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Sample records for l-arabinose isomerase ecai

  1. Crystal Structure of Escherichia coli L-Arabinose Isomerase (ECAI), The Putative Target of Biological Tagatose Production

    Energy Technology Data Exchange (ETDEWEB)

    Manjasetty,B.; Chance, M.

    2006-01-01

    Escherichia coli L-arabinose isomerase (ECAI; EC 5.3.1.4) catalyzes the isomerization of L-arabinose to L-ribulose in vivo. This enzyme is also of commercial interest as it catalyzes the conversion of D-galactose to D-tagatose in vitro. The crystal structure of ECAI was solved and refined at 2.6 Angstroms resolution. The subunit structure of ECAI is organized into three domains: an N-terminal, a central and a C-terminal domain. It forms a crystallographic trimeric architecture in the asymmetric unit. Packing within the crystal suggests the idea that ECAI can form a hexameric assembly. Previous electron microscopic and biochemical studies supports that ECAI is hexameric in solution. A comparison with other known structures reveals that ECAI adopts a protein fold most similar to E. coli fucose isomerase (ECFI) despite very low sequence identity 9.7%. The structural similarity between ECAI and ECFI with regard to number of domains, overall fold, biological assembly, and active site architecture strongly suggests that the enzymes have functional similarities. Further, the crystal structure of ECAI forms a basis for identifying molecular determinants responsible for isomerization of arabinose to ribulose in vivo and galactose to tagatose in vitro.

  2. Crystal Structure of Mn2+-bound Escherichia coli L-arabinose Isomerase (ECAI) and Implications in Protein Catalytic Mechanism and Thermo-Stability

    International Nuclear Information System (INIS)

    Zhu, W.; Manjasetty, B.; Chance, M.

    2007-01-01

    The functional properties of proteins depend on their three-dimensional shapes. Protein structures can be determined by X-ray crystallography as a tool. The three-dimensional structure of the apo form of the Escherichia coli L-arabinose isomerase (ECAI) has recently been determined. ECAI is responsible for the initial stage of L-arabinose catabolism, converting arabinose into ribulose in vivo. This enzyme also plays a crucial role in catalyzing the conversion of galactose into tagatose (low calorie natural sugar) in vitro. ECAI utilizes Mn 2+ for its catalytic activity. Crystals of the ECAI + Mn 2+ complex helps to investigate the catalytic properties of the enzyme. Therefore, crystals of ECAI + Mn 2+ complex were grown using hanging drop vapor diffusion method at room temperature. Diffraction data were collected at X4C beamline, National Synchrotron Light Source, Brookhaven National Laboratory. The structure was solved by the molecular replacement technique and has been refined to Rwork of 0.23 at 2.8 (angstrom) resolution using X3A beamline computational facility. The structure was deposited to Protein Data Bank (PDB ID 2HXG). Mn 2+ ion was localized to the previously identified putative active site with octahedral coordination. Comparison of apo and holo form of ECAI structures permits the identification of structural features that are of importance to the intrinsic activity and heat stability of AI

  3. The secreted l-arabinose isomerase displays anti-hyperglycemic effects in mice

    OpenAIRE

    Rhimi, Moez; Bermudez-Humaran, Luis G.; Huang, Yuan; Boudebbouze, Samira; Gaci, Nadia; Garnier, Alexandrine; Gratadoux, Jean-Jacques; Mkaouar, H?la; Langella, Philippe; Maguin, Emmanuelle

    2015-01-01

    Background The l-arabinose isomerase is an intracellular enzyme which converts l-arabinose into l-ribulose in living systems and d-galactose into d-tagatose in industrial processes and at industrial scales. d-tagatose is a natural ketohexose with potential uses in pharmaceutical and food industries. The d-galactose isomerization reaction is thermodynamically equilibrated, and leads to secondary subproducts at high pH. Therefore, an attractive l-arabinose isomerase should be thermoactive and a...

  4. Screening and selection of wild strains for L-arabinose isomerase production

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    R. M. Manzo

    2013-12-01

    Full Text Available The majority of L-arabinose isomerases have been isolated by recombinant techniques, but this methodology implies a reduced technological application. For this reason, 29 bacterial strains, some of them previously characterized as L-arabinose isomerase producers, were assayed as L-arabinose fermenting strains by employing conveniently designed culture media with 0.5% (w/v L-arabinose as main carbon source. From all evaluated bacterial strains, Enterococcus faecium DBFIQ ID: E36, Enterococcus faecium DBFIQ ID: ETW4 and Pediococcus acidilactici ATCC ID: 8042 were, in this order, the best L-arabinose fermenting strains. Afterwards, to assay L-arabinose metabolization and L-arabinose isomerase activity, cell-free extract and saline precipitated cell-free extract of the three bacterial cultures were obtained and the production of ketoses was determined by the cysteine carbazole sulfuric acid method. Results showed that the greater the L-arabinose metabolization ability, the higher the enzymatic activity achieved, so Enterococcus faecium DBFIQ ID: E36 was selected to continue with production, purification and characterization studies. This work thus describes a simple microbiological method for the selection of L-arabinose fermenting bacteria for the potential production of the enzyme L-arabinose isomerase.

  5. High production of D-tagatose, a potential sugar substitute, using immobilized L-arabinose isomerase.

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    Kim, P; Yoon, S H; Roh, H J; Choi, J H

    2001-01-01

    An L-arabinose isomerase of Escherichia coli was immobilized using covalent binding to agarose to produce D-tagatose, a bulking sweetener that can be economically used as a sugar substitute. The immobilized L-arabinose isomerase stably produced an average of 7.5 g-tagatose/L.day for 7 days with a productivity exceeding that of the free enzyme (0.47 vs 0.30 mg/U.day). Using a scaled-up immobilized enzyme system, 99.9 g-tagatose/L was produced from galactose with 20% equilibrium in 48 h. The process was repeated two more times with production of 104.1 and 103.5 g-tagatose/L. D-Tagatose production using an immobilized L-arabinose isomerase has a high potential for commercial application.

  6. The secreted L-arabinose isomerase displays anti-hyperglycemic effects in mice.

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    Rhimi, Moez; Bermudez-Humaran, Luis G; Huang, Yuan; Boudebbouze, Samira; Gaci, Nadia; Garnier, Alexandrine; Gratadoux, Jean-Jacques; Mkaouar, Héla; Langella, Philippe; Maguin, Emmanuelle

    2015-12-21

    The L-arabinose isomerase is an intracellular enzyme which converts L-arabinose into L-ribulose in living systems and D-galactose into D-tagatose in industrial processes and at industrial scales. D-tagatose is a natural ketohexose with potential uses in pharmaceutical and food industries. The D-galactose isomerization reaction is thermodynamically equilibrated, and leads to secondary subproducts at high pH. Therefore, an attractive L-arabinose isomerase should be thermoactive and acidotolerant with high catalytic efficiency. While many reports focused on the set out of a low cost process for the industrial production of D-tagatose, these procedures remain costly. When compared to intracellular enzymes, the production of extracellular ones constitutes an interesting strategy to increase the suitability of the biocatalysts. The L-arabinose isomerase (L-AI) from Lactobacillus sakei was expressed in Lactococcus lactis in fusion with the signal peptide of usp45 (SP(Usp45)). The L-AI protein and activity were detected only in the supernatant of the induced cultures of the recombinant L. lactis demonstrating the secretion in the medium of the intracellular L. sakei L-AI in an active form. Moreover, we showed an improvement in the enzyme secretion using either (1) L. lactis strains deficient for their two major proteases, ClpP and HtrA, or (2) an enhancer of protein secretion in L. lactis fused to the recombinant L-AI with the SP(Usp45). Th L-AI enzyme secreted by the recombinant L. lactis strains or produced intracellularly in E. coli, showed the same functional properties than the native enzyme. Furthermore, when mice are fed with the L. lactis strain secreting the L-AI and galactose, tagatose was produced in vivo and reduced the glycemia index. We report for the first time the secretion of the intracellular L-arabinose isomerase in the supernatant of food grade L. lactis cultures with hardly display other secreted proteins. The secreted L-AI originated from the food

  7. Bacterial L-arabinose isomerases: industrial application for D-tagatose production.

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    Boudebbouze, Samira; Maguin, Emmanuelle; Rhimi, Moez

    2011-12-01

    D-tagatose is a natural monosaccharide with a low caloric value and has an anti-hyperglycemiant effect. This hexose has potential applications both in pharmaceutical and agro-food industries. However, the use of D-tagatose remains limited by its production cost. Many production procedures including chemical and biological processes were developed and patented. The most profitable production way is based on the use of L-arabinose isomerase which allows the manufacture of D-tagatose with an attractive rate. Future developments are focused on the generation of L-arabinose isomerases having biochemical properties satisfying the industrial applications. This report provides a brief review of the most recent patents that have been published relating to this area.

