to Actinobacteria, Bacteroidetes, Chloroflexi, Deinococcus-Thermus, Firmicutes, and Proteobacteria. A tot...

  4. Functional characterization and crystal structure of thermostable amylase from Thermotoga petrophila, reveals high thermostability and an unusual form of dimerization

    DEFF Research Database (Denmark)

    Hameed, Uzma; Price, Ian; Ikram-Ul-Haq

    2017-01-01

    and able to hydrolyze starch into dextrin between 90 and 100°C, with optimum activity at 98°C and pH8.5. The activity increased in the presence of Rb(1+), K(1+) and Ca(2+) ions, whereas other ions inhibited activity. The crystal structure of Tp-AmyS at 1.7Å resolution showed common features of the GH-13...... of salivary amylase from a previous crystal structure, and thus could be a functional feature of some amylases....

  5. Production of thermostable glucoamylase by newly isolated Aspergillus flavus A 1.1 and Thermomyces lanuginosus A 13.37 Produção e glucoamilase por Aspergillus flavus A1.1 e Thermomyces lanuginosus A13.37

    Directory of Open Access Journals (Sweden)

    Eleni Gomes

    2005-03-01

    Full Text Available Thirteen thermophilic fungal strains were isolated from agricultural soil, tubers and compost samples in tropical Brazil. Two strains were selected based on of their ability to produce considerable glucoamylase activity while growing in liquid medium at 45ºC with starch as the only carbon source. They were identified as Aspergillus flavus A1.1 and Thermomyces lanuginosus A 13.37 Tsiklinsky. The experiment to evaluate the effect of carbon source, temperature and initial pH of the medium on enzyme production was developed in a full factorial design (2x2x3. Enzyme productivity was influenced by the type of starch used as carbon source. Cassava starch showed to be a better substrate than corn starch for glucoamylase production by A. flavus but for T. lanuginosus the difference was not significant. Enzyme activities were determined using as substrates 0.3% soluble starch, 0.3% maltose or 0.3% of starch plus 0.1% maltose. The enzymes from A. flavus A1.1 hydrolyzed soluble starch preferentially but also exhibited a significant maltase activity. Moreover higher quantities of glucose were released when the substrate used was a mixture of starch and maltose, suggesting that this fungus produced two types of enzyme. In the case T. lanuginosus A 13.37, the substrate specificity test indicated that the enzyme released also hydrolyzed starch more efficiently than maltose, but there was no increase in the liberation of glucose when a mixture of starch and maltose was used as substrate, suggesting that only one type of enzyme was secreted. Glucoamylases produced from A. flavus A1.1 and T. lanuginous A.13-37 have high optimum temperature (65ºC and 70ºC and good thermostability in the absence of substrate (maintaining 50% of activity for 5 and 8 hours, respectively, at 60ºC and are stable over in a wide pH range. These new strains offer an attractive alternative source of enzymes for industrial starch processing.Entre 13 linhagens de fungos filamentosos

  6. Molecular characterisation of the thermostability and catalytic properties of enzymes from hyperthermophiles

    NARCIS (Netherlands)

    Lebbink, J.H.G.

    1999-01-01

    Hyperthermophilic organisms are able to survive and reproduce optimally between 80°C and 113°C. Most of them belong to the domain of the Archaea, although several hyperthermophilic Bacteria have been described. One of the major questions regarding hyperthermophiles concerns the molecular

  7. PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.

    Science.gov (United States)

    Cline, J; Braman, J C; Hogrefe, H H

    1996-09-15

    The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 x 10(-6)) Pfu and UlTma (approximately 5 x 10(-5)). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2-3 mM MgSO4 and 100-300 microM each dNTP and at pH 8.5-9.1. Under these conditions, the error rate of exo- Pfu was approximately 40-fold higher (5 x 10(-5)) than the error rate of Pfu. As the reaction pH was raised from pH 8 to 9, the error rate of Pfu decreased approximately 2-fold, while the error rate of exo- Pfu increased approximately 9-fold. An increase in error rate with pH has also been noted for the exonuclease-deficient DNA polymerases Taq and exo- Klenow, suggesting that the parameters which influence replication error rates may be similar in pol l- and alpha-like polymerases. Finally, the fidelity of 'long PCR' DNA polymerase mixtures was examined. The error rates of a Taq/Pfu DNA polymerase mixture and a Klentaq/Pfu DNA polymerase mixture were found to be less than the error rate of Taq DNA polymerase, but approximately 3-4-fold higher than the error rate of Pfu DNA polymerase.

  8. PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.

    OpenAIRE

    Cline, J; Braman, J C; Hogrefe, H H

    1996-01-01

    The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 x 10(-6)) < Deep Vent (2.7 x 10(-6)) < Vent (2.8 x 10(-6)) < Taq (8.0 x 10(-6)) < < exo- Pfu and UlTma (approximately 5 x 10(-5)). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2-3 mM MgSO4 and 100-300 microM each dNTP and at p...

  9. Reconstitution of a thermostable xylan-degrading enzyme mixture from the bacterium Caldicellulosiruptor bescii.

    Science.gov (United States)

    Su, Xiaoyun; Han, Yejun; Dodd, Dylan; Moon, Young Hwan; Yoshida, Shosuke; Mackie, Roderick I; Cann, Isaac K O

    2013-03-01

    Xylose, the major constituent of xylans, as well as the side chain sugars, such as arabinose, can be metabolized by engineered yeasts into ethanol. Therefore, xylan-degrading enzymes that efficiently hydrolyze xylans will add value to cellulases used in hydrolysis of plant cell wall polysaccharides for conversion to biofuels. Heterogeneous xylan is a complex substrate, and it requires multiple enzymes to release its constituent sugars. However, the components of xylan-degrading enzymes are often individually characterized, leading to a dearth of research that analyzes synergistic actions of the components of xylan-degrading enzymes. In the present report, six genes predicted to encode components of the xylan-degrading enzymes of the thermophilic bacterium Caldicellulosiruptor bescii were expressed in Escherichia coli, and the recombinant proteins were investigated as individual enzymes and also as a xylan-degrading enzyme cocktail. Most of the component enzymes of the xylan-degrading enzyme mixture had similar optimal pH (5.5 to ∼6.5) and temperature (75 to ∼90°C), and this facilitated their investigation as an enzyme cocktail for deconstruction of xylans. The core enzymes (two endoxylanases and a β-xylosidase) exhibited high turnover numbers during catalysis, with the two endoxylanases yielding estimated k(cat) values of ∼8,000 and ∼4,500 s(-1), respectively, on soluble wheat arabinoxylan. Addition of side chain-cleaving enzymes to the core enzymes increased depolymerization of a more complex model substrate, oat spelt xylan. The C. bescii xylan-degrading enzyme mixture effectively hydrolyzes xylan at 65 to 80°C and can serve as a basal mixture for deconstruction of xylans in bioenergy feedstock at high temperatures.

  10. Characterization of novel thermostable bacterial Laccase-like multi-copper oxidases

    DEFF Research Database (Denmark)

    Brander, Søren; Mikkelsen, Jørn Dalgaard

    decolorization, bleaching of paper pulp, delignification of biomass and remediation of polluted water. In order to widen the applicability of LMCOs, it is important to establish the properties and substrate specificities of naturally occurring LMCOs. This is especially true for LMCOs from bacteria, whose role...

  11. Characterizing a thermostable Cas9 for bacterial genome editing and silencing

    NARCIS (Netherlands)

    Mougiakos, Ioannis; Mohanraju, Prarthana; Bosma, Elleke F.; Vrouwe, Valentijn; Finger Bou, Max; Naduthodi, Mihris I.S.; Gussak, Alex; Brinkman, Rudolf B.L.; Kranenburg, Van Richard; Oost, Van Der John

    2017-01-01

    CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including

  12. Fabrication of electrospun almond gum/PVA nanofibers as a thermostable delivery system for vanillin.

    Science.gov (United States)

    Rezaei, Atefe; Tavanai, Hossein; Nasirpour, Ali

    2016-10-01

    In this study, the fabrication of vanillin incorporated almond gum/polyvinyl alcohol (PVA) nanofibers through electrospinning has been investigated. Electrospinning of only almond gum was proved impossible. It was found that the aqueous solution of almond gum/PVA (80:20, concentration=7% (w/w)) containing 3% (w/w) vanillin could have successfully electrospun to uniform nanofibers with diameters as low as 77nm. According to the thermal analysis, incorporated vanillin in almond gum/PVA nanofibers showed higher thermal stability than free vanillin, making this composite especially suitable for high temperature applications. XRD and FTIR analyses proved the presence of vanillin in the almond gum/PVA nanofibers. It was also found that vanillin was dispersed as big crystallites in the matrix of almond gum/PVA nanofibers. FTIR analysis showed almond gum and PVA had chemical cross-linking by etheric bonds between COH groups of almond gum and OH groups of PVA. Also, in the nanofibers, there were no major interaction between vanillin and either almond gum or PVA. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Water isotope effect on the thermostability of a polio viral RNA hairpin: A metadynamics study

    Science.gov (United States)

    Pathak, Arup K.; Bandyopadhyay, Tusar

    2017-04-01

    Oral polio vaccine is considered to be the most thermolabile of all the common childhood vaccines. Despite heavy water (D2O) having been known for a long time to stabilise attenuated viral RNA against thermodegradation, the molecular underpinnings of its mechanism of action are still lacking. Whereas, understanding the basis of D2O action is an important step that might reform the way other thermolabile drugs are stored and could possibly minimize the cold chain problem. Here using a combination of parallel tempering and well-tempered metadynamics simulation in light water (H2O) and in D2O, we have fully described the free energy surface associated with the folding/unfolding of a RNA hairpin containing a non-canonical basepair motif, which is conserved within the 3'-untranslated region of poliovirus-like enteroviruses. Simulations reveal that in heavy water (D2O) there is a considerable increase of the stability of the folded basin as monitored through an intramolecular hydrogen bond (HB), size, shape, and flexibility of RNA structures. This translates into a higher melting temperature in D2O by 41 K when compared with light water (H2O). We have explored the hydration dynamics of the RNA, hydration shell around the RNA surface, and spatial dependence of RNA-solvent collective HB dynamics in the two water systems. Simulation in heavy water clearly showed that D2O strengthens the HB network in the solvent, lengthens inter-residue water-bridge lifetime, and weakens dynamical coupling of the hairpin to its solvation environment, which enhances the rigidity of solvent exposed sites of the native configurations. The results might suggest that like other added osmoprotectants, D2O can act as a thermostabilizer when used as a solvent.

  14. The effect of carbon sources on the expression level of thermostable ...

    African Journals Online (AJOL)

    Jane

    2011-10-12

    Oct 12, 2011 ... 1 Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. ... production of detergents, animal skin-based industry and food processing ... and minimal microbial contamination are achievable.

  15. Production, Characterization and Application of a Thermostable Tannase from Pestalotiopsis guepinii URM 7114

    Directory of Open Access Journals (Sweden)

    Amanda Reges de Sena

    2014-01-01

    Full Text Available Tannase (EC 3.1.1.20 is an enzyme that hydrolyzes the ester and depside bonds of tannic acid to gallic acid and glucose. In the production of foods and beverages, it contributes to the removal of the undesirable effects of tannins. The aim of this study is to investigate the potential of endophytic fungi isolated from jamun (Syzygium cumini (L. Skeels leaves, and identified as Pestalotiopsis guepinii, in the production of tannase. Tannase was produced extracellularly by P. guepinii under submerged, slurry-state and solid-state fermentations. The submerged fermentation was found to be the most promising (98.6 U/mL. Response surface methodology was employed to evaluate the effect of variables (pH and temperature, and the results showed that the best conditions for tannase activity were pH=6.9 and 30 °C. Km was found to be 7.18·10–4 mol/L and vmax=250.00 U/mL. The tannase activity was the highest in the presence of Ca2+ at a concentration of 5·10–3 mol/L. Moreover, the enzyme was not inhibited by the tested chelators and detergents. The stability of the enzyme was also studied, and crude enzyme was evaluated in simulation of gastrointestinal digestion of monogastric animals. The crude enzyme was highly stable under simulated conditions; it retained 87.3 % of its original activity after 6 h. The study contributes to the identification of microbial species that produce tannase, with potential application in biotechnology.

  16. A thermostable metal-tolerant laccase with bioremediation potential from a marine-derived fungus

    Digital Repository Service at National Institute of Oceanography (India)

    DeSouza-Ticlo, D.; Sharma, D.; Raghukumar, C.

    their optimum pH and temperature). From these results, K cat and specificity constant of Lac IId with ABTS were derived (Das et al., 2001). Substrate specificity and inhibitors of Lac IId. Oxygen uptake during reaction of Lac IId with substrates, non... from Q 10 values between 60 o and 70 o C at this pH was 2.5 kJ mol -1 whereas from the Arrhenius plot, it was 8.15 kJ mol -1 . The K m , V max and K cat of Lac IId were compared using ABTS and syringaldazine; maximum specificity constant (K...

  17. Modeling the thermostability of surface functionalisation by oxygen, hydroxyl, and water on nanodiamonds.

    Science.gov (United States)

    Lai, Lin; Barnard, Amanda S

    2011-06-01

    Understanding nanodiamond functionalisation is of great importance for biological and medical applications. Here we examine the stabilities of oxygen, hydroxyl, and water functionalisation of the nanodiamonds using the self-consistent charge density functional tight-binding simulations. We find that the oxygen and hydroxyl termination are thermodynamically favourable and form strong C–O covalent bonds on the nanodiamond surface in an O2 and H2 gas reservoir, which confirms previous experiments. Yet, the thermodynamic stabilities of oxygen and hydroxyl functionalisation decrease dramatically in a water vapour reservoir. In contrast, H2O molecules are found to be physically adsorbed on the nanodiamond surface, and forced chemical adsorption results in decomposition of H2O. Moreover, the functionalisation efficiency is found to be facet dependent. The oxygen functionalisation prefers the {100} facets as opposed to alternative facets in an O2 and H2 gas reservoir. The hydroxyl functionalisation favors the {111} surfaces in an O2 and H2 reservoir and the {100} facets in a water vapour reservoir, respectively. This facet selectivity is found to be largely dependent upon the environmental temperature, chemical reservoir, and morphology of the nanodiamonds.