  8. Structural insights into conserved L-arabinose metabolic enzymes reveal the substrate binding site of a thermophilic L-arabinose isomerase.

    Science.gov (United States)

    Lee, Yong-Jik; Lee, Sang-Jae; Kim, Seong-Bo; Lee, Sang Jun; Lee, Sung Haeng; Lee, Dong-Woo

    2014-03-18

    Structural genomics demonstrates that despite low levels of structural similarity of proteins comprising a metabolic pathway, their substrate binding regions are likely to be conserved. Herein based on the 3D-structures of the α/β-fold proteins involved in the ara operon, we attempted to predict the substrate binding residues of thermophilic Geobacillus stearothermophilus L-arabinose isomerase (GSAI) with no 3D-structure available. Comparison of the structures of L-arabinose catabolic enzymes revealed a conserved feature to form the substrate-binding modules, which can be extended to predict the substrate binding site of GSAI (i.e., D195, E261 and E333). Moreover, these data implicated that proteins in the l-arabinose metabolic pathway might retain their substrate binding niches as the modular structure through conserved molecular evolution even with totally different structural scaffolds. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  9. Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase that increases the production rate of D-tagatose.

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    Kim, H-J; Kim, J-H; Oh, H-J; Oh, D-K

    2006-07-01

    Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase used to increase the production rate of D-tagatose. A mutated gene was obtained by an error-prone polymerase chain reaction using L-arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in Escherichia coli. The expressed mutated L-arabinose isomerase exhibited the change of three amino acids (Met322-->Val, Ser393-->Thr, and Val408-->Ala), compared with the wild-type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerization activity at pH 8.0, 65 degrees C, and 1.0 mM Co2+, while the wild-type enzyme had a maximum activity at pH 8.0, 60 degrees C, and 1.0-mM Mn2+. The mutated L-arabinose isomerase exhibited increases in D-galactose isomerization activity, optimum temperature, catalytic efficiency (kcat/Km) for D-galactose, and the production rate of D-tagatose from D-galactose. The mutated L-arabinose isomerase from G. stearothermophilus is valuable for the commercial production of D-tagatose. This work contributes knowledge on the characterization of a mutated L-arabinose isomerase, and allows an increased production rate for D-tagatose from D-galactose using the mutated enzyme.

  10. Heterologous expression and characterization of Bacillus coagulans L-arabinose isomerase.

    Science.gov (United States)

    Zhou, Xingding; Wu, Jin Chuan

    2012-05-01

    Bacillus coagulans has been of great commercial interest over the past decade owing to its strong ability of producing optical pure L: -lactic acid from both hexose and pentose sugars including L: -arabinose with high yield, titer and productivity under thermophilic conditions. The L: -arabinose isomerase (L-AI) from Bacillus coagulans was heterologously over-expressed in Escherichia coli. The open reading frame of the L-AI has 1,422 nucleotides encoding a protein with 474 amino acid residues. The recombinant L-AI was purified to homogeneity by one-step His-tag affinity chromatography. The molecular mass of the enzyme was estimated to be 56 kDa by SDS-PAGE. The enzyme was most active at 70°C and pH 7.0. The metal ion Mn(2+) was shown to be the best activator for enzymatic activity and thermostability. The enzyme showed higher activity at acidic pH than at alkaline pH. The kinetic studies showed that the K (m), V (max) and k (cat)/K (m) for the conversion of L: -arabinose were 106 mM, 84 U/mg and 34.5 mM(-1)min(-1), respectively. The equilibrium ratio of L: -arabinose to L: -ribulose was 78:22 under optimal conditions. L: -ribulose (97 g/L) was obtained from 500 g/l of L: -arabinose catalyzed by the enzyme (8.3 U/mL) under the optimal conditions within 1.5 h, giving at a substrate conversion of 19.4% and a production rate of 65 g L(-1) h(-1).

  11. Characterization of an L-arabinose isomerase from Bacillus thermoglucosidasius for D-tagatose production.

    Science.gov (United States)

    Seo, Myung-Ji

    2013-01-01

    L-Arabinose isomerase from Bacillus thermoglucosidasius KCTC 1828 (BTAI) was expressed in Escherichia coli. The optimal temperature and pH for the activity of the purified BTAI were 40 °C and pH 7.0. The Mn(2+) ion was an activator of BTAI activity. The kinetic parameters of BTAI for D-galactose were a K(m) of 175 mM and a k(cat)/K(m) of 2.8 mM(-1)min(-1). The conversion ratio by BTAI to D-tagatose reached 45.6% at 40 °C.

  12. Mechanism of ultraviolet light induced catabolite repression of L-arabinose isomerase

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    Bhatnagar, D; Bhattacharya, A K [Banaras Hindu Univ. (India). Inst. of Medical Sciences

    1982-12-01

    An attempt has been made to find out how U.V. irradiation of E.coli B/r cells causes catabolite repression to inhibit L-arabinose isomerase synthesis. The results presented show that U.V. irradiation leads to a lowering of the cellular cyclic AMP level and of the cyclic AMP binding activity. Unlike catabolite repression by glucose, no small molecular weight compound is involved in U.V. light induced inhibition of the binding activity. It is therefore concluded that the mechanism of catabolite repression induced by U.V. appears to be different from that of the catabolite repression by glucose.

  13. Bioconversion of D-galactose into D-tagatose by expression of L-arabinose isomerase.

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    Roh, H J; Kim, P; Park, Y C; Choi, J H

    2000-02-01

    D-Tagatose is a potential bulking agent in food as a non-calorific sweetener. To produce D-tagatose from cheaper resources, plasmids harbouring the L-arabinose isomerase gene (araA) from Escherichia coli, Bacillus subtilis and Salmonella typhimurium were constructed because L-arabinose isomerase was suggested previously as an enzyme that mediates the bioconversion of galactose into tagatose as well as that of arabinose to ribulose. The constructed plasmids were named pTC101, pTC105 and pTC106, containing araA from E. coli, B. subtilis and S. typhimurium respectively. In the cultures of recombinant E. coli with pTC101, pTC105 and pTC106, tagatose was produced from galactose in 9.9, 7.1 and 6.9% yields respectively. The enzyme extract of E. coli with the plasmid pTC101 also converted galactose into tagatose with a 96.4% yield.

  14. L-Arabinose isomerase and its use for biotechnological production of rare sugars.

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    Xu, Zheng; Li, Sha; Feng, Xiaohai; Liang, Jinfeng; Xu, Hong

    2014-11-01

    L-Arabinose isomerase (AI), a key enzyme in the microbial pentose phosphate pathway, has been regarded as an important biological catalyst in rare sugar production. This enzyme could isomerize L-arabinose into L-ribulose, as well as D-galactose into D-tagatose. Both the two monosaccharides show excellent commercial values in food and pharmaceutical industries. With the identification of novel AI family members, some of them have exhibited remarkable potential in industrial applications. The biological production processes for D-tagatose and L-ribose (or L-ribulose) using AI have been developed and improved in recent years. Meanwhile, protein engineering techniques involving rational design has effectively enhanced the catalytic properties of various AIs. Moreover, the crystal structure of AI has been disclosed, which sheds light on the understanding of AI structure and catalytic mechanism at molecular levels. This article reports recent developments in (i) novel AI screening, (ii) AI-mediated rare sugar production processes, (iii) molecular modification of AI, and (iv) structural biology study of AI. Based on previous reports, an analysis of the future development has also been initiated.

  15. Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis.

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    de Sousa, Marylane; Manzo, Ricardo M; García, José L; Mammarella, Enrique J; Gonçalves, Luciana R B; Pessela, Benevides C

    2017-12-06

    l-Arabinose isomerase (EC 5.3.1.4) (l-AI) from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N -His-l-AI and C -His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C -His-l-AI was preferentially hexameric in solution, whereas N -His-l-AI was mainly monomeric. The specific activity of the N -His-l-AI at acidic pH was higher than that of C -His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg -1 , respectively. However, C -His-l-AI was more active and stable at alkaline pH than N -His-l-AI. N -His-l-AI follows a Michaelis-Menten kinetic, whereas C -His-l-AI fitted to a sigmoidal saturation curve.

  16. Enhanced activity and stability of L-arabinose isomerase by immobilization on aminopropyl glass.

    Science.gov (United States)

    Zhang, Ye-Wang; Jeya, Marimuthu; Lee, Jung-Kul

    2011-03-01

    Immobilization of Bacillus licheniformis L: -arabinose isomerase (BLAI) on aminopropyl glass modified with glutaraldehyde (4 mg protein g support⁻¹) was found to enhance the enzyme activity. The immobilization yield of BLAI was proportional to the quantity of amino groups on the surface of support. Reducing particle size increased the adsorption capacity (q(m)) and affinity (k(a)). The pH and temperature for immobilization were optimized to be pH 7.1 and 33 °C using response surface methodology (RSM). The immobilized enzyme was characterized and compared to the free enzyme. There is no change in optimal pH and temperature before and after immobilization. However, the immobilized BLAI enzyme achieved 145% of the activity of the free enzyme. Correspondingly, the catalytic efficiency (k(cat)/K(m)) was improved 1.47-fold after immobilization compared to the free enzyme. The thermal stability was improved 138-fold (t₁/₂) increased from 2 to 275 h) at 50 °C following immobilization.

  17. Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis

    Directory of Open Access Journals (Sweden)

    Marylane de Sousa

    2017-12-01

    Full Text Available l-Arabinose isomerase (EC 5.3.1.4 (l-AI from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N-His-l-AI and C-His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C-His-l-AI was preferentially hexameric in solution, whereas N-His-l-AI was mainly monomeric. The specific activity of the N-His-l-AI at acidic pH was higher than that of C-His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg−1, respectively. However, C-His-l-AI was more active and stable at alkaline pH than N-His-l-AI. N-His-l-AI follows a Michaelis-Menten kinetic, whereas C-His-l-AI fitted to a sigmoidal saturation curve.

  18. Rational design of Bacillus stearothermophilus US100 L-arabinose isomerase: potential applications for D-tagatose production.

    Science.gov (United States)

    Rhimi, Moez; Aghajari, Nushin; Juy, Michel; Chouayekh, Hichem; Maguin, Emmanuelle; Haser, Richard; Bejar, Samir

    2009-05-01

    L-arabinose isomerases catalyze the bioconversion of D-galactose into D-tagatose. With the aim of producing an enzyme optimized for D-tagatose production, three Bacillus stearothermophilus US100 L-arabinose isomerase mutants were constructed, purified and characterized. Our results indicate that mutant Q268K was significantly more acidotolerant and more stable at acidic pH than the wild-type enzyme. The N175H mutant has a broad optimal temperature range from 50 to 65 degrees C. With the aim of constructing an acidotolerant mutant working at relatively low temperatures we generated the Q268K/N175H construct. This double mutant displays an optimal pH in the range 6.0-7.0 and an optimal activity around 50-65 degrees C, temperatures at which the enzyme was stable without addition of metal ions.

  19. Biochemical properties of L-arabinose isomerase from Clostridium hylemonae to produce D-tagatose as a functional sweetener.

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    Nguyen, Tien-Kieu; Hong, Moon-Gi; Chang, Pahn-Shick; Lee, Byung-Hoo; Yoo, Sang-Ho

    2018-01-01

    d-Tagatose has gained substantial interest due to its potential functionalities as a sucrose substitute. In this study, the gene araA, encoding l-arabinose isomerase (l-AI) from Clostridium hylemonae (DSM 15053), was cloned and expressed in Escherichia coli BL21 (DE3). This gene consists of 1,506 nucleotides and encodes a protein of 501 amino acid residues with a calculated molecular mass of 56,554 Da. Since l-AI was expressed as an intracellular inclusion body, this enzyme was solubilized with guanidine hydrochloride, refolded, and activated with a descending concentration gradient of urea. The purified enzyme exhibited the greatest activity at 50°C, pH 7-7.5, and required 1 mM of Mg2+ as a cofactor. Notably, the catalytic efficiency (3.69 mM-1sec-1) of l-AI from C. hylemonae on galactose was significantly greater than that of other previously reported enzymes. The bioconversion yield of d-tagatose using the C. hylemonae l-arabinose isomerase at 60°C reached approximately 46% from 10 mM of d-galactose after 2 h. From these results, it is suggested that the l-arabinose isomerase from C. hylemonae could be utilized as a potential enzyme for d-tagatose production due to its high conversion yield at an industrially competitive temperature.

  20. Creation of metal-independent hyperthermophilic L-arabinose isomerase by homologous recombination.

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    Hong, Young-Ho; Lee, Dong-Woo; Pyun, Yu-Ryang; Lee, Sung Haeng

    2011-12-28

    Hyperthermophilic L-arabinose isomerases (AIs) are useful in the commercial production of D-tagatose as a low-calorie bulk sweetener. Their catalysis and thermostability are highly dependent on metals, which is a major drawback in food applications. To study the role of metal ions in the thermostability and catalysis of hyperthermophilic AI, four enzyme chimeras were generated by PCR-based hybridization to replace the variable N- and C-terminal regions of hyperthermophilic Thermotoga maritima AI (TMAI) and thermophilic Geobacillus stearothermophilus AI (GSAI) with those of the homologous mesophilic Bacillus halodurans AI (BHAI). Unlike Mn(2+)-dependent TMAI, the GSAI- and TMAI-based hybrids with the 72 C-terminal residues of BHAI were not metal-dependent for catalytic activity. By contrast, the catalytic activities of the TMAI- and GSAI-based hybrids containing the N-terminus (residues 1-89) of BHAI were significantly enhanced by metals, but their thermostabilities were poor even in the presence of Mn(2+), indicating that the effects of metals on catalysis and thermostability involve different structural regions. Moreover, in contrast to the C-terminal truncate (Δ20 residues) of GSAI, the N-terminal truncate (Δ7 residues) exhibited no activity due to loss of its native structure. The data thus strongly suggest that the metal dependence of the catalysis and thermostability of hyperthermophilic AIs evolved separately to optimize their activity and thermostability at elevated temperatures. This may provide effective target regions for engineering, thereby meeting industrial demands for the production of d-tagatose.

  1. A single and two step isomerization process for d-tagatose and l-ribose bioproduction using l-arabinose isomerase and d-lyxose isomerase.

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    Patel, Manisha J; Akhani, Rekha C; Patel, Arti T; Dedania, Samir R; Patel, Darshan H

    2017-02-01

    l-ribose and d-tagatose are biochemically synthesized using sugar isomerases. The l-arabinose isomerase gene from Shigella flexneri (Sf-AI) was cloned and expressed in Escherichia coli BL-21. Sf-AI was applied for the bioproduction of d-tagatose from d-galactose. l-ribose synthesis was performed by two step isomerization using Sf-AI and d-lyxose/ribose isomerase from Cohnella laevoribosii. The overall 22.3% and 25% conversion rate were observed for d-tagatose and l-ribose production from d-galactose and l-arabinose respectively. In the present manuscript, synthesis of rare sugars from naturally available sugars is discussed along with the biochemical characterization of Sf-AI and its efficiency. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Increase in D-tagatose production rate by site-directed mutagenesis of L-arabinose isomerase from Geobacillus thermodenitrificans.