  18. Evaluation of flocculating performance of a thermostable bioflocculant produced by marine Bacillus sp.

    Science.gov (United States)

    Okaiyeto, Kunle; Nwodo, Uchechukwu U; Mabinya, Leonard V; Okoli, Arinze S; Okoh, Anthony I

    2016-01-01

    This study assessed the bioflocculant (named MBF-W7) production potential of a bacterial isolate obtained from Algoa Bay, Eastern Cape Province of South Africa. The 16S ribosomal deoxyribonucleic acids gene sequence analysis showed 98% sequence similarity to Bacillus licheniformis strain W7. Optimum culture conditions for MBF-W7 production include 5% (v/v) inoculum size, maltose and NH4NO3 as carbon and nitrogen sources of choice, medium pH of 6 as the initial pH of the growth medium. Under these optimal conditions, maximum flocculating activity of 94.9% was attained after 72 h of cultivation. Chemical composition analyses showed that the purified MBF-W7 was a glycoprotein which was predominantly composed of polysaccharides 73.7% (w/w) and protein 6.2% (w/w). Fourier transform infrared spectroscopy revealed the presence of hydroxyl, carboxyl and amino groups as the main functional groups identified in the bioflocculant molecules. Thermogravimetric analyses showed the thermal decomposition profile of MBF-W7. Scanning electron microscopy imaging revealed that bridging played an important role in flocculation. MBF-W7 exhibited excellent flocculating activity for kaolin clay suspension at 0.2 mg/ml over a wide pH range of 3-11; with the maximal flocculation rate of 85.8% observed at pH 3 in the presence of Mn(2+). It maintained and retained high flocculating activity of over 70% after heating at 100°C for 60 min. MBF-W7 showed good turbidity removal potential (86.9%) and chemical oxygen demand reduction efficiency (75.3%) in Tyume River. The high flocculating rate of MBF-W7 makes it an attractive candidate to replace chemical flocculants utilized in water treatment.

  19. Identification of a haloalkaliphilic and thermostable cellulase with improved ionic liquid tolerance

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Tao; Datta, Supratim; Eichler, Jerry; Ivanova, Natalia; Axen, Seth D.; Kerfeld, Cheryl A.; Chen, Feng; Kyrpides, Nikos; Hugenholtz, Philip; Cheng, Jan-Fang; Sale, Kenneth L.; Simmons, Blake; Rubin, Eddy

    2011-02-17

    Some ionic liquids (ILs) have been shown to be very effective solvents for biomass pretreatment. It is known that some ILs can have a strong inhibitory effect on fungal cellulases, making the digestion of cellulose inefficient in the presence of ILs. The identification of IL-tolerant enzymes that could be produced as a cellulase cocktail would reduce the costs and water use requirements of the IL pretreatment process. Due to their adaptation to high salinity environments, halophilic enzymes are hypothesized to be good candidates for screening and identifying IL-resistant cellulases. Using a genome-based approach, we have identified and characterized a halophilic cellulase (Hu-CBH1) from the halophilic archaeon, Halorhabdus utahensis. Hu-CBH1 is present in a gene cluster containing multiple putative cellulolytic enzymes. Sequence and theoretical structure analysis indicate that Hu-CBH1 is highly enriched with negatively charged acidic amino acids on the surface, which may form a solvation shell that may stabilize the enzyme, through interaction with salt ions and/or water molecules. Hu-CBH1 is a heat tolerant haloalkaliphilic cellulase and is active in salt concentrations up to 5 M NaCl. In high salt buffer, Hu-CBH1 can tolerate alkali (pH 11.5) conditions and, more importantly, is tolerant to high levels (20percent w/w) of ILs, including 1-allyl-3-methylimidazolium chloride ([Amim]Cl). Interestingly, the tolerances to heat, alkali and ILs are found to be salt-dependent, suggesting that the enzyme is stabilized by the presence of salt. Our results indicate that halophilic enzymes are good candidates for the screening of IL-tolerant cellulolytic enzymes.

  20. A Novel Highly Thermostable Multifunctional Beta-Glycosidase from Crenarchaeon Acidilobus saccharovorans

    Directory of Open Access Journals (Sweden)

    Vadim M. Gumerov

    2015-01-01

    Full Text Available We expressed a putative β-galactosidase Asac_1390 from hyperthermophilic crenarchaeon Acidilobus saccharovorans in Escherichia coli and purified the recombinant enzyme. Asac_1390 is composed of 490 amino acid residues and showed high sequence similarity to family 1 glycoside hydrolases from various thermophilic Crenarchaeota. The maximum activity was observed at pH 6.0 and 93°C. The half-life of the enzyme at 90°C was about 7 hours. Asac_1390 displayed high tolerance to glucose and exhibits hydrolytic activity towards cellobiose and various aryl glucosides. The hydrolytic activity with p-nitrophenyl (pNP substrates followed the order pNP-β-D-galactopyranoside (328 U mg−1, pNP-β-D-glucopyranoside (246 U mg−1, pNP-β-D-xylopyranoside (72 U mg−1, and pNP-β-D-mannopyranoside (28 U mg−1. Thus the enzyme was actually a multifunctional β-glycosidase. Therefore, the utilization of Asac_1390 may contribute to facilitating the efficient degradation of lignocellulosic biomass and help enhance bioconversion processes.

  1. Influence of ligand binding on structure and thermostability of human alpha(1)-acid glycoprotein

    Czech Academy of Sciences Publication Activity Database

    Kopecký, V. Jr.; Ettrich, Rüdiger; Pazderka, T.; Hofbauerová, Kateřina; Řeha, David; Baumruk, V.

    2016-01-01

    Roč. 29, č. 2 (2016), s. 70-79 ISSN 0952-3499 Institutional support: RVO:61388971 Keywords : orosomucoid * binding site * Raman spectroscopy Subject RIV: CE - Biochemistry Impact factor: 2.175, year: 2016

  2. Cloning and characterization of a thermostable 2- deoxy-D-ribose-5 ...

    African Journals Online (AJOL)

    Analysis of the presumptive 2-deoxy-D-ribose 5-phosphate aldolase gene from Aciduliprofundum boonei revealed an open reading frame (ORF) encoding 222 amino acids, which was subcloned and then expressed in Escherichia coli. The recombinant DERA protein was purified to apparent homogeneity. The enzyme ...

  3. Structure-function relationships governing activity and stability of a DNA alkylation damage repair thermostable protein.

    Science.gov (United States)

    Perugino, Giuseppe; Miggiano, Riccardo; Serpe, Mario; Vettone, Antonella; Valenti, Anna; Lahiri, Samarpita; Rossi, Franca; Rossi, Mosè; Rizzi, Menico; Ciaramella, Maria

    2015-10-15

    Alkylated DNA-protein alkyltransferases repair alkylated DNA bases, which are among the most common DNA lesions, and are evolutionary conserved, from prokaryotes to higher eukaryotes. The human ortholog, hAGT, is involved in resistance to alkylating chemotherapy drugs. We report here on the alkylated DNA-protein alkyltransferase, SsOGT, from an archaeal species living at high temperature, a condition that enhances the harmful effect of DNA alkylation. The exceptionally high stability of SsOGT gave us the unique opportunity to perform structural and biochemical analysis of a protein of this class in its post-reaction form. This analysis, along with those performed on SsOGT in its ligand-free and DNA-bound forms, provides insights in the structure-function relationships of the protein before, during and after DNA repair, suggesting a molecular basis for DNA recognition, catalytic activity and protein post-reaction fate, and giving hints on the mechanism of alkylation-induced inactivation of this class of proteins. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Production of a Thermostable Α-Amylase and its Assay using ...

    African Journals Online (AJOL)

    Screening for amylolytic properties from obtained isolates was carried out on starch agar plates while production and characterization of α-amylase was carried out using submerged fermentation. The isolated organism was identified as Bacillus licheniformis. Physiological studies on the isolate showed that temperature of ...

  5. Different thermostabilities of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPases from rabbit and trout muscles.

    Science.gov (United States)

    de Toledo, F G; Albuquerque, M C; Goulart, B H; Chini, E N

    1995-05-01

    Trout and rabbit (Ca2+ + Mg2+)-ATPases from sarcoplasmic reticulum were compared for differences in thermal inactivation and susceptibility to trypsin digestion. The trout ATPase is more heat-sensitive than the rabbit ATPase and is stabilized by Ca2+, Na+, K+ and nucleotides. Solubilization of both ATPases shows that the two ATPases have different protein-intrinsic inactivation kinetics. When digested by trypsin, the two ATPases display different cleavage patterns. The present results indicate that the trout and rabbit ATPases have dissimilarities in protein structure that may explain the differences in thermal inactivation kinetics.

  6. Contact materials for thermostable resistors on the base of Ni-Re alloy

    International Nuclear Information System (INIS)

    Yusipov, H.Yu.; Glasman, L.I.; Arskaya, E.P.; Lazarev, Eh.M.; Korotkov, N.A.

    1979-01-01

    Given are the electron diffraction analysis results and the operational characteristics of the contact materials, used in the heat-resistant thin-filmed resistors (TFR), made on the basis of the Ni-Re system alloy. The results are compared with the pure nickel. Operational tests of the thin-filmed resistors, having (NR10-VP) alloy contacts, showed that the departure of the resistors nominals is almost twice as small as that for the resistors, having pure nickel contacts. The use of this alloy permits to increase the thermal stability and durability of the TFRs, if they are used under extreme conditions

  7. Optical bistability of a thin film of resonant atoms in a phase-sensitive thermostate

    International Nuclear Information System (INIS)

    Basharov, A.M.

    1995-01-01

    It is shown theoretically that when a thin film of two-level atoms interacting with a resonant coherent electromagnetic wave is additionally illuminated with a squeezed field, a bistable transmission/reflection regime for coherent waves is obtained. This regime depends strongly on the phase difference between the coherent and the squeezed fields. New regimes, including a bistable regime, for the interaction of a coherent field with a film of resonant atoms are predicted based on this phenomenon. 14 refs., 5 figs

  8. Thermostability of the coating, antigen and immunostimulator in an adjuvanted oral capsule vaccine formulation.

    Science.gov (United States)

    Longet, Stephanie; Aversa, Vincenzo; O'Donnell, Daire; Tobias, Joshua; Rosa, Monica; Holmgren, Jan; Coulter, Ivan S; Lavelle, Ed C

    2017-12-20

    Oral vaccines present an attractive alternative to injectable vaccines for enteric diseases due to ease of delivery and the induction of intestinal immunity at the site of infection. However, susceptibility to gastrointestinal proteolysis, limited transepithelial uptake and a lack of clinically acceptable adjuvants present significant challenges. A further challenge to mass vaccination in developing countries is the very expensive requirement to maintain the cold chain. We recently described the effectiveness of a Single Multiple Pill ® (SmPill ® ) adjuvanted capsule approach to enhance the effectiveness of a candidate enterotoxigenic Escherichia coli (ETEC) oral vaccine. Here it was demonstrated that this delivery system maintains the antigenicity of ETEC colonisation factor antigen I (CFA/I) and the immunostimulatory activity of the orally active α-Galactosylceramide (α-GalCer) adjuvant after storage of SmPill ® minispheres under room temperature and extreme storage conditions for several months. In addition, the internal structure of the cores of SmPill ® minispheres and antigen release features at intestinal pH were found to be preserved under all these conditions. However, changes in the surface morphology of SmPill ® minispheres leading to the antigen release at gastric pH were observed after a few weeks of storage under extreme conditions. Those modifications were prevented by the introduction of an Opadry ® White film coating layer between the core of SmPill ® minispheres and the enteric coating. Under these conditions, protection against antigen release at gastric pH was maintained even under high temperature and humidity conditions. These results support the potential of the SmPill ® minisphere approach to maintain the stability of an adjuvanted whole cell killed oral vaccine formulation. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Generalized Langevin dynamics of a nanoparticle using a finite element approach: Thermostating with correlated noise

    Science.gov (United States)

    Uma, B.; Swaminathan, T. N.; Ayyaswamy, P. S.; Eckmann, D. M.; Radhakrishnan, R.

    2011-09-01

    A direct numerical simulation (DNS) procedure is employed to study the thermal motion of a nanoparticle in an incompressible Newtonian stationary fluid medium with the generalized Langevin approach. We consider both the Markovian (white noise) and non-Markovian (Ornstein-Uhlenbeck noise and Mittag-Leffler noise) processes. Initial locations of the particle are at various distances from the bounding wall to delineate wall effects. At thermal equilibrium, the numerical results are validated by comparing the calculated translational and rotational temperatures of the particle with those obtained from the equipartition theorem. The nature of the hydrodynamic interactions is verified by comparing the velocity autocorrelation functions and mean square displacements with analytical results. Numerical predictions of wall interactions with the particle in terms of mean square displacements are compared with analytical results. In the non-Markovian Langevin approach, an appropriate choice of colored noise is required to satisfy the power-law decay in the velocity autocorrelation function at long times. The results obtained by using non-Markovian Mittag-Leffler noise simultaneously satisfy the equipartition theorem and the long-time behavior of the hydrodynamic correlations for a range of memory correlation times. The Ornstein-Uhlenbeck process does not provide the appropriate hydrodynamic correlations. Comparing our DNS results to the solution of an one-dimensional generalized Langevin equation, it is observed that where the thermostat adheres to the equipartition theorem, the characteristic memory time in the noise is consistent with the inherent time scale of the memory kernel. The performance of the thermostat with respect to equilibrium and dynamic properties for various noise schemes is discussed.

  10. The use of thermostable bacterial hemicellulases improves the conversion of lignocellulosic biomass to valuable molecules.

    Science.gov (United States)

    Rakotoarivonina, Harivony; Revol, Pierre-Vincent; Aubry, Nathalie; Rémond, Caroline

    2016-09-01

    The hydrolysis of xylans, one of the main classes of carbohydrates that constitute lignocellulosic biomass, requires the synergistic action of several enzymes. The development of efficient enzymatic strategies for hydrolysis remains a challenge in the pursuit of viable biorefineries, particularly with respect to the valorisation of pentoses. The approach developed in this work is based on obtaining and characterising hemicellulasic cocktails from Thermobacillus xylanilyticus after culturing this bacterium on the hemicellulose-rich substrates wheat bran and wheat straw, which differ in their chemistries. The two obtained cocktails (WSC and WBC, for cocktails obtained from wheat straw and wheat bran, respectively) were resistant to a broad range of temperature and pH conditions. At 60 °C, both cocktails efficiently liberated pentoses and phenolic acids from wheat bran (liberating more than 60, 30 and 40 % of the total xylose, arabinose and ferulic acid in wheat bran, respectively). They acted to a lesser extent on the more recalcitrant wheat straw, with hydrolytic yields of more than 30 % of the total arabinose and xylose content and 22 % of the ferulic acid content. Hydrolysis is associated with a high rate of sugar monomerisation. When associated with cellulases, high quantities of glucose were also obtained. On wheat bran, total glucose yields were improved by 70 % compared to the action of cellulases alone. This improvement was obtained by cellulase complementation either with WSC or with WBC. On wheat straw, similar levels of total glucose were obtained for cellulases alone or complemented with WSC or WBC. Interestingly, the complementation of cellulases with WSC or WBC induced an increase in the monomeric glucose yield of more than 20 % compared to cellulases alone.