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    Oh, Hyo-Jung; Kim, Hye-Jung; Oh, Deok-Kun

    2006-02-01

    Among single-site mutations of L-arabinose isomerase derived from Geobacillus thermodenitrificans, two mutants were produced having the lowest and highest activities of D-tagatose production. Site-directed mutagenesis at these sites showed that the aromatic ring at amino acid 164 and the size of amino acid 475 were important for D-tagatose production. Among double-site mutations, one mutant converted D-galactose into D-tagatose with a yield of 58% whereas the wild type gave 46% D-tagatose conversion after 300 min at 65 degrees C.

  3. Purification and characterization of an L-arabinose isomerase from an isolated strain of Geobacillus thermodenitrificans producing D-tagatose.

    Science.gov (United States)

    Kim, Hye-Jung; Oh, Deok-Kun

    2005-11-04

    The araA gene, encoding l-arabinose isomerase (AI), from the thermophilic bacterium Geobacillus thermodenitrificans was cloned and expressed in Escherichia coli. Recombinant AI was isolated with a final purity of about 97% and a final specific activity of 2.10 U/mg. The molecular mass of the purified AI was estimated to be about 230 kDa to be a tetramer composed of identical subunits. The AI exhibited maximum activity at 70 degrees C and pH 8.5 in the presence of Mn2+. The enzyme was stable at temperatures below 60 degrees C and within the pH range 7.5-8.0. d-Galactose and l-arabinose as substrate were isomerized with high activities. Ribitol was the strongest competitive inhibitor of AI with a Ki of 5.5mM. The apparent Km and Vmax for L-arabinose were 142 mM and 86 U/mg, respectively, whereas those for d-galactose were 408 mM and 6.9 U/mg, respectively. The catalytic efficiency (kcat/Km) was 48 mM(-1)min(-1) for L-arabinose and 0.5mM(-1)min(-1) for D-galactose. Mn2+ was a competitive activator and increased the thermal stability of the AI. The D-tagatose yield produced by AI from d-galactose was 46% without the addition of Mn2+ and 48% with Mn2+ after 300 min at 65 degrees C.

  4. Production of D-tagatose, a low caloric sweetener during milk fermentation using L-arabinose isomerase.

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    Rhimi, Moez; Chouayekh, Hichem; Gouillouard, Isabelle; Maguin, Emmanuelle; Bejar, Samir

    2011-02-01

    Lactobacillusdelbrueckii subsp. bulgaricus and Streptococcus thermophilus are used for the biotransformation of milk in yoghurt. During milk fermentation, these lactic acid bacteria (LAB) hydrolyze lactose producing a glucose moiety that is further metabolized and a galactose moiety that they are enable to metabolize. We investigated the ability of L. bulgaricus and S. thermophilus strains expressing a heterologous L-arabinose isomerase to convert residual D-galactose to D-tagatose. The Bacillus stearothermophilus US100l-arabinose isomerase (US100l-AI) was expressed in both LAB, using a new shuttle vector where the araA US100 gene is under the control of the strong and constitutive promoter of the L. bulgaricus ATCC 11842 hlbA gene. The production of L-AI by these LAB allowed the bioconversion of D-galactose to D-tagatose during fermentation in laboratory media and milk. We also established that the addition of L-AI to milk also allowed the conversion of D-galactose into D-tagatose during the fermentation process. Copyright © 2010 Elsevier Ltd. All rights reserved.

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

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    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.

  6. 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.

  7. 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.

  8. The acid-tolerant L-arabinose isomerase from the mesophilic Shewanella sp. ANA-3 is highly active at low temperatures

    Science.gov (United States)

    2011-01-01

    Background L-arabinose isomerases catalyse the isomerization of L-arabinose into L-ribulose at insight biological systems. At industrial scale of this enzyme is used for the bioconversion of D-galactose into D-tagatose which has many applications in pharmaceutical and agro-food industries. The isomerization reaction is thermodynamically equilibrated, and therefore the bioconversion rates is shifted towards tagatose when the temperature is increased. Moreover, to prevent secondary reactions it will be of interest to operate at low pH. The profitability of this D-tagatose production process is mainly related to the use of lactose as cheaper raw material. In many dairy products it will be interesting to produce D-tagatose during storage. This requires an efficient L-arabinose isomerase acting at low temperature and pH values. Results The gene encoding the L-arabinose isomerase from Shewanella sp. ANA-3 was cloned and overexpressed in Escherichia coli. The purified protein has a tetrameric arrangement composed by four identical 55 kDa subunits. The biochemical characterization of this enzyme showed that it was distinguishable by its maximal activity at low temperatures comprised between 15-35°C. Interestingly, this biocatalyst preserves more than 85% of its activity in a broad range of temperatures from 4.0 to 45°C. Shewanella sp. ANA-3 L-arabinose isomerase was also optimally active at pH 5.5-6.5 and maintained over 80% of its activity at large pH values from 4.0 to 8.5. Furthermore, this enzyme exhibited a weak requirement for metallic ions for its activity evaluated at 0.6 mM Mn2+. Stability studies showed that this protein is highly stable mainly at low temperature and pH values. Remarkably, T268K mutation clearly enhances the enzyme stability at low pH values. Use of this L-arabinose isomerase for D-tagatose production allows the achievement of attractive bioconversion rates of 16% at 4°C and 34% at 35°C. Conclusions Here we reported the purification and the

  9. The acid-tolerant L-arabinose isomerase from the mesophilic Shewanella sp. ANA-3 is highly active at low temperatures

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    Rhimi Moez

    2011-11-01

    Full Text Available Abstract Background L-arabinose isomerases catalyse the isomerization of L-arabinose into L-ribulose at insight biological systems. At industrial scale of this enzyme is used for the bioconversion of D-galactose into D-tagatose which has many applications in pharmaceutical and agro-food industries. The isomerization reaction is thermodynamically equilibrated, and therefore the bioconversion rates is shifted towards tagatose when the temperature is increased. Moreover, to prevent secondary reactions it will be of interest to operate at low pH. The profitability of this D-tagatose production process is mainly related to the use of lactose as cheaper raw material. In many dairy products it will be interesting to produce D-tagatose during storage. This requires an efficient L-arabinose isomerase acting at low temperature and pH values. Results The gene encoding the L-arabinose isomerase from Shewanella sp. ANA-3 was cloned and overexpressed in Escherichia coli. The purified protein has a tetrameric arrangement composed by four identical 55 kDa subunits. The biochemical characterization of this enzyme showed that it was distinguishable by its maximal activity at low temperatures comprised between 15-35°C. Interestingly, this biocatalyst preserves more than 85% of its activity in a broad range of temperatures from 4.0 to 45°C. Shewanella sp. ANA-3 L-arabinose isomerase was also optimally active at pH 5.5-6.5 and maintained over 80% of its activity at large pH values from 4.0 to 8.5. Furthermore, this enzyme exhibited a weak requirement for metallic ions for its activity evaluated at 0.6 mM Mn2+. Stability studies showed that this protein is highly stable mainly at low temperature and pH values. Remarkably, T268K mutation clearly enhances the enzyme stability at low pH values. Use of this L-arabinose isomerase for D-tagatose production allows the achievement of attractive bioconversion rates of 16% at 4°C and 34% at 35°C. Conclusions Here we

  10. Identification and characterization of a novel L-arabinose isomerase from Anoxybacillus flavithermus useful in D-tagatose production.