  11. Purification and characterization of a thermostable alkaline cellulase produced by Bacillus licheniformis 380 isolated from compost

    Directory of Open Access Journals (Sweden)

    ÉVILIN G. DE MARCO

    2017-10-01

    Full Text Available ABSTRACT During composting processes, the degradation of organic waste is accomplished and driven by a succession of microbial populations exhibiting a broad range of functional competencies. A total of 183 bacteria, isolated from a composting process, were evaluated for cellulase activity at different temperatures (37, 50, 60, and 70°C and pH values. Out of the 22 isolates that showed activity, isolate 380 showed the highest cellulase activity. Its ability to produce cellulase was evaluated in culture medium supplemented with carboxymethyl cellulose, microcrystalline cellulose, wheat straw, and rice husk. The culture medium supplemented with carboxymethyl cellulose induced higher enzyme activity after 6 hours of incubation (0.12 UEA mL-1 min-1. For wheat straw and rice husk, the results were 0.08 UEA mL-1 min-1 for both, while for microcrystalline cellulose, 0.04 UEA mL-1 min-1 were observed. The highest carboxymethyl cellulase activity was observed at 60°C (0.14 UEA mL-1 min-1 for both crude and partially purified enzyme after 30 and 120 min of incubation, respectively. Alkalinization of the medium was observed during cultivation in all substrates. The cellulase had a molecular mass of 20 kDa determined by SDS-Page. Isolate 380 was identified as Bacillus licheniformis. This work provides a basis for further studies on composting optimization.

  12. Thermostable phycocyanin from the red microalga Cyanidioschyzon merolae, a new natural blue food colorant

    NARCIS (Netherlands)

    Rahman, D. Y.; Sarian, F. D.; van Wijk, A.; Martinez-Garcia, M.; van der Maarel, M. J. E. C.

    The demand for natural food colorants is growing as consumers question the use of artificial colorants more and more. The phycobiliprotein C-phycocyanin of Arthospira platensis is used as a natural blue colorant in certain food products. The thermoacidophilic red microalga Cyanidioschyzon merolae

  13. Improvement of thermostability and activity of firefly luciferase through [TMG][Ac] ionic liquid mediator.

    Science.gov (United States)

    Ebrahimi, Mehdi; Hosseinkhani, Saman; Heydari, Akbar; Khavari-Nejad, Ramazan Ali; Akbari, Jafar

    2012-10-01

    Firefly luciferase catalyzes production of light from luciferin in the presence of Mg(2+)-ATP and oxygen. This enzyme has wide range of applications in biotechnology and development of biosensors. The low thermal stability of wild-type firefly luciferase is a limiting factor in most applications. Improvements in activity and stability of few enzymes in the presence of ionic liquids were shown in many reports. In this study, kinetic and thermal stability of firefly luciferase from Photinus pyralis in the presence of three tetramethylguanidine-based ionic liquids was investigated. The enzyme has shown improved activity in the presence of [1, 1, 3, 3-tetramethylguanidine][acetate], but in the presence of [TMG][trichloroacetate] and [TMG][triflouroacetate] activity, it decreased or unchanged significantly. Among these ionic liquids, only [TMG][Ac] has increased the thermal stability of luciferase. Incubation of [TMG][Ac] with firefly luciferase brought about with decrease of K(m) for ATP.

  14. A thermostable Gloeophyllum trabeum xylanase with potential for the brewing industry.

    Science.gov (United States)

    Wang, Xiaoyu; Luo, Huiying; Yu, Wangning; Ma, Rui; You, Shuai; Liu, Weina; Hou, Lingyu; Zheng, Fei; Xie, Xiangming; Yao, Bin

    2016-05-15

    A xylanase gene of glycoside hydrolase family 10, GtXyn10, was cloned from Gloeophyllum trabeum CBS 900.73 and expressed in Pichia pastoris GS115. Purified recombinant GtXyn10 exhibited significant activities to xylan (100.0%), lichenan (11.2%), glucan (15.2%) and p-nitrophenol-β-cellobiose (18.6%), demonstrated the maximum xylanase and glucanase activities at pH 4.5-5.0 and 75°C, retained stability over the pH range of 2.0-7.5 and at 70°C, and was resistant to pepsin and trypsin, most metal ions and SDS. Multiple sequence alignment and modeled-structure analysis identified a unique Gly48 in GtXyn10, and site-directed mutagenesis of Gly48 to Lys improved the temperature optimum up to 80°C. Under simulated mashing conditions, GtXyn10 (80U) reduced the mash viscosity by 12.8% and improved the filtration rate by 31.3%. All these properties above make GtXyn10 attractive for potential applications in the feed and brewing industries. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Antimicrobial activity and high thermostability of a novel blissecreted by enterococcus mundtiiisolated from Lebanese cow's milk

    International Nuclear Information System (INIS)

    Al Kassaa, I.; Safourim, N.; Mostafa, N.; Hamze, M.

    2016-01-01

    Lactic acid bacteria (LAB) are used in many fields such as fermentation agents, increasing nutritional value and improving organoleptic quality of food. Also they are use das probiotics and preservatives against pathogensand spoilage microbes by producing antimicrobial substances such as bacteriocins. Fifty cow's milk samples were collected and 175 LAB isolates were isolated and identified by using biochemical method. Fifteen isolates showedan antimicrobial activity against Listeria monocytogenesATCC® 19115™.One strain, BL4 which showed the strongest activity, was chosen to extract and characterize its antimicrobial substance in order to evaluate its potential use as a new food protective agent. This strain was identified as Enterococcus mundtiibypyrosequencing method.The active substance was extracted using solvent method.This Bacteriocin likeInhibitory Substances “BLIS”can support a high temperature (121 °C) for a long time and resist pH variation.The BLIS BL4 can be considered as a peptide active against many food pathogen and food-spoilage microbes, such as Listeriamonocytogenes and Penicillium spp. BLISBL4can be used in food application as bio-preservative to reduce food-spoilage and food-borne diseases in food product. (author

  16. Purification, crystallization and preliminary crystallographic analysis of a thermostable endonuclease IV from Thermotoga maritima

    International Nuclear Information System (INIS)

    Hughes, Ronny C.; Tomanicek, Stephen J.; Ng, Joseph D.; Coates, Leighton

    2009-01-01

    The overexpression, purification and crystallization of endonuclease IV from T. maritima are reported. The crystals belonged to the hexagonal space group P6 1 and diffracted to 2.36 Å resolution. The DNA-repair enzyme endonuclease IV from the thermophilic bacterium Thermotoga maritima MSB8 (reference sequence NC-000853) has been expressed in Escherichia coli and crystallized for X-ray analysis. T. maritima endonuclease IV is a 287-amino-acid protein with 32% sequence identity to E. coli endonuclease IV. The protein was purified to homogeneity and was crystallized using the sitting-drop vapor-diffusion method. The protein crystallized in space group P6 1 , with one biological molecule in the asymmetric unit, corresponding to a Matthews coefficient of 2.39 Å 3 Da −1 and 47% solvent content. The unit-cell parameters of the crystals were a = b = 123.2, c = 35.6 Å. Microseeding and further optimization yielded crystals with an X-ray diffraction limit of 2.36 Å. A single 70° data set was collected and processed, resulting in an overall R merge and a completeness of 9.5% and 99.3%, respectively

  17. TtMCO: A highly thermostable laccase-like multicopper oxidase from the thermophilic Thermobaculum terrenum

    DEFF Research Database (Denmark)

    Brander, Søren; Mikkelsen, Jørn Dalgaard; Kepp, Kasper Planeta

    2015-01-01

    This paper reports the identification, heterologous expression in Escherichia coli and characterization of TtMCO from the thermophilic bacterium Thermobaculum terrenum, the first laccase-like multi-copper oxidase (LMCO) from the distinct Phylum Chloroflexi. TtMCO has only 39% identity to its...... closest characterized homologue, CotA from Bacillus subtilis, but sequence and spectrophotometry confirmed copper coordination similar to that of LMCOs. TtMCO is extremely thermophilic with a half-time of inactivation of 2.24 days at 70 degrees C and 350 min at 80°C and pH 7, consistent...

  18. Bioprospecting of Thermostable Cellulolytic Enzymes through Modeling and Virtual Screening Method

    Directory of Open Access Journals (Sweden)

    R. Navanietha Krishnaraj

    2017-04-01

    Full Text Available Cellulolytic enzymes are promising candidates for the use of cellulose in any bioprocess operations and for the disposal of the cellulosic wastes in an environmentally benign manner. Cellulases from thermophiles have the advantage of hydrolyzing cellulose at wider range of operating conditions unlike the normal enzymes. Herein we report the modeled structures of cellulolytic enzymes (endoglucanase, cellobiohydrolase and ß-glucosidase from a thermophilic bacterium,Clostridium thermocellumand their validation using Root Mean Square Deviation (RMSD and Ramachandran plot analyses. Further, the molecular interactions of the modeled enzyme with cellulose were analyzed using molecular docking technique. The results of molecular docking showed that the endoglucanase, cellobiohydrolase and ß-glucosidase had the binding affinities of -10.7, -9.0 and -10.8 kcal/mol, respectively. A correlation between the binding affinity of the endoglucanase with cellulose and the enzyme activity was also demonstrated. The results showed that the binding affinities of cellulases with cellulose could be used as a tool to assess the hydrolytic activity of cellulases. The results obtained could be used in virtual screening of cellulolytic enzymes based on the molecular interactions with the substrate, and aid in developing systems biology models of thermophiles for industrial biotechnology applications.

  19. Thermostable amylolytic enzymes from a cellulolytic fungus Myceliophthora thermophila D14 (ATCC 48 104)

    Energy Technology Data Exchange (ETDEWEB)

    Sadhukhan, R K; Manna, S; Roy, S K; Chakrabarty, S L [Bose Research Inst., Calcutta (India). Dept. of Microbiology

    1990-09-01

    The production of amylolytic enzymes by a thermophilic cellulolytic fungus, Myceliophthora thermophila D14 was investigated by batch cultivation in Czapek-Dox medium at 45deg C. Among various nitrogenous compounds used, NaNO{sub 3} and KNO{sub 3} were found to be the best for amylase production. Starch, cellobiose and maltose induced the synthesis of amylase while glucose, fructose, galactose, lactose, arabinose, xylose, sorbitol, mesoinositol and sucrose did not. Calcium ions had the most stimulating effect on enzyme formation amongst many ions investigated. The synthesis of amylolytic enzymes was dependent on growth and occurred predominantly in the mid-stationary phase. The enzyme was active in a broad temperature range (50deg C-60deg C) and displayed activity optima at 60deg C and pH 5.6. (orig.).

  20. Production of a thermostable B-glucosidase by a mesophilic fungus aspergillus niger NIAB 280

    International Nuclear Information System (INIS)

    Iqbal, M.J.; Rajoka, M.I.; Malik, K.A.

    1991-01-01

    Aspergillus niger NIAB 280 produced extracellular B- glucosidease when grown on different ligno cellulosic substrates. Wheat bran was found to be the best inducer of this enzyme. Maximum enzyme production was found at initial pH 5. When the organisms was grown on easily available carbohydrates, the enzyme was induced only to a basic level essential for microbial growth. The optimum pH for enzyme activity was found to be 3 whereas optimum temperature was 55 degree C. The enzyme retained 100% activity when exposed to a temperature of 50 degree C for 5 days. When this enzyme was added to FPase from T. reesei and celluclast (a commercial cellulase from Novazyme), the %age saccharification as well as glucose production from lignocellulosic biomass was doubled. (author)

  1. Recombinant thermostable AP exonuclease from Thermoanaerobacter tengcongensis: cloning, expression, purification, properties and PCR application

    DEFF Research Database (Denmark)

    Dabrowski, Slawomir; Brillowska-Dabrowska, Anna; Ahring, Birgitte Kiær

    2013-01-01

    Apurinic/apyrimidinic (AP) sites in DNA are considered to be highly mutagenic and must be corrected to preserve genetic integrity, especially at high temperatures. The gene encoding a homologue of AP exonuclease was cloned from the thermophilic anaerobic bacterium Thermoanaerobacter tengcongensis......) of fully active and soluble His6-tagged Tte AP enzyme with His6-tag on C-terminal end was obtained in Escherichia coli Rosetta (DE3) pLysS. The active enzyme was purified up to 98% homogeneity in one chromatographic step using metal-affinity chromatography on Ni(2+)-IDA-Sepharose resin. The yield was 90 mg......, pH 8.0 and at low Mg2+ concentration (0.5 mM). Higher Mg2+ concentration (> 1 mM) enhanced 3'-5' exonuclease activity and at Mg2+ concentration > 2.0 mM 3' nuclease activity was observed. Because of the endonuclease activity of Tte AP exonuclease, the enzyme was applied in PCR amplification of long...

  2. Effect of mutations on the thermostability of Aspergillus aculeatus β-1, 4-galactanase

    DEFF Research Database (Denmark)

    Terpenholt, Søs; De Maria, Leonardo; Olsson, Mats H.M.

    2015-01-01

    New variants of β-1,4-galactanase from the mesophilic organism Aspergillus aculeatus were designed using the structure of β-1,4-galactanase from the thermophile organism Myceliophthora thermophila as a template. Some of the variants were generated using PROPKA 3.0, a validated pKa prediction tool......, to test its usefulness as an enzyme design tool. The PROPKA designed variants were D182N and S185D/Q188T, G104D/A156R. Variants Y295F and G306A were designed by a consensus approach, as a complementary and validated design method. D58N was a stabilizing mutation predicted by both methods. The predictions......R. The Tm changes were in the range predicted by PROPKA. Given the experimental errors, only the D58N and G306A show significant increase in thermodynamic stability. Given the practical importance of kinetic stability, the kinetics of the irreversible enzyme inactivation process were also...