    Science.gov (United States)

    Li, Yanjun; Zhu, Yueming; Liu, Anjun; Sun, Yuanxia

    2011-05-01

    D-Tagatose is a highly functional rare ketohexose and many attempts have been made to convert D-galactose into the valuable D-tagatose using L-arabinose isomerase (L-AI). In this study, a thermophilic strain possessing L-AI gene was isolated from hot spring sludge and identified as Anoxybacillus flavithermus based on its physio-biochemical characterization and phylogenetic analysis of its 16s rRNA gene. Furthermore, the gene encoding L-AI from A. flavithermus (AFAI) was cloned and expressed at a high level in E. coli BL21(DE3). L-AI had a molecular weight of 55,876 Da, an optimum pH of 10.5 and temperature of 95°C. The results showed that the conversion equilibrium shifted to more D-tagatose from D-galactose by raising the reaction temperatures and adding borate. A 60% conversion of D-galactose to D-tagatose was observed at an isomerization temperature of 95°C with borate. The catalytic efficiency (k (cat) /K (m)) for D-galactose with borate was 9.47 mM(-1) min(-1), twice as much as that without borate. Our results indicate that AFAI is a novel hyperthermophilic and alkaliphilic isomerase with a higher catalytic efficiency for D-galactose, suggesting its great potential for producing D-tagatose.

  11. Cloning, expression and characterization of L-arabinose isomerase from Thermotoga neapolitana: bioconversion of D-galactose to D-tagatose using the enzyme.

    Science.gov (United States)

    Kim, Byoung-Chan; Lee, Yoon-Hee; Lee, Han-Seung; Lee, Dong-Woo; Choe, Eun-Ah; Pyun, Yu-Ryang

    2002-06-18

    Gene araA encoding an L-arabinose isomerase (AraA) from the hyperthermophile, Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 496 residues with a calculated molecular mass of 56677 Da. The deduced amino acid sequence has 94.8% identical amino acids compared with the residues in a putative L-arabinose isomerase of Thermotoga maritima. The recombinant enzyme expressed in E. coli was purified to homogeneity by heat treatment, ion exchange chromatography and gel filtration. The thermophilic enzyme had a maximum activity of L-arabinose isomerization and D-galactose isomerization at 85 degrees C, and required divalent cations such as Co(2+) and Mn(2+) for its activity and thermostability. The apparent K(m) values of the enzyme for L-arabinose and D-galactose were 116 mM (v(max), 119 micromol min(-1) mg(-1)) and 250 mM (v(max), 14.3 micromol min(-1) mg(-1)), respectively, that were determined in the presence of both 1 mM Co(2+) and 1 mM Mn(2+). A 68% conversion of D-galactose to D-tagatose was obtained using the recombinant enzyme at the isomerization temperature of 80 degrees C.

  12. Production of D-tagatose at high temperatures using immobilized Escherichia coli cells expressing L-arabinose isomerase from Thermotoga neapolitana.

    Science.gov (United States)

    Hong, Young-Ho; Lee, Dong-Woo; Lee, Sang-Jae; Choe, Eun-Ah; Kim, Seong-Bo; Lee, Yoon-Hee; Cheigh, Chan-Ick; Pyun, Yu-Ryang

    2007-04-01

    Escherichia coli cells expressing L-arabinose isomerase from Thermotoga neapolitana (TNAI) were immobilized in calcium alginate beads. The resulting cell reactor (2.4 U, t (1/2) = 43 days at 70 degrees C) in a continuous recycling mode at 70 degrees C produced 49 and 38 g D-tagatose/l from 180 and 90 g D-galactose/l, respectively, within 12 h.

  13. Characterization of an L-arabinose isomerase from Bacillus coagulans NL01 and its application for D-tagatose production.

    Science.gov (United States)

    Mei, Wending; Wang, Lu; Zang, Ying; Zheng, Zhaojuan; Ouyang, Jia

    2016-06-30

    L-arabinose isomerase (AI) is a crucial catalyst for the biotransformation of D-galactose to D-tagatose. In previous reports, AIs from thermophilic bacterial strains had been wildly researched, but the browning reaction and by-products formed at high temperatures restricted their applications. By contrast, AIs from mesophilic Bacillus strains have some different features including lower optimal temperatures and lower requirements of metallic cofactors. These characters will be beneficial to the development of a more energy-efficient and safer production process. However, the relevant data about the kinetics and reaction properties of Bacillus AIs in D-tagatose production are still insufficient. Thus, in order to support further applications of these AIs, a comprehensive characterization of a Bacillus AI is needed. The coding gene (1422 bp) of Bacillus coagulans NL01 AI (BCAI) was cloned and overexpressed in the Escherichia coli BL21 (DE3) strain. The enzymatic property test showed that the optimal temperature and pH of BCAI were 60 °C and 7.5 respectively. The raw purified BCAI originally showed high activity in absence of outsourcing metallic ions and its thermostability did not change in a low concentration (0.5 mM) of Mn(2+) at temperatures from 70 °C to 90 °C. Besides these, the catalytic efficiencies (k cat/K m) for L-arabinose and D-galactose were 8.7 mM(-1) min(-1) and 1.0 mM(-1) min(-1) respectively. Under optimal conditions, the recombinant E. coli cell containing BCAI could convert 150 g L(-1) and 250 g L(-1) D-galactose to D-tagatose with attractive conversion rates of 32 % (32 h) and 27 % (48 h). In this study, a novel AI from B. coagulans NL01was cloned, purified and characterized. Compared with other reported AIs, this AI could retain high proportions of activity at a broader range of temperatures and was less dependent on metallic cofactors such as Mn(2+). Its substrate specificity was understood deeply by carrying out molecular

  14. Enzymatic conversion of D-galactose to D-tagatose: cloning, overexpression and characterization of L-arabinose isomerase from Pediococcus pentosaceus PC-5.

    Science.gov (United States)

    Men, Yan; Zhu, Yueming; Zhang, Lili; Kang, Zhenkui; Izumori, Ken; Sun, Yuanxia; Ma, Yanhe

    2014-01-01

    The gene encoding L-arabinose isomerase from food-grade strain Pediococcus pentosaceus PC-5 was cloned and overexpressed in Escherichia coli. The recombinant protein was purified and characterized. It was optimally active at 50 °C and pH 6.0. Furthermore, this enzyme exhibited a weak requirement for metallic ions for its maximal activity evaluated at 0.6 mM Mn(2+) or 0.8 mM Co(2+). Interestingly, this enzyme was distinguished from other L-AIs, it could not use L-arabinose as its substrate. In addition, a three-dimensional structure of L-AI was built by homology modeling and L-arabinose and D-galactose were docked into the active site pocket of PPAI model to explain the interaction between L-AI and its substrate. The purified P. pentosaceus PC-5 L-AI converted D-galactose into D-tagatose with a high conversion rate of 52% after 24 h at 50 °C, suggesting its excellent potential in D-tagatose production. Crown Copyright © 2013. Published by Elsevier GmbH. All rights reserved.

  15. Cloning of araA Gene Encoding L-Arabinose Isomerase from Marine Geobacillus stearothermophilus Isolated from Tanjung Api, Poso, Indonesia

    Directory of Open Access Journals (Sweden)

    DEWI FITRIANI

    2010-06-01

    Full Text Available L-arabinose isomerase is an enzyme converting D-galactose to D-tagatose. D-tagatose is a potential sweetener-sucrose substitute which has low calorie. This research was to clone and sequence araA gene from marine bacterial strain Geobacillus stearothermophilus isolated from Tanjung Api Poso Indonesia. The amplified araA gene consisted of 1494 bp nucleotides encoding 497 amino acids. DNA alignment analysis showed that the gene had high homology with that of G. stearothermophilus T6. The enzyme had optimum activity at high temperature and alkalin condition.

  16. Coexpression of β-D-galactosidase and L-arabinose isomerase in the production of D-tagatose: a functional sweetener.

    Science.gov (United States)

    Zhan, Yijing; Xu, Zheng; Li, Sha; Liu, Xiaoliu; Xu, Lu; Feng, Xiaohai; Xu, Hong

    2014-03-19

    The functional sweetener, d-tagatose, is commonly transformed from galactose by l-arabinose isomerase. To make use of a much cheaper starting material, lactose, hydrolization, and isomerization are required to take place collaboratively. Therefore, a single-step method involving β-d-galactosidase was explored for d-tagatose production. The two vital genes, β-d-galactosidase gene (lacZ) and l-arabinose isomerase mutant gene (araA') were extracted separately from Escherichia coli strains and incorporated into E. coli simultaneously. This gave us E. coli-ZY, a recombinant producing strain capable of coexpressing the two key enzymes. The resulted cells exhibited maximum d-tagatose producing activity at 34 °C and pH 6.5 and in the presence of borate, 10 mM Fe(2+), and 1 mM Mn(2+). Further monitoring showed that the recombinant cells could hydrolyze more than 95% lactose and convert 43% d-galactose into d-tagatose. This research has verified the feasibility of single-step d-tagatose fermentation, thereby laying down the foundation for industrial usage of lactose.

  17. 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.

  18. A method for the production of D-tagatose using a recombinant Pichia pastoris strain secreting β-D-galactosidase from Arthrobacter chlorophenolicus and a recombinant L-arabinose isomerase from Arthrobacter sp. 22c.

    Science.gov (United States)

    Wanarska, Marta; Kur, Józef

    2012-08-23

    D-Tagatose is a natural monosaccharide which can be used as a low-calorie sugar substitute in food, beverages and pharmaceutical products. It is also currently being tested as an anti-diabetic and obesity control drug. D-Tagatose is a rare sugar, but it can be manufactured by the chemical or enzymatic isomerization of D-galactose obtained by a β-D-galactosidase-catalyzed hydrolysis of milk sugar lactose and the separation of D-glucose and D-galactose. L-Arabinose isomerases catalyze in vitro the conversion of D-galactose to D-tagatose and are the most promising enzymes for the large-scale production of D-tagatose. In this study, the araA gene from psychrotolerant Antarctic bacterium Arthrobacter sp. 22c was isolated, cloned and expressed in Escherichia coli. The active form of recombinant Arthrobacter sp. 22c L-arabinose isomerase consists of six subunits with a combined molecular weight of approximately 335 kDa. The maximum activity of this enzyme towards D-galactose was determined as occurring at 52°C; however, it exhibited over 60% of maximum activity at 30°C. The recombinant Arthrobacter sp. 22c L-arabinose isomerase was optimally active at a broad pH range of 5 to 9. This enzyme is not dependent on divalent metal ions, since it was only marginally activated by Mg2+, Mn2+ or Ca2+ and slightly inhibited by Co2+ or Ni2+. The bioconversion yield of D-galactose to D-tagatose by the purified L-arabinose isomerase reached 30% after 36 h at 50°C. In this study, a recombinant Pichia pastoris yeast strain secreting β-D-galactosidase Arthrobacter chlorophenolicus was also constructed. During cultivation of this strain in a whey permeate, lactose was hydrolyzed and D-glucose was metabolized, whereas D-galactose was accumulated in the medium. Moreover, cultivation of the P. pastoris strain secreting β-D-galactosidase in a whey permeate supplemented with Arthrobacter sp. 22c L-arabinose isomerase resulted in a 90% yield of lactose hydrolysis, the complete utilization

  19. A method for the production of D-tagatose using a recombinant Pichia pastoris strain secreting β-D-galactosidase from Arthrobacter chlorophenolicus and a recombinant L-arabinose isomerase from Arthrobacter sp. 22c

    Directory of Open Access Journals (Sweden)

    Wanarska Marta

    2012-08-01

    Full Text Available Abstract Background D-Tagatose is a natural monosaccharide which can be used as a low-calorie sugar substitute in food, beverages and pharmaceutical products. It is also currently being tested as an anti-diabetic and obesity control drug. D-Tagatose is a rare sugar, but it can be manufactured by the chemical or enzymatic isomerization of D-galactose obtained by a β-D-galactosidase-catalyzed hydrolysis of milk sugar lactose and the separation of D-glucose and D-galactose. L-Arabinose isomerases catalyze in vitro the conversion of D-galactose to D-tagatose and are the most promising enzymes for the large-scale production of D-tagatose. Results In this study, the araA gene from psychrotolerant Antarctic bacterium Arthrobacter sp. 22c was isolated, cloned and expressed in Escherichia coli. The active form of recombinant Arthrobacter sp. 22c L-arabinose isomerase consists of six subunits with a combined molecular weight of approximately 335 kDa. The maximum activity of this enzyme towards D-galactose was determined as occurring at 52°C; however, it exhibited over 60% of maximum activity at 30°C. The recombinant Arthrobacter sp. 22c L-arabinose isomerase was optimally active at a broad pH range of 5 to 9. This enzyme is not dependent on divalent metal ions, since it was only marginally activated by Mg2+, Mn2+ or Ca2+ and slightly inhibited by Co2+ or Ni2+. The bioconversion yield of D-galactose to D-tagatose by the purified L-arabinose isomerase reached 30% after 36 h at 50°C. In this study, a recombinant Pichia pastoris yeast strain secreting β-D-galactosidase Arthrobacter chlorophenolicus was also constructed. During cultivation of this strain in a whey permeate, lactose was hydrolyzed and D-glucose was metabolized, whereas D-galactose was accumulated in the medium. Moreover, cultivation of the P. pastoris strain secreting β-D-galactosidase in a whey permeate supplemented with Arthrobacter sp. 22c L-arabinose isomerase resulted in a 90% yield

  20. 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%.

  1. [Screening of food-grade microorganisms for biotransformation of D-tagatose and cloning and expression of L-arabinose isomerase].

    Science.gov (United States)

    Men, Yan; Zhu, Yueming; Guan, Yuping; Zhang, Tongcun; Izumori, Ken; Sun, Yuanxia

    2012-05-01

    L-Arabinose isomerase (L-AI) is an intracellular enzyme that catalyzes the reversible isomerization of D-galactose and D-tagatose. Given the widespread use of D-tagatose in the food industry, food-grade microorganisms and the derivation of L-AI for the production of D-tagatose is gaining increased attention. In the current study, food-grade strains from different foods that can convert D-galactose to D-tagatose were screened. According to physiological, biochemical, and 16S rDNA gene analyses, the selected strain was found to share 99% identity with Pediococcus pentosaceus, and was named as Pediococcus pentosaceus PC-5. The araA gene encoding L-AI from Pediococcus pentosaceus PC-5 was cloned and overexpressed in E. coli BL21. The yield of D-tagatose using D-galactose as the substrate catalyzed by the crude enzyme in the presence of Mn2+ was found to be 33% at 40 degrees C.

  2. D-Tagatose production in the presence of borate by resting Lactococcus lactis cells harboring Bifidobacterium longum L-arabinose isomerase.

    Science.gov (United States)

    Salonen, Noora; Salonen, Kalle; Leisola, Matti; Nyyssölä, Antti

    2013-04-01

    Bifidobacterium longum NRRL B-41409 L-arabinose isomerase (L-AI) was overexpressed in Lactococcus lactis using a phosphate depletion inducible expression system. The resting L. lactis cells harboring the B. longum L-AI were used for production of D-tagatose from D-galactose in the presence of borate buffer. Multivariable analysis suggested that high pH, temperature and borate concentration favoured the conversion of D-galactose to D-tagatose. Almost quantitative conversion (92 %) was achieved at 20 g L⁻¹ substrate and at 37.5 °C after 5 days. The D-tagatose production rate of 185 g L⁻¹ day ⁻¹ was obtained at 300 g L⁻¹ galactose, at 1.15 M borate, and at 41 °C during 10 days when the production medium was changed every 24 h. There was no significant loss in productivity during ten sequential 24 h batches. The initial D-tagatose production rate was 290 g L⁻¹ day⁻¹ under these conditions.

  3. The acid tolerant L-arabinose isomerase from the food grade Lactobacillus sakei 23K is an attractive D-tagatose producer.