  3. Investigation of the Thermostability of Bovine Submaxillary Mucin (BSM) and its Impact on Lubrication

    DEFF Research Database (Denmark)

    Madsen, Jan Busk; Pakkanen, Kirsi I.; Lee, Seunghwan

    2013-01-01

    Bovine Submaxillary Mucin (BSM) generates thin film layers via spontaneous adsorption onto hydrophobic surfaces such as Poly(dimethylsiloxane) (PDMS) and High Density Polyethylene (HDPE). A characteristic feature of mucin is its tribological- or lubricating properties. Circular dichroismspectrosc......Bovine Submaxillary Mucin (BSM) generates thin film layers via spontaneous adsorption onto hydrophobic surfaces such as Poly(dimethylsiloxane) (PDMS) and High Density Polyethylene (HDPE). A characteristic feature of mucin is its tribological- or lubricating properties. Circular...

  4. Characterizing a thermostable Cas9 for bacterial genome editing and silencing

    DEFF Research Database (Denmark)

    Mougiakos, Ioannis; Mohanraju, Prarthana; Bosma, Elleke Fenna

    2017-01-01

    CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including...

  5. ASP53, a thermostable protein from Acacia erioloba seeds that protects target proteins against thermal denaturation

    CSIR Research Space (South Africa)

    Mtwisha, L

    2007-02-01

    Full Text Available ) and the Typha pollen D7 protein was found to stabilise sugar glasses in an in vitro system (Wolkers et al. 2001). The cupin family of proteins comprises a wide variety of proteins from both prokaryotes and eukaryotes and includes the seed storage proteins...–268. Garay-Arroyo A, Colmenero-Flores JM, Garciarrubio A, Covarrubias AA (2000) Highly hydrophilic proteins in prokaryotes and eukaryotes are common during conditions of water deficit. Journal of Biological Chemistry 275, 5668–5674. doi: 10.1074/jbc.275...

  6. SAT2 Foot-and-Mouth Disease Virus Structurally Modified for Increased Thermostability.

    Science.gov (United States)

    Scott, Katherine A; Kotecha, Abhay; Seago, Julian; Ren, Jingshan; Fry, Elizabeth E; Stuart, David I; Charleston, Bryan; Maree, Francois F

    2017-05-15

    Foot-and-mouth disease virus (FMDV), particularly strains of the O and SAT serotypes, is notoriously unstable. Consequently, vaccines derived from heat-labile SAT viruses have been linked to the induction of immunity with a poor duration and hence require more frequent vaccinations to ensure protection. In silico calculations predicted residue substitutions that would increase interactions at the interpentamer interface, supporting increased stability. We assessed the stability of the 18 recombinant mutant viruses in regard to their growth kinetics, antigenicity, plaque morphology, genetic stability, and temperature, ionic, and pH stability by using Thermofluor and inactivation assays in order to evaluate potential SAT2 vaccine candidates with improved stability. The most stable mutant for temperature and pH stability was the S2093Y single mutant, while other promising mutants were the E3198A, L2094V, and S2093H single mutants and the F2062Y-H2087M-H3143V triple mutant. Although the S2093Y mutant had the greatest stability, it exhibited smaller plaques, a reduced growth rate, a change in monoclonal antibody footprint, and poor genetic stability properties compared to those of the wild-type virus. However, these factors affecting production can be overcome. The addition of 1 M NaCl was found to further increase the stability of the SAT2 panel of viruses. The S2093Y and S2093H mutants were selected for future use in stabilizing SAT2 vaccines. IMPORTANCE Foot-and-mouth disease virus (FMDV) causes a highly contagious acute vesicular disease in cloven-hoofed livestock and wildlife. The control of the disease by vaccination is essential, especially at livestock-wildlife interfaces. The instability of some serotypes, such as SAT2, affects the quality of vaccines and therefore the duration of immunity. We have shown that we can improve the stability of SAT2 viruses by mutating residues at the capsid interface through predictive modeling. This is an important finding for the potential use of such mutants in improving the stability of SAT2 vaccines in countries where FMD is endemic, which rely heavily on the maintenance of the cold chain, with potential improvement to the duration of immune responses. Copyright © 2017 American Society for Microbiology.

  7. Glycoforms of B-Lactoglobulin with improved thermostability and preserved structural packing.

    NARCIS (Netherlands)

    Broersen, K.; Voragen, A.G.J.; Hamer, R.J.; Jongh, de H.H.J.

    2004-01-01

    In this article we show how various degrees of glycosylation can be used to control the thermal stability of proteins. The primary amines of -lactoglobulin were glycosylated with glucose or fructose within a range of non-denaturing reaction parameters. The modified fractions were characterized and

  8. Functional and structural characterization of a thermostable acetyl esterase from Thermotoga maritima

    NARCIS (Netherlands)

    Levisson, M.; Han, G.W.; Deller, M.C.; Hendriks, S.N.A.; Oost, van der J.; Kengen, S.W.M.

    2012-01-01

    TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100°C and pH 7.5. The positional specificity

  9. Production, Characterization and Application of a Thermostable Tannase from Pestalotiopsis guepinii URM 7114.

    Science.gov (United States)

    Reges de Sena, Amanda; Claúdia de Barros Dos Santos, Ana; Gouveia, Miquéas Jamesse; Figueira de Mello, Marcelo Rodrigues; Leite, Tonny Cley Campos; Moreira, Keila Aparecida; Aparecida de Assis, Sandra

    2014-12-01

    Tannase (EC 3.1.1.20) is an enzyme that hydrolyzes the ester and depside bonds of tannic acid to gallic acid and glucose. In the production of foods and beverages, it contributes to the removal of the undesirable effects of tannins. The aim of this study is to investigate the potential of endophytic fungi isolated from jamun ( Syzygium cumini (L.) Skeels) leaves, and identified as Pestalotiopsis guepinii , in the production of tannase. Tannase was produced extracellularly by P. guepinii under submerged, slurry-state and solid-state fermentations. The submerged fermentation was found to be the most promising (98.6 U/mL). Response surface methodology was employed to evaluate the effect of variables (pH and temperature), and the results showed that the best conditions for tannase activity were pH=6.9 and 30 °C. K m was found to be 7.18·10 -4 mol/L and v max =250.00 U/mL. The tannase activity was the highest in the presence of Ca 2+ at a concentration of 5·10 -3 mol/L. Moreover, the enzyme was not inhibited by the tested chelators and detergents. The stability of the enzyme was also studied, and crude enzyme was evaluated in simulation of gastrointestinal digestion of monogastric animals. The crude enzyme was highly stable under simulated conditions; it retained 87.3% of its original activity after 6 h. The study contributes to the identification of microbial species that produce tannase, with potential application in biotechnology.

  10. Purification and biochemical characterization of a highly thermostable bacteriocin isolated from Brevibacillus brevis strain GM100.

    Science.gov (United States)

    Ghadbane, Mouloud; Harzallah, Daoud; Laribi, Atef Ibn; Jaouadi, Bassem; Belhadj, Hani

    2013-01-01

    A bacteriocin-producing (11,000 AU mL(-1)) strain was isolated from the rhizosphere of healthy Algerian plants Ononis angustissima Lam., and identified as Brevibacillus brevis strain GM100. The bacteriocin, called Bac-GM100, was purified to homogeneity from the culture supernatant, and, based on MALDI-TOF/MS analysis, was a monomer protein with a molecular mass of 4375.66 Da. The 21 N-terminal residues of Bac-GM100 displayed 65% homology with thurincin H from Bacillus thuringiensis. Bac-GM100 was extremely heat-stable (20 min at 120 °C), and was stable within a pH range of 3-10. It proved sensitive to various proteases, which demonstrated its protein nature. It was also found to display a bactericidal mode of action against gram-negative (Salmonella enteric ATCC 43972, Pseudomonas aeruginosa ATCC 49189, and Agrobacterium tumefaciens C58) and gram-positive (Enterococcus faecalis ENSAIA 631 and Staphylococcus aureus ATCC 6538) bacteria, and a fungistatic mode of action against the pathogenic fungus Candida tropicalis R2 CIP 203.

  11. Influence of drying method on the material properties of nanocellulose I: thermostability and crystallinity

    Science.gov (United States)

    Yucheng Peng; Douglas J. Gardner; Yousoo Han; Alper Kiziltas; Zhiyong Cai; Mandla A. Tshabalala

    2013-01-01

    The effect of drying method on selected material properties of nanocellulose was investigated. Samples of nanofibrillated cellulose (NFC) and cellulose nanocrystals (CNC) were each subjected to four separate drying methods: air-drying, freeze-drying, spray-drying, and supercritical-drying. The thermal stability and crystallinity of the dried nanocellulose were...

  12. Dissipative particle dynamics: Effects of thermostating schemes on nano-colloid electrophoresis

    Science.gov (United States)

    Hassanzadeh Afrouzi, Hamid; Moshfegh, Abouzar; Farhadi, Mousa; Sedighi, Kurosh

    2018-05-01

    A novel fully explicit approach using dissipative particle dynamics (DPD) method is introduced in the present study to model the electrophoretic transport of nano-colloids in an electrolyte solution. Slater type charge smearing function included in 3D Ewald summation method is employed to treat electrostatic interaction. Performance of various thermostats are challenged to control the system temperature and study the dynamic response of colloidal electrophoretic mobility under practical ranges of external electric field (0 . 072 relationships respectively with electric field and colloidal repulsion; although they each respectively behave direct and inverse trends with salt concentration under various thermostats. Nosé-Hoover-Lowe-Andersen and Lowe-Andersen thermostats are found to function more effectively under high electric fields (E > 0 . 145[v/nm ]) while thermal equilibrium is maintained. Reasonable agreements are achieved by benchmarking the system radial distribution function with available EW3D modellings, as well as comparing reduced mobility against conventional Smoluchowski and Hückel theories, and numerical solution of Poisson-Boltzmann equation.

  13. RNA versatility, flexibility, and thermostability for practice in RNA nanotechnology and biomedical applications.

    Science.gov (United States)

    Haque, Farzin; Pi, Fengmei; Zhao, Zhengyi; Gu, Shanqing; Hu, Haibo; Yu, Hang; Guo, Peixuan

    2018-01-01

    In recent years, RNA has attracted widespread attention as a unique biomaterial with distinct biophysical properties for designing sophisticated architectures in the nanometer scale. RNA is much more versatile in structure and function with higher thermodynamic stability compared to its nucleic acid counterpart DNA. Larger RNA molecules can be viewed as a modular structure built from a combination of many 'Lego' building blocks connected via different linker sequences. By exploiting the diversity of RNA motifs and flexibility of structure, varieties of RNA architectures can be fabricated with precise control of shape, size, and stoichiometry. Many structural motifs have been discovered and characterized over the years and the crystal structures of many of these motifs are available for nanoparticle construction. For example, using the flexibility and versatility of RNA structure, RNA triangles, squares, pentagons, and hexagons can be constructed from phi29 pRNA three-way-junction (3WJ) building block. This review will focus on 2D RNA triangles, squares, and hexamers; 3D and 4D structures built from basic RNA building blocks; and their prospective applications in vivo as imaging or therapeutic agents via specific delivery and targeting. Methods for intracellular cloning and expression of RNA molecules and the in vivo assembly of RNA nanoparticles will also be reviewed. WIREs RNA 2018, 9:e1452. doi: 10.1002/wrna.1452 This article is categorized under: RNA Methods > RNA Nanotechnology RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA in Disease and Development > RNA in Disease Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs. © 2017 Wiley Periodicals, Inc.

  14. Thermostability of IFN-γ and IP-10 release assays for latent infection with Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Blauenfeldt, Thomas; Wagner, Dirk; Aabye, Martine Grosos

    2016-01-01

    accuracy of IP-10 release assay and IGRAs. RESULTS: We included 65 patients with confirmed pulmonary tuberculosis and 160 healthy controls from 6 European centres collaborating in the TBnet. In patients, IP-10 responses increased 1.07 (IQR 0.90-1.36) fold and IFN-γ responses decreased 0.88 (IQR 0......INTRODUCTION: Interferon-γ (IFN-γ) inducible protein 10kD (IP-10) and IFN-γ release assays (IGRAs) are immunodiagnostic tests aiming to identify the presence of specific cellular immune responses, interpreted as markers for latent infection with Mycobacterium tuberculosis. Incubation at higher...

  15. Cell membrane thermo-stability studies through joint segregation analysis in various wheat populations

    International Nuclear Information System (INIS)

    Ullah, K.; Khan, N.U.; Gul, S.; Khan, S.J.

    2014-01-01

    Using joint segregation analysis (JSA) technique as statistical approach, mixed inheritance analysis for cell plasma membrane as membrane thermal stability (MTS) was assayed in two parental lines (P1, P2) and their four populations (F1, BC1, BC2, F2) of four wheat crosses, viz., Hashim-08 * LU-26, Farid-06 * Shafaq, Parula * Blue Silver and TD-1 * D-97603 at Faculty of Agriculture, Gomal University, Dera Ismail Khan, Pakistan during crop season 2011-12. Results revealed that MTS was under control of two mixed groups of genes i.e., additive-dominant-epistatic major genes plus additive-dominant-epistasis of polygenes (model E) in Hashim-08 * LU-26 and Farid-06 * Shafaq crosses, respectively. In cross Parula * Blue Silver, it was governed by mixed genes i.e. one major-gene and additive-dominance-epistatic polygenes (model D). However, in cross TD-1 * D-97603, the MTS was under the influence of mixed epistasis of two major genes plus polygenes (model E-1). Polygene variation and polygene heritability were higher than major gene variation and heritability in crosses Hashim-08 * LU-26 and Farid-06 * Shafaq. In crosses Parula * Blue Silver and TD-1 * D-97603, the major gene variation and heritability were higher than polygene variation and heritability, indicating maximum contribution of the major genes. While in cross TD-1 * D-97603, epistatic components were also positive and due to which the polygene heritability was almost zero. Moderate to high environmental variation in the MTS for segregating generations revealed that the said trait was highly persuaded by the environment. However, the genetic behavior of the MTS suggested that early selection for MTS in the crosses Hashim-08 * LU-26 and Farid-06 * Shafaq would be efficient. Whereas, the delayed selection in crosses Parula * Blue Silver and TD-1 * D-97603 until the accumulation of maximum favorable genes will be effective. (author)

  16. Purification and characterization of a thermostable hypothetical xylanase from Aspergillus oryzae HML366.

    Science.gov (United States)

    He, Haiyan; Qin, Yongling; Li, Nan; Chen, Guiguang; Liang, Zhiqun

    2015-03-01

    In the current study, fermentation broth of Aspergillus oryzae HML366 in sugar cane bagasse was subjected to ultrafiltration and ion exchange chromatography, and two xylanases, XynH1 and XynH2, were purified. Time-of-flight mass spectrometry coupled with SDS-PAGE analysis revealed that XynH1 is identical to the hypothetical A. oryzae RIB40 protein XP_001826985.1, with a molecular weight of 33.671 kDa. Likewise, XynH2 was identified as xylanase XynF1 with a molecular weight of 35.402 kDa. Sequence analysis indicated that XynH1 belongs to glycosyl hydrolases family 10. The specific activity of XynH1 was measured at 476.9 U/mg. Optimal xylanase activity was observed at pH 6.0, and enzyme remained active within pH 4.0-10.0 and at a temperature below 70 °C. Mg(2+), Mn(2+), Ca(2+), and K(+) enhanced the XynH1 xylanase activity to 146, 122, 114, and 108%, respectively. XynH1 hydrolyzed Birchwood xylan and Larchwood xylan effectively. The K m and V max of XynH1 values determined were 1.16 mM and 336 μmol/min/mg with Birchwood xylan as the substrate. A. oryzae HML366 xylanase XynH1 showed superior heat and pH tolerance, therefore may have significant applications in paper and biofuel industries. These studies constitute the first investigation of the xylanase activities of the hypothetical protein XP_001826985.1 form A. oryzae.

  17. Characterization and kinetic analysis of a thermostable GH3 ß-glucosidase from Penicillium brasilianum

    DEFF Research Database (Denmark)

    Krogh, Kristian Bertel Rømer; Harris, P.V.; Olsen, C.L.

    2010-01-01

    A GH3 beta-glucosidase (BGL) from Penicillium brasilianum was purified to homogeneity after cultivation on a cellulose and xylan rich medium. The BGL was identified in a genomic library, and it was successfully expressed in Aspergillus oryzae. The BGL had excellent stability at elevated...

  18. Phylogenetic and Comparative Sequence Analysis of Thermostable Alpha Amylases of kingdom Archea, Prokaryotes and Eukaryotes.

    Science.gov (United States)

    Huma, Tayyaba; Maryam, Arooma; Rehman, Shahid Ur; Qamar, Muhammad Tahir Ul; Shaheen, Tayyaba; Haque, Asma; Shaheen, Bushra

    2014-01-01

    Alpha amylase family is generally defined as a group of enzymes that can hydrolyse and transglycosylase α-(1, 4) or α-(1, 6) glycosidic bonds along with the preservation of anomeric configuration. For the comparative analysis of alpha amylase family, nucleotide sequences of seven thermo stable organisms of Kingdom Archea i.e. Pyrococcus furiosus (100-105°C), Kingdom Prokaryotes i.e. Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C), Bacillus amyloliquefaciens (72°C), Bacillus subtilis (70°C) and Bacillus KSM K38 (55°C) and Eukaryotes i.e. Aspergillus oryzae (60°C) were selected from NCBI. Primary structure composition analysis and Conserved sequence analysis were conducted through Bio Edit tools. Results from BioEdit shown only three conserved regions of base pairs and least similarity in MSA of the above mentioned alpha amylases. In Mega 5.1 Phylogeny of thermo stable alpha amylases of Kingdom Archea, Prokaryotes and Eukaryote was handled by Neighbor-Joining (NJ) algorithm. Mega 5.1 phylogenetic results suggested that alpha amylases of thermo stable organisms i.e. Pyrococcus furiosus (100-105°C), Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C) and Bacillus amyloliquefaciens (72°C) are more distantly related as compared to less thermo stable organisms. By keeping in mind the characteristics of most thermo stable alpha amylases novel and improved features can be introduced in less thermo stable alpha amylases so that they become more thermo tolerant and productive for industry.

  19. Isolation and Screening of Thermo-Stable Cellulase Enzyme Fungal Producer at Different Temperature

    International Nuclear Information System (INIS)

    Noor Ashiqin Jamroo; Noor Azrimi Umor; Kamsani

    2015-01-01

    Thermo stable cellulase from fungi has high potential for industrial application. In this study, wild -type of fungal were isolate from different sources such as hot spring water, sea water, soft wood, rice straw and cow dung. The isolates were characterized by cultural and morphological observation. Based on morphological characteristics, the genera of all fungal cultures were identified namely Aspergillus fumigatus. The screening for thermo stable cellulase were done using 2 % carboxymethyl cellulose and congo red as an indicator at temperature 30, 37, 45 and 50 degree Celsius respectively. Out of 26 fungal isolates, only eight isolates were selected for further screening and showed the abilities to secrete cellulases by forming distinct halo zones on selective agar plate. The maximum halo zone ranging from 32 mm to 35 mm were obtained after 72 hour incubation at 50 degree Celsius by H2, SW1 and C1 isolates. As contrary other isolates showed halo zone range from 22 mm to 29 mm at same temperature. All the isolates showed the abilities to secrete cellulase enzyme at other temperature but lower when compared to 50 degree Celsius referred to the halo zone obtained. The SW1 isolates showed highest cellulolytic index which was 2.93 measured at 37 degree Celsius and 2.67 at 50 degree Celsius respectively. (author)

  20. Production and application of a thermostable lipase from Serratia marcescens in detergent formulation and biodiesel production.

    Science.gov (United States)

    García-Silvera, Edgar Edurman; Martínez-Morales, Fernando; Bertrand, Brandt; Morales-Guzmán, Daniel; Rosas-Galván, Nashbly Sarela; León-Rodríguez, Renato; Trejo-Hernández, María R

    2018-03-01

    In this study, extracellular lipase was produced by Serratia marcescens wild type and three mutant strains. The maximum lipase activity (80 U/mL) was obtained with the SMRG4 mutant strain using soybean oil. Using a 2 2 factorial design, the lipase production increased 1.55-fold (124 U/mL) with 4% and 0.05% of soybean oil and Triton X-100, respectively. The optimum conditions for maximum lipase activity were 50 °C and pH 8. However, the enzyme was active in a broad range of pH (6-10) and temperatures (5-55 °C). This lipase was stable in organic solvents and in the presence of oxidizing agents. The enzyme also proved to be efficient for the removal of triacylglycerol from olive oil in cotton cloth. A Box-Behnken experimental design was used to evaluate the effects of the interactions between total lipase activity, buffer pH, and wash temperatures on oil removal. The model obtained suggested that all selected factors had a significant impact on oil removal, with optimum conditions of 550 U lipase, 45 °C, pH 9.5, with 79.45% removal. Biotransformation of waste frying oil using the enzyme and in presence of methanol resulted in the synthesis of methyl esters such as methyl oleate, methyl palmitate, and methyl stearate. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

  1. Thermostable, alkaline and detergent-tolerant lipase from a newly isolated thermophilic Bacillus stearothermophilus.

    Science.gov (United States)

    Ben Bacha, Abir; Moubayed, Nadine M S; Abid, Islam

    2015-04-01

    Lipases are the enzymes of choice for laundry detergent industries, owing to their triglyceride removing ability from the soiled fabric, which eventually reduces the usage of phosphate-based chemical cleansers in the detergent formulation. In this study, a novel thermo-alkaline lipase-producing strain identified as Bacillus stearothermophilus was isolated from the soil samples of olive oil mill. Enhanced lipase production was observed at 55 degrees C, pH 11 and after 48 h of incubation. Among the substrates tested, xylose (a carbon source), peptone (a nitrogen source) and olive oil at a concentration of 1% were suitable substrates for enhancing lipase production. MgSO4 and Tween-80 were suitable substrates for maximizing lipase production. The enzyme was purified to homogeneity by a single CM-Sephadex column chromatography and revealed molecular mass of 67 kDa. The enzyme (BL1) was active over a wide range of pH from 9.0 to 13.0, with an optimum at pH 11.0, exhibited maximal activity at 55 degreesC and retained more than 70% of its activity after incubation at 70 degrees C or pH 13 for 0.5 h or 24 h, respectively. The enzyme hydrolyzed both short and long-chain triacylglycerols at comparable rates. BL1 was studied in a preliminary evaluation for use in detergent formulation solutions. This novel lipase showed extreme stability towards non-ionic and anionic surfactants after pre-incubation for 1 h at 40 degrees C, and good stability towards oxidizing agents. Additionally, the enzyme showed excellent stability and compatibility with various commercial detergents, suggesting its potential as an additive in detergent formulations.

  2. Kinetics, improved activity and thermostability of endoglucanase and beta glucosidase from a mutant-derivative of aspergillus niger ms82

    International Nuclear Information System (INIS)

    Sohail, M.; Ahmad, A.; Khan, S.A.; Uddin, F.

    2013-01-01

    A mutant MS301 of Aspergillus niger MS82 showed 1.5 to 2.5-fold improved endoglucanase and beta-glucosidase activity when grown on crude lignocellulosic substrates under solid-state and submerged conditions. Indicators of thermal stability of enzymes (Tm and T1/2) showed that the wild type and mutant endoglucanase was more heat-resistant compared to beta-glucosidase. However, mutant and parent enzymes shared almost the same values for melting temperatures and half-lives. Endoglucanase and beta-glucosidase from both the strains showed optimum activity under acidic pH. Energy of activation (Ea) of mutant beta-glucosidase was substantially lower than the parent enzyme while Ea of mutant endoglucanase was slightly less than the parent. The lowered Ea values can be attributed to the improved beta-glucosidase activity of the mutant strain. Moreover, the MS301 enzymes were better in hydrolyzing purified and crude cellulosic materials than the parent MS82. (author)

  3. Participatory role of zinc in structural and functional characterization of bioremediase: a unique thermostable microbial silica leaching protein.

    Science.gov (United States)

    Chowdhury, Trinath; Sarkar, Manas; Chaudhuri, Biswadeep; Chattopadhyay, Brajadulal; Halder, Umesh Chandra

    2015-07-01

    A unique protein, bioremediase (UniProt Knowledgebase Accession No.: P86277), isolated from a hot spring bacterium BKH1 (GenBank Accession No.: FJ177512), has shown to exhibit silica leaching activity when incorporated to prepare bio-concrete material. Matrix-assisted laser desorption ionization mass spectrometry analysis suggests that bioremediase is 78% homologous to bovine carbonic anhydrase II though it does not exhibit carbonic anhydrase-like activity. Bioinformatics study is performed for understanding the various physical and chemical parameters of the protein which predicts the involvement of zinc encircled by three histidine residues (His94, His96 and His119) at the active site of the protein. Isothermal titration calorimetric-based thermodynamic study on diethyl pyrocarbonate-modified protein recognizes the presence of Zn(2+) in the enzyme moiety. Exothermic to endothermic transition as observed during titration of the protein with Zn(2+) discloses that there are at least two binding sites for zinc within the protein moiety. Addition of Zn(2+) regains the activity of EDTA chelated bioremediase confirming the presence of extra binding site of Zn(2+) in the protein moiety. Revival of folding pattern of completely unfolded urea-treated protein by Zn(2+) explains the participatory role of zinc in structural stability of the protein. Restoration of the λ max in intrinsic fluorescence emission study of the urea-treated protein by Zn(2+) similarly confirms the involvement of Zn in the refolding of the protein. The utility of bioremediase for silica nanoparticles preparation is observed by field emission scanning electron microscopy.

  4. Purification and Properties of a Novel Thermostable Galacto-Oligosaccharide-Producing (beta)-Galactosidase from Sterigmatomyces elviae CBS8119

    OpenAIRE

    Onishi, N.; Tanaka, T.

    1996-01-01

    Volume 61, no. 11, p. 4028, column 1, line 37: "p-nitrophenyl-(beta)-d-galactopyranoside" should read "p-nitrophenyl-(beta)-d-glucopyranoside." Page 4028, column 1, line 38: "p-nitrophenyl-(beta)-l-arabinopyranoside" should read "p-nitrophenyl-(alpha)-l-arabinopyranoside." Page 4028, column 2, Table 2, last line: "PNP-(beta)-l-arabinopyranoside" should read "PNP-(alpha)-l-arabinopyranoside." Page 4028, column 2, Table 2, footnote a, line 4: "PNP-(alpha)-l-arabinopyranoside" should read "PNP-(...

  5. Production, purification and characterization of thermostable α-amylase from soil isolate Bacillus sp. strain B-10

    Directory of Open Access Journals (Sweden)

    Ravindra Nath Singh

    2016-04-01

    Full Text Available A bacterial strain B-10 that produces α-amylase was isolated from compost and kitchen waste receiving agricultural soil. Based on microbiological and biochemical tests the isolate B-10 was identified as Bacillus sp. Alpha-amylase produced by this isolate was purified by (NH42SO4 precipitation and DEAE cellulose ion-exchange chromatography showing 15.91 and 48.21 fold purification, respectively. SDS-PAGE of the purified enzyme confirmed the purification and monomeric nature of the enzyme. The purified α-amylase showed maximum activity at pH 7 and temperature 50°C. The enzyme was significantly active in the temperature range of 30-60°C for the studied period of 2 h. During the incubation of purified enzyme at pH ranging from 5 to 10 for 24 h the maximum stability was observed at pH 7 followed by pH 8, whereas at extreme pH, the stability was very poor. Km and Vmax were found to be 1.4 mg/mL and 6.2 U/mL, respectively.

  6. The role of salt bridges on the temperature adaptation of aqualysin I, a thermostable subtilisin-like proteinase.

    Science.gov (United States)

    Jónsdóttir, Lilja B; Ellertsson, Brynjar Ö; Invernizzi, Gaetano; Magnúsdóttir, Manuela; Thorbjarnardóttir, Sigríður H; Papaleo, Elena; Kristjánsson, Magnús M

    2014-12-01

    Differences in salt bridges are believed to be a structural hallmark of homologous enzymes from differently temperature-adapted organisms. Nevertheless, the role of salt bridges on structural stability is still controversial. While it is clear that most buried salt bridges can have a functional or structural role, the same cannot be firmly stated for ion pairs that are exposed on the protein surface. Salt bridges, found in X-ray structures, may not be stably formed in solution as a result of high flexibility or high desolvation penalty. More studies are thus needed to clarify the picture on salt bridges and temperature adaptation. We contribute here to this scenario by combining atomistic simulations and experimental mutagenesis of eight mutant variants of aqualysin I, a thermophilic subtilisin-like proteinase, in which the residues involved in salt bridges and not conserved in a psychrophilic homolog were systematically mutated. We evaluated the effects of those mutations on thermal stability and on the kinetic parameters. Overall, we show here that only few key charged residues involved in salt bridges really contribute to the enzyme thermal stability. This is especially true when they are organized in networks, as here attested by the D17N mutation, which has the most remarkable effect on stability. Other mutations had smaller effects on the properties of the enzyme indicating that most of the isolated salt bridges are not a distinctive trait related to the enhanced thermal stability of the thermophilic subtilase. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Enzymatic production of dietary nucleotides from low-soluble purine bases by an efficient, thermostable and alkali-tolerant biocatalyst.