    Science.gov (United States)

    Rhimi, Moez; Ilhammami, Rimeh; Bajic, Goran; Boudebbouze, Samira; Maguin, Emmanuelle; Haser, Richard; Aghajari, Nushin

    2010-12-01

    The araA gene encoding an L-arabinose isomerase (L-AI) from the psychrotrophic and food grade Lactobacillus sakei 23K was cloned, sequenced and over-expressed in Escherichia coli. The recombinant enzyme has an apparent molecular weight of nearly 220 kDa, suggesting it is a tetramer of four 54 kDa monomers. The enzyme is distinguishable from previously reported L-AIs by its high activity and stability at temperatures from 4 to 40 degrees C, and pH from 3 to 8, and by its low metal requirement of only 0.8 mM Mn(2+) and 0.8 mM Mg(2+) for its maximal activity and thermostability. Enzyme kinetic studies showed that this enzyme displays a high catalytic efficiency allowing D-galactose bioconversion rates of 20% and 36% at 10 and 45 degrees C, respectively, which are useful for commercial production of D-tagatose. 2010 Elsevier Ltd. All rights reserved.

  4. Rational Design of Bacillus coagulans NL01 l-Arabinose Isomerase and Use of Its F279I Variant in d-Tagatose Production.

    Science.gov (United States)

    Zheng, Zhaojuan; Mei, Wending; Xia, Meijuan; He, Qin; Ouyang, Jia

    2017-06-14

    d-Tagatose is a prospective functional sweetener that can be produced by l-arabinose isomerase (AI) from d-galactose. To improve the activity of AI toward d-galactose, the AI of Bacillus coagulans was rationally designed on the basis of molecular modeling and docking. After alanine scanning and site-saturation mutagenesis, variant F279I that exhibited improved activity toward d-galactose was obtained. The optimal temperature and pH of F279I were determined to be 50 °C and 8.0, respectively. This variant possessed 1.4-fold catalytic efficiency compared with the wild-type (WT) enzyme. The recombinant Escherichia coli overexpressing F279I also showed obvious advantages over the WT in biotransformation. Under optimal conditions, 67.5 and 88.4 g L -1 d-tagatose could be produced from 150 and 250 g L -1 d-galactose, respectively, in 15 h. The biocatalyst constructed in this study presents a promising alternative for large-scale d-tagatose production.

  5. l-Arabinose Isomerase and d-Xylose Isomerase from Lactobacillus reuteri: Characterization, Coexpression in the Food Grade Host Lactobacillus plantarum, and Application in the Conversion of d-Galactose and d-Glucose

    Science.gov (United States)

    2014-01-01

    The l-arabinose isomerase (l-AI) and the d-xylose isomerase (d-XI) encoding genes from Lactobacillus reuteri (DSMZ 17509) were cloned and overexpressed in Escherichia coli BL21 (DE3). The proteins were purified to homogeneity by one-step affinity chromatography and characterized biochemically. l-AI displayed maximum activity at 65 °C and pH 6.0, whereas d-XI showed maximum activity at 65 °C and pH 5.0. Both enzymes require divalent metal ions. The genes were also ligated into the inducible lactobacillal expression vectors pSIP409 and pSIP609, the latter containing a food grade auxotrophy marker instead of an antibiotic resistance marker, and the l-AI- and d-XI-encoding sequences/genes were coexpressed in the food grade host Lactobacillus plantarum. The recombinant enzymes were tested for applications in carbohydrate conversion reactions of industrial relevance. The purified l-AI converted d-galactose to d-tagatose with a maximum conversion rate of 35%, and the d-XI isomerized d-glucose to d-fructose with a maximum conversion rate of 48% at 60 °C. PMID:24443973

  6. L-arabinose fermenting yeast

    Science.gov (United States)

    Zhang, Min; Singh, Arjun; Knoshaug, Eric; Franden, Mary Ann; Jarvis, Eric; Suominen, Pirkko

    2010-12-07

    An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. Methods of producing ethanol include utilizing these modified yeast strains. ##STR00001##

  7. Construction of genetically engineered Candida tropicalis for conversion of l-arabinose to l-ribulose.

    Science.gov (United States)

    Yeo, In-Seok; Shim, Woo-Yong; Kim, Jung Hoe

    2018-05-20

    For the biological production of l-ribulose, conversion by enzymes or resting cells has been investigated. However, expensive or concentrated substrates, an additional purification step to remove borate and the requirement for cell cultivation and harvest steps before utilization of resting cells make the production process complex and unfavorable. Microbial fermentation may help overcome these limitations. In this study, we constructed a genetically engineered Candida tropicalis strain to produce l-ribulose by fermentation with a glucose/l-arabinose mixture. For the uptake of l-arabinose as a substrate and conversion of l-arabinose to l-ribulose, two heterologous genes coding for l-arabinose transporter and l-arabinose isomerase, were constitutively expressed in C. tropicalis under the GAPDH promoter. The Arabidopsis thaliana-originated l-arabinose transporter gene (STP2)-expressing strain exhibited a high l-arabinose uptake rate of 0.103 g/g cell/h and the expression of l-arabinose isomerase from Lactobacillus sakei 23 K showed 30% of conversion (9 g/L) from 30 g/L of l-arabinose. This genetically engineered strain can be used for l-ribulose production by fermentation using mixed sugars of glucose and l-arabinose. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. L-arabinose fermenting yeast

    Science.gov (United States)

    Zhang, Min; Singh, Arjun; Suominen, Pirkko; Knoshaug, Eric; Franden, Mary Ann; Jarvis, Eric

    2013-02-12

    An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. A yeast strain engineered to metabolize arabinose through a novel pathway is also disclosed. Methods of producing ethanol include utilizing these modified yeast strains.

  9. L-arabinose metabolism in Herbaspirillum seropedicae.

    Science.gov (United States)

    Mathias, A L; Rigo, L U; Funayama, S; Pedrosa, F O

    1989-01-01

    The pathway for L-arabinose metabolism in Herbaspirillum seropedicae was shown to involve nonphosphorylated intermediates and to produce alpha-ketoglutarate. The activities of the enzymes and the natures of several intermediates were determined. The pathway was inducible by L-arabinose, and two key enzymes, L-arabinose dehydrogenase and 2-keto-glutarate semialdehyde dehydrogenase, were present in all strains of H. seropedicae tested. PMID:2768202

  10. Metabolic control analysis of Aspergillus niger L-arabinose catabolism

    DEFF Research Database (Denmark)

    de Groot, M.J.L.; Prathumpai, Wai; Visser, J.

    2005-01-01

    A mathematical model of the L-arabinose/D-xylose catabolic pathway of Aspergillus niger was constructed based on the kinetic properties of the enzymes. For this purpose L-arabinose reductase, L-arabitol dehydrogenase and D-xylose reductase were purified using dye-affinity chromatography...... aiming at either flux or metabolite level optimization of the L-arabinose catabolic pathway of A. niger. Faster L-arabinose utilization may enhance utilization of readily available organic waste containing hemicelluloses to be converted into industrially interesting metabolites or valuable enzymes...

  11. Nutritional implications of L-arabinose in pigs

    NARCIS (Netherlands)

    Schutte, J.B.; Jong, J. de; Weerden, E.J. van; Tamminga, S.

    1992-01-01

    The pentose sugar L-arabinose is one of the most abundant components released by complete hydrolysis of non-starch polysaccharides of feed ingredients of vegetable origin. Two studies were conducted to investigate the apparent ileal digestibility and urinary excretion of L-arabinose at dietary

  12. Metabolic control analysis of Aspergillus niger L-arabinose catabolism

    NARCIS (Netherlands)

    Groot, de M.J.L.; Prathumpai, W.; Visser, J.; Ruijter, G.J.G.