    Science.gov (United States)

    Del Arco, J; Cejudo-Sanches, J; Esteban, I; Clemente-Suárez, V J; Hormigo, D; Perona, A; Fernández-Lucas, J

    2017-12-15

    Traditionally, enzymatic synthesis of nucleoside-5'-monophosphates (5'-NMPs) using low water-soluble purine bases has been described as less efficient due to their low solubility in aqueous media. The use of enzymes from extremophiles, such as thermophiles or alkaliphiles, offers the potential to increase solubilisation of these bases by employing high temperatures or alkaline pH. This study describes the cloning, expression and purification of hypoxanthine-guanine-xanthine phosphoribosyltransferase from Thermus thermophilus (TtHGXPRT). Biochemical characterization indicates TtHGXPRT as a homotetramer with excellent activity and stability across a broad range of temperatures (50-90°C) and ionic strengths (0-500mMNaCl), but it also reveals an unusually high activity and stability under alkaline conditions (pH range 8-11). In order to explore the potential of TtHGXPRT as an industrial biocatalyst, enzymatic production of several dietary 5'-NMPs, such as 5'-GMP and 5'-IMP, was carried out at high concentrations of guanine and hypoxanthine. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Thermostability enhancement of an endo-1,4-β-galactanase from Talaromyces stipitatus by site-directed mutagenesis

    DEFF Research Database (Denmark)

    Larsen, Dorte Møller; Nyffenegger, Christian; Swiniarska, Malgorzata Maria

    2015-01-01

    -life of TSGAL, nine single amino acid residues were selected for site-directed mutagenesis on the basis of semi-rational design. Of these nine mutants, G305A showed half-lives of 114 min at 55 °C and 15 min at 60 °C, respectively. This is 8.6-fold higher than that of the TSGAL at 55 °C, whereas the other...

  9. Thermosetting materials from the radiation-modified polymer compositions, 2. Development of adhesion-active and thermostable thermosetting materials

    International Nuclear Information System (INIS)

    Kalkis, V.; Maksimovs, R.D.; Zicans, J.; Bocoka, T.; Revjakins, O.

    1999-01-01

    Methods that improve the adhesion and thermal stability of the thermosetting materials are considered. Experimental studies of the blends composed of polyethylene, ethylene-propylene-diene copolymer (with characteristic specific for rubbers) and mesomorphic copolyesters, using rheological, spectrometric, and thermomechanical methods, have shown that radiation modification improves the adhesion and deformation properties as well as thermal stability of these blends. Therefore, materials of such system can be successfully used, e.g., as elastic and adhesion-active thermosetting materials at temperature above 473 K. (author)

  10. Preparation and characterization of novel zwitterionic poly(arylene ether sulfone) ultrafiltration membrane with good thermostability and excellent antifouling properties

    Science.gov (United States)

    Rong, Guolong; Zhou, Di; Han, Xiaocui; Pang, Jinhui

    2018-01-01

    Zwitterionic poly(arylene ether sulfone) (PAES-NS) was synthesized via copolymerization by using a bisphenol monomer with a pyridine group. The chemical structures of the copolymers were confirmed by using Fourier transform infrared (FTIR) and 1H nuclear magnetic resonance (NMR) spectroscopy; the copolymers showed good thermal stability. A series of polyphenysulfone (PPSU)/PAES-NS blend ultrafiltration (UF) membranes was prepared via conventional immersion precipitation phase inversion methods The morphologies of the modified membranes were investigated by scanning electron microscopy (SEM). The surface hydrophilicity of the UF membranes was studied by water contact angle measurement, indicating that the zwitterionic group increased the membrane hydrophilicity. UF of solvated model pollutants using the membranes showed a significant reduction of the irreversible adsorption of the foulants, illustrating the excellent anti-fouling properties of the membrane. The water flux of the PAES-NS membrane was significantly enhanced, being almost three times higher than that of the pristine PPSU membrane, with retention of a high rejection level. After three UF cycles, the water flux recovery of the PAES-NS membrane was as high as 96%.

  11. α,β-D-constrained nucleic acids are strong terminators of thermostable DNA polymerases in polymerase chain reaction.

    Directory of Open Access Journals (Sweden)

    Olivier Martínez

    Full Text Available (S(C5', R(P α,β-D- Constrained Nucleic Acids (CNA are dinucleotide building blocks that can feature either B-type torsional angle values or non-canonical values, depending on their 5'C and P absolute stereochemistry. These CNA are modified neither on the nucleobase nor on the sugar structure and therefore represent a new class of nucleotide with specific chemical and structural characteristics. They promote marked bending in a single stranded DNA so as to preorganize it into a loop-like structure, and they have been shown to induce rigidity within oligonucleotides. Following their synthesis, studies performed on CNA have only focused on the constraints that this family of nucleotides introduced into DNA. On the assumption that bending in a DNA template may produce a terminator structure, we investigated whether CNA could be used as a new strong terminator of polymerization in PCR. We therefore assessed the efficiency of CNA as a terminator in PCR, using triethylene glycol phosphate units as a control. Analyses were performed by denaturing gel electrophoresis and several PCR products were further analysed by sequencing. The results showed that the incorporation of only one CNA was always skipped by the polymerases tested. On the other hand, two CNA units always stopped proofreading polymerases, such as Pfu DNA polymerase, as expected for a strong replication terminator. Non-proofreading enzymes, e.g. Taq DNA polymerase, did not recognize this modification as a strong terminator although it was predominantly stopped by this structure. In conclusion, this first functional use of CNA units shows that these modified nucleotides can be used as novel polymerization terminators of proofreading polymerases. Furthermore, our results lead us to propose that CNA and their derivatives could be useful tools for investigating the behaviour of different classes of polymerases.

  12. Molecular and biochemical characterization of a new thermostable bacterial laccase from Meiothermus ruber DSM 1279

    DEFF Research Database (Denmark)

    Kalyani, D. C.; Munk, L.; Mikkelsen, J. D.

    2016-01-01

    . Spectroscopic analysis of the purified enzyme by UV/visible and electron paramagnetic resonance spectroscopy confirmed that the Mrlac was a multicopper oxidase. The Mrlac had a molecular weight of ∼ 50 kDa and exhibited activity towards the canonical laccase substrates 2,2'-azino-bis(3-ethylbenzothiazoline-6...

  13. The Chemistry of Hexaazatriphenylene Hexanitrile, a Polyfunctional Heterocycle with Potential Utility in the Formulation of Thermostable Polymers

    Science.gov (United States)

    1989-06-01

    Kapton polamicacids incorporating trianhydride I processability. While the trend in stability is not what we (o 0 mol, I -- ). to mo -". - --- ) and 5O mof ...HN03 (150 mL) over a period of 10 min. The temperature lyzed esterification with methanol yields the hexamethyl of the vigorous reaction was controlled

  14. A Highly Thermostable In2O3/ITO Thin Film Thermocouple Prepared via Screen Printing for High Temperature Measurements

    Directory of Open Access Journals (Sweden)

    Yantao Liu

    2018-03-01

    Full Text Available An In2O3/ITO thin film thermocouple was prepared via screen printing. Glass additives were added to improve the sintering process and to increase the density of the In2O3/ITO films. The surface and cross-sectional images indicate that both the grain size and densification of the ITO and In2O3 films increased with the increase in annealing time. The thermoelectric voltage of the In2O3/ITO thermocouple was 53.5 mV at 1270 °C at the hot junction. The average Seebeck coefficient of the thermocouple was calculated as 44.5 μV/°C. The drift rate of the In2O3/ITO thermocouple was 5.44 °C/h at a measuring time of 10 h at 1270 °C.

  15. Structural and Functional Studies of A. oryzae Cutinase: Enhanced Thermostability and Hydrolytic Activity of Synthetic Ester and Polyester Degradation

    OpenAIRE

    Liu, Zhiqiang; Gosser, Yuying; Baker, Peter James; Ravee, Yaniv; Lu, Ziying; Alemu, Girum; Li, Huiguang; Butterfoss, Glenn L.; Kong, Xiang-Peng; Gross, Richard; Montclare, Jin Kim

    2009-01-01

    Cutinases are responsible for hydrolysis of the protective cutin lipid polyester matrix in plants and thus have been exploited for hydrolysis of small molecule esters and polyesters. Here we explore the reactivity, stability, and structure of Aspergillus oryzae cutinase and compare it to the well-studied enzyme from Fusarium solani. Two critical differences are highlighted in the crystallographic analysis of the A. oryzae structure: (i) an additional disulfide bond and (ii) a topologically fa...

  16. Production of thermostable β-glucosidase and CMCase by Penicillium sp. LMI01 isolated from the Amazon region

    Directory of Open Access Journals (Sweden)

    Pamella S. Santa-Rosa

    2018-01-01

    Conclusions: The effectiveness and characteristics of these enzymes indicate that LMI01 can be an alternative for the hydrolysis of lignocellulosic materials and should be tested in commercial formulations.

  17. Enhanced Production and Characterization of a Solvent Stable Amylase from Solvent Tolerant Bacillus tequilensis RG-01: Thermostable and Surfactant Resistant

    Directory of Open Access Journals (Sweden)

    Soni Tiwari

    2014-01-01

    Full Text Available Ten bacterial strains isolated from the soil samples in the presence of cyclohexane were screened for amylase production. Among them, culture RG-01 was adjudged as the best amylase producer and was identified as Bacillus tequilensis from MTCC, Chandigarh. The isolate showed maximum amylase production (8100 U/mL in the presence of starch, peptone, and Ca2+ ions at 55°C pH 7.0 within 24 h of incubation. The enzyme was stable in the presence of n-dodecane, isooctane, n-decane, xylene, toluene, n-hexane, n-butanol, and cyclohexane, respectively. The presence of benzene, methanol, and ethanol marginally reduced the amylase stability, respectively. The enzyme was showed it 100% activity at 55°C and pH 7.0 with 119% and 127% stability at 55°C and pH 7.0, respectively. The enzyme was also stable in the presence of SDS, Tween-40, Tween-60, and Tween-80 (1% and was found stimulatory effect, respectively. Only Triton-X-100 showed a moderate inhibitory effect (5% on amylase activity. This isolate (Bacillus tequilensis RG-01 may be useful in several industrial applications owing to its thermotolerant and organic solvents and surfactants resistance characteristics.

  18. Effect of mutations on the thermostability of Aspergillus aculeatus β-1,4-galactanase

    DEFF Research Database (Denmark)

    Torpenholt, Søs Katja; De Maria, Leonardo; Olsson, Mats Henrik Mikael

    2015-01-01

    New variants of β-1,4-galactanase from the mesophilic organism Aspergillus aculeatus were designed using the structure of β-1,4-galactanase from the thermophile organism Myceliophthora thermophila as a template. Some of the variants were generated using PROPKA 3.0, a validated pKa prediction tool......, to test its usefulness as an enzyme design tool. The PROPKA designed variants were D182N and S185D/Q188T, G104D/A156R. Variants Y295F and G306A were designed by a consensus approach, as a complementary and validated design method. D58N was a stabilizing mutation predicted by both methods. The predictions...... were experimentally validated by measurements of the melting temperature (Tm) by differential scanning calorimetry. We found that the Tm is elevated by 1.1 °C for G306A, slightly increased (in the range of 0.34 to 0.65 °C) for D182N, D58N, Y295F and unchanged or decreased for S185D/Q188T and G104D/A156...

  19. Pharmacokinetics/Pharmacodynamics findings after repeated administration of ARTESUNATE thermostable suppositories (RECTOCAPS) in Vietnamese patients with uncomplicated malaria.

    Science.gov (United States)

    Benakis, A; Binh, Tran Quang; Keundjian, A; Scheiwe, M W

    2006-01-01

    In recent years, Artemisinin and particularly one of its derivatives--Artesunate (ART--has become an essential alternative for treatment of both uncomplicated and severe falciparum malaria in Asia and Africa as well. Therefore, these compounds are still and inccreasingly in the focus of interest because of quick acting of this drug, is able to help even unconscious to overcome the malaria attack, when administered by injection. As an alternative, RECTOCAPS have been developed and their use is meanwhile well established. From earlier studies in children, suffering from plasmodium falciparum malaria, we obtained a high level of DHART in the blood, but as expected also a rapid decline in the levels of both DHART and ART. A second administration of ART was additionally applied 4 hours after the first administration. DHART and ART plasma levels were found to last longer on an assumed therapeutic level than those obtained after one administration only. The fever clearance and the parasitemia reduction rates were found to be effective according to this dosing regimen. In view of these findings, we decided to conduct the actual described study by administering 200 mg of ART every 3 hours (0, 3, 6 and 9 h) by the rectal route. Soft geiatine capsules (RECTOCAPS) containing 200 mg of ART GMP--type each (Artesunic acid) were administered by rectal route. Each patient received four RECTOCAPS capsules (4 x 200 mg of ART) over a 3 h period. 12 adult patients with uncomplicated malaria were selected. Age, weight, height, body temperature, parasite counts before treatment and their evolution until 96 h are determined. Blood samples were taken at short time intervals after starting with the first medication: 0, 30 min, 60 min, 3 h, 6 h, 9 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h and 108 h. The aliquots of all the blood samples were used for performing parasite counts. Plasma obtained following the traditional procedure was kept at -40 degrees C until analysis. HPLC technique with electrochemical detector was used for quantification of ART and DHART. From the blood concentration values of ART and DHART, the following observation can be derived: the onset of action is observed within the first half hours, therapeutic levels of the drug obtained (89 microg/ml ART compared to 84 microg/ml DHART). The DHART levels are somewhat higher than those of ART (a peak concentration after 6 h starting medication of 151 microg/ml ART as compared to 276 microg/ml DHART). The variations as a function of frequency of DHART uptake are much less marked than those observed for ART. Another finding is that after the administration, some sort of a plateau of DHART and ART is built up, lasting at least from 9 to 12 hours with DHART level of about 190 microg/ml and ART of 90 microg/ml. In the case of single-dose administration, the levels of both compounds were below the detection threshold after three hours. With regard to the parasite counts, although there were inter-individual variations, it should be noted that after 48 hours a high proportion of the patients (8 out of 12) was completely clear of parasites. Similar results were observed with regard to the body temperature (7 out of 12 returned to normal temperature 36 hours after starting the therapy). The findings of the study support the RECTOCAPS application principle resulting in effectiveness both for the velocity of drug uptake as well as for the height of plasma levels. Repeated administration of ART can extend the duration of therapeutic plasma levels of the drug.

  20. Improving enzymatic activities and thermostability of a tri-functional enzyme with SOD, catalase and cell-permeable activities.

    Science.gov (United States)

    Luangwattananun, Piriya; Eiamphungporn, Warawan; Songtawee, Napat; Bülow, Leif; Isarankura Na Ayudhya, Chartchalerm; Prachayasittikul, Virapong; Yainoy, Sakda

    2017-04-10

    Synergistic action of major antioxidant enzymes, e.g., superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) is known to be more effective than the action of any single enzyme. Recently, we have engineered a tri-functional enzyme, 6His-MnSOD-TAT/CAT-MnSOD (M-TAT/CM), with SOD, CAT and cell-permeable activities. The protein actively internalized into the cells and showed superior protection against oxidative stress-induced cell death over native enzymes fused with TAT. To improve its molecular size, enzymatic activity and stability, in this study, MnSOD portions of the engineered protein were replaced by CuZnSOD, which is the smallest and the most heat resistant SOD isoform. The newly engineered protein, CAT-CuZnSOD/6His-CuZnSOD-TAT (CS/S-TAT), had a 42% reduction in molecular size and an increase in SOD and CAT activities by 22% and 99%, respectively. After incubation at 70°C for 10min, the CS/S-TAT retained residual SOD activity up to 54% while SOD activity of the M-TAT/CM was completely abolished. Moreover, the protein exhibited a 5-fold improvement in half-life at 70°C. Thus, this work provides insights into the design and synthesis of a smaller but much more stable multifunctional antioxidant enzyme with ability to enter mammalian cells for further application as protective/therapeutic agent against oxidative stress-related conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Phenylalanine hydroxylase from Legionella pneumophila is a thermostable enzyme with a major functional role in pyomelanin synthesis.

    Directory of Open Access Journals (Sweden)

    Marte I Flydal

    Full Text Available Legionella pneumophila is a pathogenic bacterium that can cause Legionnaires' disease and other non-pneumonic infections in humans. This bacterium produces a pyomelanin pigment, a potential virulence factor with ferric reductase activity. In this work, we have investigated the role of phenylalanine hydroxylase from L. pneumophila (lpPAH, the product of the phhA gene, in the synthesis of the pyomelanin pigment and the growth of the bacterium in defined compositions.Comparative studies of wild-type and phhA mutant corroborate that lpPAH provides the excess tyrosine for pigment synthesis. phhA and letA (gacA appear transcriptionally linked when bacteria were grown in buffered yeast extract medium at 37°C. phhA is expressed in L. pneumophila growing in macrophages. We also cloned and characterized lpPAH, which showed many characteristics of other PAHs studied so far, including Fe(II requirement for activity. However, it also showed many particular properties such as dimerization, a high conformational thermal stability, with a midpoint denaturation temperature (T(m = 79 ± 0.5°C, a high specific activity at 37°C (10.2 ± 0.3 µmol L-Tyr/mg/min and low affinity for the substrate (K(m (L-Phe = 735 ± 50 µM.lpPAH has a major functional role in the synthesis of pyomelanin and promotes growth in low-tyrosine media. The high thermal stability of lpPAH might reflect the adaptation of the enzyme to withstand relatively high survival temperatures.

  2. Cor a 14, the allergenic 2S albumin from hazelnut, is highly thermostable and resistant to gastrointestinal digestion

    Science.gov (United States)

    Pfeifer, Sabine; Bublin, Merima; Dubiela, Pawel; Hummel, Karin; Wortmann, Judith; Hofer, Gerhard; Keller, Walter; Radauer, Christian

    2015-01-01

    Scope Allergens from nuts frequently induce severe allergic reactions in sensitive individuals. The aim of this study was to elucidate the physicochemical characteristics of natural Cor a 14, the 2S albumin from hazelnut. Methods and results Cor a 14 was purified from raw hazelnuts using a combination of precipitation and chromatographic techniques. The protein was analyzed using gel electrophoresis, MS, and far‐UV circular dichroism (CD) analyses. The immunoglobulin E (IgE) binding of native, heat‐treated, and in vitro digested Cor a 14 was studied. We identified two different Cor a 14 isoforms and showed microclipping at the C‐terminus. CD spectra at room temperature showed the typical characteristics of 2S albumins, and temperatures of more than 80°C were required to start unfolding of Cor a 14 demonstrating its high stability to heat treatment. In vitro digestion experiments revealed that Cor a 14 is resistant to proteolytic degradation. Native and heat‐treated protein was recognized by sera from hazelnut allergic patients. However, denaturation of the allergen led to significantly reduced IgE binding. Conclusion We identified two different isoforms of Cor a 14 displaying high stability under heating and gastric and duodenal conditions. Data from IgE‐binding experiments revealed the existence of both, linear and conformational epitopes. PMID:26178695

  3. High-level expression, secretion, and purification of the thermostable aqualysin I from Thermus aquaticus YT-1 in Pichia pastoris

    DEFF Research Database (Denmark)

    Oledzka, G.; Dabrowski, Slawomir; Kur, J.

    2003-01-01

    Aqualysin I is a heat-stable subtilisin-type serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extreme thermophile. We report the high-level expression of an aqualysin I protein using its native signal sequence for secretion in the methylotrophic yeast, Pichia...... to that of the native enzyme. We also explored the possibility of secreting the GAP expressed aqualysin I in P. pastoris by in-frame fusion of the Saccharomyces cerevisiae alpha-factor secretion signal. However, the levels of secreted pro-aqualysin I particles were approximately 10 times lower, possibly...

  4. Purification and characterization of a thermostable glutamate dehydrogenase from a thermophilic bacterium isolated from a sterilization drying oven

    Directory of Open Access Journals (Sweden)

    Maximiliano J. Amenábar

    2012-02-01

    Full Text Available Glutamate dehydrogenase from axenic bacterial cultures of anew microorganism, called GWE1, isolated from the interior ofa sterilization drying oven, was purified by anion-exchange andmolecular-exclusion liquid chromatography. The apparent molecularmass of the native enzyme was 250.5 kDa and wasshown to be an hexamer with similar subunits of molecularmass 40.5 kDa. For glutamate oxidation, the enzyme showedan optimal pH and temperature of 8.0 and 70oC, respectively.In contrast to other glutamate dehydrogenases isolated frombacteria, the enzyme isolated in this study can use both NAD+and NADP+ as electron acceptors, displaying more affinity forNADP+ than for NAD+. No activity was detected with NADHor NADPH, 2-oxoglutarate and ammonia. The enzyme was exceptionallythermostable, maintaining more than 70% of activityafter incubating at 100oC for more than five hours suggestingbeing one of the most thermoestable enzymes reported inthe family of dehydrogenases. [BMB reports 2012; 45(2: 91-95

  5. Alcohol Selectivity in a Synthetic Thermophilic n-Butanol Pathway Is Driven by Biocatalytic and Thermostability Characteristics of Constituent Enzymes.

    Science.gov (United States)

    Loder, Andrew J; Zeldes, Benjamin M; Garrison, G Dale; Lipscomb, Gina L; Adams, Michael W W; Kelly, Robert M

    2015-10-01

    n-Butanol is generated as a natural product of metabolism by several microorganisms, but almost all grow at mesophilic temperatures. A synthetic pathway for n-butanol production from acetyl coenzyme A (acetyl-CoA) that functioned at 70°C was assembled in vitro from enzymes recruited from thermophilic bacteria to inform efforts for engineering butanol production into thermophilic hosts. Recombinant versions of eight thermophilic enzymes (β-ketothiolase [Thl], 3-hydroxybutyryl-CoA dehydrogenase [Hbd], and 3-hydroxybutyryl-CoA dehydratase [Crt] from Caldanaerobacter subterraneus subsp. tengcongensis; trans-2-enoyl-CoA reductase [Ter] from Spirochaeta thermophila; bifunctional acetaldehyde dehydrogenase/alcohol dehydrogenase [AdhE] from Clostridium thermocellum; and AdhE, aldehyde dehydrogenase [Bad], and butanol dehydrogenase [Bdh] from Thermoanaerobacter sp. strain X514) were utilized to examine three possible pathways for n-butanol. These pathways differed in the two steps required to convert butyryl-CoA to n-butanol: Thl-Hbd-Crt-Ter-AdhE (C. thermocellum), Thl-Hbd-Crt-Ter-AdhE (Thermoanaerobacter X514), and Thl-Hbd-Crt-Ter-Bad-Bdh. n-Butanol was produced at 70°C, but with different amounts of ethanol as a coproduct, because of the broad substrate specificities of AdhE, Bad, and Bdh. A reaction kinetics model, validated via comparison to in vitro experiments, was used to determine relative enzyme ratios needed to maximize n-butanol production. By using large relative amounts of Thl and Hbd and small amounts of Bad and Bdh, >70% conversion to n-butanol was observed in vitro, but with a 60% decrease in the predicted pathway flux. With more-selective hypothetical versions of Bad and Bdh, >70% conversion to n-butanol is predicted, with a 19% increase in pathway flux. Thus, more-selective thermophilic versions of Bad, Bdh, and AdhE are needed to fully exploit biocatalytic n-butanol production at elevated temperatures. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  6. Biocatalysis with thermostable enzymes: structure and properties of a thermophilic 'ene'-reductase related to old yellow enzyme.

    Science.gov (United States)

    Adalbjörnsson, Björn V; Toogood, Helen S; Fryszkowska, Anna; Pudney, Christopher R; Jowitt, Thomas A; Leys, David; Scrutton, Nigel S

    2010-01-25

    We report the crystal structure of a thermophilic "ene" reductase (TOYE) isolated from Thermoanaerobacter pseudethanolicus E39. The crystal structure reveals a tetrameric enzyme and an active site that is relatively large compared to most other structurally determined and related Old Yellow Enzymes. The enzyme adopts higher order oligomeric states (octamers and dodecamers) in solution, as revealed by sedimentation velocity and multiangle laser light scattering. Bead modelling indicates that the solution structure is consistent with the basic tetrameric structure observed in crystallographic studies and electron microscopy. TOYE is stable at high temperatures (T(m)>70 degrees C) and shows increased resistance to denaturation in water-miscible organic solvents compared to the mesophilic Old Yellow Enzyme family member, pentaerythritol tetranitrate reductase. TOYE has typical ene-reductase properties of the Old Yellow Enzyme family. There is currently major interest in using Old Yellow Enzyme family members in the preparative biocatalysis of a number of activated alkenes. The increased stability of TOYE in organic solvents is advantageous for biotransformations in which water-miscible organic solvents and biphasic reaction conditions are required to both deliver novel substrates and minimize product racemisation.

  7. Development of Reverse Transcription Thermostable Helicase-Dependent DNA Amplification for the Detection of Tomato Spotted Wilt Virus.

    Science.gov (United States)

    Wu, Xinghai; Chen, Chanfa; Xiao, Xizhi; Deng, Ming Jun

    2016-11-01

    A protocol for the reverse transcription-helicase-dependent amplification (RT-HDA) of isothermal DNA was developed for the detection of tomato spotted wilt virus (TSWV). Specific primers, which were based on the highly conserved region of the N gene sequence in TSWV, were used for the amplification of virus's RNA. The LOD of RT-HDA, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP), and reverse transcriptase-polymerase chain reaction (RT-PCR) assays were conducted using 10-fold serial dilution of RNA eluates. TSWV sensitivity in RT-HDA and RT-LAMP was 4 pg RNA compared with 40 pg RNA in RT-PCR. The specificity of RT-HDA for TSWV was high, showing no cross-reactivity with other tomato and Tospovirus viruses including cucumber mosaic virus (CMV), tomato black ring virus (TBRV), tomato mosaic virus (ToMV), or impatiens necrotic spot virus (INSV). The RT-HDA method is effective for the detection of TSWV in plant samples and is a potential tool for early and rapid detection of TSWV.

  8. Enhancement of water soluble wheat bran polyphenolic compounds using different steviol glucosides prepared by thermostable β-galactosidase

    Directory of Open Access Journals (Sweden)

    Hee-jung Lim

    2016-10-01

    Full Text Available Background: Production of wheat bran (WB for human consumption is estimated to be about 90 million tons per year. WB contains an abundant source of dietary fiber, minerals, vitamins, and bioactive compounds. WB is a by-product of milling and contains an abundant source of carbohydrate (60%, protein (12%, fat (0.5%, minerals (2%, and bioactive compounds such as phenolic acids, arabinoxylans, flavonoids, caroteinoids alkylresorcinol and phytosterols. These are known for health promoting properties such as controlling glycemic index, reducing plasma cholesterol level, antioxidant, anti-inflammatory, and anticarcinogenic activities. Several terpene glycosides such as mogroside V, paenoiflorin, geniposide, rubusoside (Ru, stevioside (Ste, rebaudioside A (RebA, steviol monoside, and stevioside glucoside have been discovered to enhance the solubility of a number of pharmaceutically and medically important compounds that normally show poor solubility in water. Context and purpose of this study: In this study, in order to increase soluble extraction of polyphenol compounds of WB using Ru, the expression of β-galactosidase from Thermus thermophilus (T. thermophilus was optimized using different E. coli hosts and a different concentration of lactose inducer rather than of isopropyl-1- thio-β-D-galactopyranoside (IPTG for industrial production. Additionally, the effect of different steviol glucosides (Ru, Ste, RebA, and SG on the enhancement of polyphenol compounds extraction from wheat bran was studied. Results: β-galactosidase from T. thermophilus was used for the specific conversion of stevioside (Ste to rubusoside (Ru with 92% productivity. The enzyme was optimized to be expressed in E. coli. With 7 mM lactose, the β-galactosidase activity expressed was 34.3, 14.2, or 34.4 ± 0.5 U/mL in E. coli BL21(DE3pLysS, Rosetta(DE3pLysS, or BL21(DE3 at 37°C, and 9.8 ± 0.2, 7.0 ± 0.5, or 7.4 ± 0.2 U/mL at 28°C respectively. The expression of β-galactosidase was dependent on the lactose concentration and the highest activity was obtained with the conditions of 5 mM lactose in E. coli Rosetta(DE3pLysS, 53.3 ± 1.5 U/mL. 78% of the mesophilic proteins was eliminated by heating at 70°C for 15 min with 89% β-galactosidase activity recovery. The total polyphenol content of WB extracted by water, Ru, Ste, rebaudioside A (RebA, and steviol glucosides (SG were 533.8 ± 9.6 µg/mL, 633.3 ± 1.25 µg/mL, 604.4 ± 10.1 µg/mL, 654.8 ± 26.5 µg/mL, and 601.2 ± 33.4 µg/mL, respectively. The DPPH radical scavenging activity prepared by water, Ru, Ste, RebA, and SG extraction were 8.76 ± 0.3 mg/mL, 4.87 ± 0.3 mg/mL, 5.34 ± 0.22 mg/mL, 7.27 ± 0.1 mg/mL, and 7.82 ± 0.02 mg/mL respectively. Conclusions: To increase soluble extraction of polyphenol compounds of WB using Ru, the expression of β-galactosidase from T. thermophilus was optimized using different E. coli hosts and a different concentration of lactose inducer rather than isopropyl-1- thio-β-D- galactopyranoside (IPTG for industrial production of the enzyme. The highest antioxidant activity was shown in WB extracted by Ru. The number of glucosyl units attached to steviol can possibly affect the efficiency of antioxidant activity of WB extracted by steviol glucosides.

  9. Enzyme engineering through evolution: thermostable recombinant group II intron reverse transcriptases provide new tools for RNA research and biotechnology.

    Science.gov (United States)

    Collins, Kathleen; Nilsen, Timothy W

    2013-08-01

    Current investigation of RNA transcriptomes relies heavily on the use of retroviral reverse transcriptases. It is well known that these enzymes have many limitations because of their intrinsic properties. This commentary highlights the recent biochemical characterization of a new family of reverse transcriptases, those encoded by group II intron retrohoming elements. The novel properties of these enzymes endow them with the potential to revolutionize how we approach RNA analyses.

  10. Structural Analysis and Bioengineering of Thermostable Pyrococcus furiosus Prolidase for the Optimization of Organophosphorus Nerve Agent Detoxification

    Science.gov (United States)

    2012-04-26

    organophosphorus acid anhydrase from a halophilic bacterial isolate. J Bacteriol, 173, 1938-1943. Du, X., Tove, S., Kast -Hutcheson, K. & Grunden, A. M...1938-1943. Du, X., Tove, S., Kast -Hutcheson, K. & Grunden, A. M. 2005. Characterization of the dinuclear metal center of Pyrococcus furiosus

  11. Improved thermostable polyvinyl alcohol electrospun nanofibers with entangled naringinase used in a novel mini-packed bed reactor.

    Science.gov (United States)

    Nunes, Mário A P; Martins, Samuel; Rosa, M Emilia; Gois, Pedro M P; Fernandes, Pedro C B; Ribeiro, Maria H L

    2016-08-01

    Polyvinyl alcohol (PVA) electrospun nanofibers were produced using an electrospinning technique. Key parameters (e.g. collectors, distance from needle tip to collector, among others) that influence the structure and morphology of fibers were optimized. The naringinase entrapped in PVA nanofibers retained over 100% of its initial activity after 212h of operation, at 25°C. Chemical crosslinking with several boronic acids further increased the hydrolysis temperature (up to 85°C) and yielded nanofibers with thermal stability up to 121°C. A mini packed bed reactor (PBR) developed to establish the feasibility for continuous enzymatic operation, ran for 16days at 45°C. Highest naringenin biosynthesis was attained at a flow rate of 10mLh(-1). Highest volumetric (78molL(-1)h(-1)) and specific (26molh(-1)genzyme(-1)) productivities were attained at 30mLh(-1). The activity of NGase in electrospun nanofibers remained constant for almost 16days of operation at 10mLh(-1). Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Secretory production of tetrameric native full-length streptavidin with thermostability using Streptomyces lividans as a host.

    Science.gov (United States)

    Noda, Shuhei; Matsumoto, Takuya; Tanaka, Tsutomu; Kondo, Akihiko

    2015-01-13

    Streptavidin is a tetrameric protein derived from Streptomyces avidinii, and has tight and specific biotin binding affinity. Applications of the streptavidin-biotin system have been widely studied. Streptavidin is generally produced using protein expression in Escherichia coli. In the present study, the secretory production of streptavidin was carried out using Streptomyces lividans as a host. In this study, we used the gene encoding native full-length streptavidin, whereas the core region is generally used for streptavidin production in E. coli. Tetrameric streptavidin composed of native full-length streptavidin monomers was successfully secreted in the culture supernatant of S. lividans transformants, and had specific biotin binding affinity as strong as streptavidin produced by E. coli. The amount of Sav using S. lividans was about 9 times higher than using E. coli. Surprisingly, streptavidin produced by S. lividans exhibited affinity to biotin after boiling, despite the fact that tetrameric streptavidin is known to lose its biotin binding ability after brief boiling. We successfully produced a large amount of tetrameric streptavidin as a secretory-form protein with unique thermotolerance.

  13. Production and characterization of a thermostable alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily

    NARCIS (Netherlands)

    Machielsen, M.P.; Uria, A.R.; Kengen, S.W.M.; Oost, van der J.

    2006-01-01

    The gene encoding a novel alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The gene, referred to as adhD, was functionally expressed in Escherichia coli and subsequently purified to homogeneity. The

  14. Thermo-stability, dose effects and shelf-life of antifungal compounds produced by the symbiotic bacterium Xenorhabdus szentirmaii

    Science.gov (United States)

    Xenorhabdus spp bacteria are associated with Steinernematid nematodes and produce antifungal metabolites that protect nematode-infected cadavers from fungal colonization. Previous work demonstrated concentrated or cell-free metabolites of X. szentirmaii were toxic to fungal phytopathogens. We prepar...

  15. Design and introduction of a disulfide bridge in firefly luciferase: increase of thermostability and decrease of pH sensitivity.

    Science.gov (United States)

    Imani, Mehdi; Hosseinkhani, Saman; Ahmadian, Shahin; Nazari, Mahboobeh

    2010-08-01

    The thermal sensitivity and pH-sensitive spectral properties of firefly luciferase have hampered its application in a variety of fields. It is proposed that the stability of a protein can be increased by introduction of disulfide bridge that decreases the configurational entropy of unfolding. A disulfide bridge is introduced into Photinus pyralis firefly luciferase to make two separate mutant enzymes with a single bridge. Even though the A103C/S121C mutant showed remarkable thermal stability, its specific activity decreased, whereas the A296C/A326C mutant showed tremendous thermal stability, relative pH insensitivity and 7.3-fold increase of specific activity. Moreover, the bioluminescence emission spectrum of A296C/A326C was resistant against higher temperatures (37 degrees C). Far-UV CD analysis showed slight secondary structure changes for both mutants. Thermal denaturation analysis showed that conformational stabilities of A103C/S121C and A296C/A326C are more than native firefly luciferase. It is proposed that since A296 and A326 are situated in the vicinity of the enzyme active site microenvironment in comparison with A103 and S121, the formation of a disulfide bridge in this region has more impact on enzyme kinetic characteristics.

  16. Production, characterization, and immobilization of partially purified surfactant-detergent and alkali-thermostable protease from newly isolated Aeromonas caviae.

    Science.gov (United States)

    Datta, Sumitra; Menon, Gopalakrishnan; Varughese, Bincy

    2017-04-21

    Proteolytic Aeromonas caviae P-1-1 growing at wide-ranging pH (7.0-11.0) and moderate salinity (0-5% NaCl) was isolated from cattle shed of Thanjavur, India. It produced lipase, gelatinase, and polyhydroxybutyrate. Different culture conditions, incubation time, carbon and nitrogen sources, vitamins, amino acids, surfactants, and metal ions for optimal growth and protease production of P-1-1 were examined. Maximum protease (0.128 U/mL) production was achieved with 1% fructose, 1% yeast extract, 0.1% ammonium sulfate, 3% NaCl, 0.1% CaCl 2  · 2H 2 O, 1% glycine, 0.1% vitamin E, and 0.1% Tween-40 at pH 8.0 after 42 hr of incubation at 37°C. It was active over broad range of pH (7.0-12.0), temperature (15-100°C), and salinity (0-9% NaCl) with optima at pH 10.0, 55°C, and 3% NaCl. It retained 65 and 48% activities at pH 12.0 and 100°C, respectively. Partially purified protease was highly stable (100%) within pH range 7.0-12.0 and salinities of 0-5% NaCl for 48 hr. Cu 2+ , Mn 2+ , Co 2+ , and Ca 2+ did not inhibit its activity. Its stability at extreme pHs, temperatures, and in the presence of surfactants and commercial detergents suggests its possible application in laundry detergents. Partially purified protease was immobilized and reused. This is the first report of alkali-thermotolerant, surfactant-detergent-stable partially purified extracellular protease from A. caviae.

  17. 13C High-Resolution Nuclear Magnetic Resonance Studies of Enzyme-Substrate Reactions at Equilibrium. Substrate Strain Studies of Chymotrypsin-N-Acetyltyrosine Semicarbazide Complexes

    NARCIS (Netherlands)

    Robillard, George; Shaw, Elliott; Shulman, R.G.

    1974-01-01

    N-Acetyl-L-tyrosine semicarbazide is hydrolyzed by chymotrypsin (EC 3.4.21.1) to N-acetyl-L-tyrosine and semicarbazide. If a high concentration of semicarbazide is present, the equilibrium for the reaction can be shifted from hydrolysis to synthesis. Using N-acetyl-L-[13C]tyrosine enriched at the

  18. Enantioselectivity and Thermostability of a Novel Hyperhermotolerant Lipase from Geobacillus Thermodenitrificans nr68 (Lip.nr-68) on Secondary Racemic Alcohols Acetylation

    Science.gov (United States)

    Nik Him, N. R.; Ibrahim, D.

    2018-05-01

    In our previous work, a new lipase enzyme has been purified from a species identified as a Gram negative Geobacillus thermodenitrificans nr68, isolated from a hot spring in Malaysia with growth temperature of 48°C. This new lipase, called Lip.nr-68 has been characterized as a hyperthermotolerant protein with high stability at 65°C and has been showing excellent characteristics that are very much comparable yet better than some of those of well-known industrially-used lipases. It shows high activity against long-chain triglycerides with molecular weight of the purified enzyme estimated to be 33.5 kDa using SDS-PAGE analysis. This paper is focusing on hyperthermotolerant Lip.nr-68 performance in promoting for enantioselectivity activities towards three secondary racemic alcohols namely 1-phenylethanol, 1-cyclohexilethanol and 1-(naft-2-il) ethanol by acetylation with vinyl acetate. Lip.nr-68 has been confirmed to show high and usual enantioselectivitiy according to the Kazlauskas Rule towards all secondary racemic alcohols and has significantly approved as an enantiomer selective biocatalyst towards 1-phenylethanol and 1-cyclohexylethanol at 65°C. Lip.nr-68 has showed a reduction of (R) and (S) enantiomers as well as the production of 68-98% ee and almost 94% yield of 3-4 mg/ml for 1-cyclohexilethanol.

  19. Spectroscopic study and enhanced thermostability of combustion-derived BaMgAl10O17:Eu2+ blue phosphors for solid-state lighting

    Science.gov (United States)

    Pradal, Nathalie; Potdevin, Audrey; Chadeyron, Geneviève; Bonville, Pierre; Caillier, Bruno; Mahiou, Rachid

    2017-02-01

    Blue-emitting BaMgAl10O17:Eu2+ (BAM:Eu), suitable for applications in a next generation of Hg-free lamps based on UV LEDs, was prepared by a microwave induced solution combustion synthesis, using urea as combustion fuel and nitrates as oxidizers. Purity control of the as-synthesized blue phosphor was undertaken by a washing step followed by a reduction one. Structural and morphological properties of the outcoming phosphors have been considered. Synthesis process allows producing a well-crystallized and nanostructured BAM phase within only few minutes. The influence of reduction treatment on the relative amounts of Eu2+/Eu3+ in our samples has been investigated through an original study by magnetization and Mössbauer spectroscopy. Furthermore, a complete optical study has been carried out and allowed us to determine the europium localization in the three possible sites in BAM matrix. The percentage of Eu2+ increased twofold after the reduction treatment, entailing an increase in the luminescence efficiency upon UV excitation. Finally, temperature-dependent luminescence of combustion-derived powders has been studied till 170 °C and compared to that of commercial BAM:Eu. MISCS-derived phosphors present a higher thermal stability than commercial one: whereas the emission efficiency of this last was reduced by 64%, the one of combustion-derived BAM:Eu experienced an only 12% decline. Furthermore, while commercial BAM suffered from a severe blue-shift with increasing temperature, our phosphors keep its color quality with a good stability of the photometric parameters.

  20. Enhanced energy density and thermostability in polyimide nanocomposites containing core-shell structured BaTiO3@SiO2 nanofibers

    Science.gov (United States)

    Wang, Junchuan; Long, Yunchen; Sun, Ying; Zhang, Xueqin; Yang, Hong; Lin, Baoping

    2017-12-01

    High energy density polymer nanocomposites with high-temperature resistance are quite desirable for film capacitors and many other power electronics. In this study, polyimide-based (PI) nanocomposite films containing the core-shell structured barium titanate@silicon dioxide (BT@SiO2) nanofibers have been successfully synthesized by the solution casting method. In the BT@SiO2/PI nanocomposite films, the dielectric permittivity as well as the breakdown strength increase significantly. The SiO2 shell layers with moderate dielectric permittivity could effectively mitigate the local field concentration induced by the large mismatch between the dielectric permittivity of BT and PI, which contributes to the enhancement of the breakdown strength of the PI nanocomposite films. As a result, the PI nanocomposite film filled with 3 vol% BT@SiO2 nanofibers exhibits a maximal energy density of 2.31 J cm-3 under the field of 346 kV/mm, which is 62% over the pristine PI (1.42 J cm-3 at 308 kV/mm) and about 200% greater than the best commercial polymer, i.e. biaxially oriented polypropylenes (BOPP) (≈1.2 J cm-3). The thermogravimetric analysis results indicate that the BT@SiO2/PI nanocomposite films have good thermal stability below 500 °C.