    2005-01-01

    A mathematical model of the L-arabinose/D-xylose catabolic pathway of Aspergillus niger was constructed based on the kinetic properties of the enzymes. For this purpose L-arabinose reductase, L-arabitol dehydrogenase and D-xylose reductase were purified using dye-affinity chromatography, and their

  13. Improving L-arabinose utilization of pentose fermenting Saccharomyces cerevisiae cells by heterologous expression of L-arabinose transporting sugar transporters

    Directory of Open Access Journals (Sweden)

    Boles Eckhard

    2011-10-01

    Full Text Available Abstract Background Hydrolysates of plant biomass used for the production of lignocellulosic biofuels typically contain sugar mixtures consisting mainly of D-glucose and D-xylose, and minor amounts of L-arabinose. The yeast Saccharomyces cerevisiae is the preferred microorganism for the fermentative production of ethanol but is not able to ferment pentose sugars. Although D-xylose and L-arabinose fermenting S. cerevisiae strains have been constructed recently, pentose uptake is still a limiting step in mixed sugar fermentations. Results Here we described the cloning and characterization of two sugar transporters, AraT from the yeast Scheffersomyces stipitis and Stp2 from the plant Arabidopsis thaliana, which mediate the uptake of L-arabinose but not of D-glucose into S. cerevisiae cells. A yeast strain lacking all of its endogenous hexose transporter genes and expressing a bacterial L-arabinose utilization pathway could no longer take up and grow with L-arabinose as the only carbon source. Expression of the heterologous transporters supported uptake and utilization of L-arabinose especially at low L-arabinose concentrations but did not, or only very weakly, support D-glucose uptake and utilization. In contrast, the S. cerevisiae D-galactose transporter, Gal2, mediated uptake of both L-arabinose and D-glucose, especially at high concentrations. Conclusions Using a newly developed screening system we have identified two heterologous sugar transporters from a yeast and a plant which can support uptake and utilization of L-arabinose in L-arabinose fermenting S. cerevisiae cells, especially at low L-arabinose concentrations.

  14. Enhancing ethanol yields through d-xylose and l-arabinose co-fermentation after construction of a novel high efficient l-arabinose-fermenting Saccharomyces cerevisiae strain.

    Science.gov (United States)

    Caballero, Antonio; Ramos, Juan Luis

    2017-04-01

    Lignocellulose contains two pentose sugars, l-arabinose and d-xylose, neither of which is naturally fermented by first generation (1G) ethanol-producing Saccharomyces cerevisiae yeast. Since these sugars are inaccessible to 1G yeast, a significant percentage of the total carbon in bioethanol production from plant residues, which are used in second generation (2G) ethanol production, remains unused. Recombinant Saccharomyces cerevisiae strains capable of fermenting d-xylose are available on the market; however, there are few examples of l-arabinose-fermenting yeasts, and commercially, there are no strains capable of fermenting both d-xylose and l-arabinose because of metabolic incompatibilities when both metabolic pathways are expressed in the same cell. To attempt to solve this problem we have tested d-xylose and l-arabinose co-fermentation. To find efficient alternative l-arabinose utilization pathways to the few existing ones, we have used stringent methodology to screen for new genes (metabolic and transporter functions) to facilitate l-arabinose fermentation in recombinant yeast. We demonstrate the feasibility of this approach in a successfully constructed yeast strain capable of using l-arabinose as the sole carbon source and capable of fully transforming it to ethanol, reaching the maximum theoretical fermentation yield (0.43 g g-1). We demonstrate that efficient co-fermentation of d-xylose and l-arabinose is feasible using two different co-cultured strains, and observed no fermentation delays, yield drops or accumulation of undesired byproducts. In this study we have identified a technically efficient strategy to enhance ethanol yields by 10 % in 2G plants in a process based on C5 sugar co-fermentation.

  15. Engineering of Saccharomyces cerevisiae for Efficient Anaerobic Alcoholic Fermentation of L-Arabinose

    NARCIS (Netherlands)

    Wisselink, H.W.; Toirkens, M.J.; Del Rosario Franco Berriel, M.; Winkler, A.A.; Van Dijken, J.P.; Pronk, J.T.; Van Maris, A.J.A.

    2007-01-01

    For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as L-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the

  16. A novel method to prepare L-Arabinose from xylose mother liquor by yeast-mediated biopurification

    Directory of Open Access Journals (Sweden)

    Lin Shuangjun

    2011-06-01

    Full Text Available Abstract Background L-arabinose is an important intermediate for anti-virus drug synthesis and has also been used in food additives for diets-controlling in recent years. Commercial production of L-arabinose is a complex progress consisting of acid hydrolysis of gum arabic, followed by multiple procedures of purification, thus making high production cost. Therefore, there is a biotechnological and commercial interest in the development of new cost-effective and high-performance methods for obtaining high purity grade L-arabinose. Results An alternative, economical method for purifying L-arabinose from xylose mother liquor was developed in this study. After screening 306 yeast strains, a strain of Pichia anomala Y161 was selected as it could effectively metabolize other sugars but not L-arabinose. Fermentation in a medium containing xylose mother liquor permitted enrichment of L-arabinose by a significant depletion of other sugars. Biochemical analysis of this yeast strain confirmed that its poor capacity for utilizing L-arabinose was due to low activities of the enzymes required for the metabolism of this sugar. Response surface methodology was employed for optimization the fermentation conditions in shake flask cultures. The optimum conditions were: 75 h fermentation time, at 32.5°C, in a medium containing 21% (v/v xylose mother liquor. Under these conditions, the highest purity of L-arabinose reached was 86.1% of total sugar, facilitating recovery of white crystalline L-arabinose from the fermentation medium by simple methods. Conclusion Yeast-mediated biopurification provides a dynamic method to prepare high purity of L-arabinose from the feedstock solution xylose mother liqour, with cost-effective and high-performance properties.

  17. Identification of Important Amino Acids in Gal2p for Improving the L-arabinose Transport and Metabolism in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Chengqiang Wang

    2017-07-01

    Full Text Available Efficient and cost-effective bioethanol production from lignocellulosic materials requires co-fermentation of the main hydrolyzed sugars, including glucose, xylose, and L-arabinose. Saccharomyces cerevisiae is a glucose-fermenting yeast that is traditionally used for ethanol production. Fermentation of L-arabinose is also possible after metabolic engineering. Transport into the cell is the first and rate-limiting step for L-arabinose metabolism. The galactose permease, Gal2p, is a non-specific, endogenous monosaccharide transporter that has been shown to transport L-arabinose. However, Gal2p-mediated transport of L-arabinose occurs at a low efficiency. In this study, homologous modeling and L-arabinose docking were used to predict amino acids in Gal2p that are crucial for L-arabinose transport. Nine amino acid residues in Gal2p were identified and were the focus for site-directed mutagenesis. In the Gal2p transport-deficient chassis cells, the capacity for L-arabinose transport of the different Gal2p mutants was compared by testing growth rates using L-arabinose as the sole carbon source. Almost all the tested mutations affected L-arabinose transport capacity. Among them, F85 is a unique site. The F85S, F85G, F85C, and F85T point mutations significantly increased L-arabinose transport activities, while, the F85E and F85R mutations decreased L-arabinose transport activities compared to the Gal2p-expressing wild-type strain. These results verified F85 as a key residue in L-arabinose transport. The F85S mutation, having the most significant effect, elevated the exponential growth rate by 40%. The F85S mutation also improved xylose transport efficiency and weakened the glucose transport preference. Overall, enhancing the L-arabinose transport capacity further improved the L-arabinose metabolism of engineered S. cerevisiae.

  18. Effects of L-arabinose efflux on λ Red recombination-mediated gene knockout in multiple-antimicrobial-resistant Salmonella enterica serovar Choleraesuis.

    Science.gov (United States)

    Liao, Shi-Wei; Lee, Jen-Jie; Ptak, Christopher P; Wu, Ying-Chen; Hsuan, Shih-Ling; Kuo, Chih-Jung; Chen, Ter-Hsin

    2018-03-01

    In this study, six swine-derived multiple-antimicrobial-resistant (MAR) strains of Salmonella Choleraesuis (S. Choleraesuis) were demonstrated to possess higher efflux pump activity than the wild-type (WT). L-Arabinose, a common inducer for gene expression, modulated S. Choleraesuis efflux pump activity in a dose-dependent manner. At low L-arabinose concentrations, increasing L-arabinose led to a corresponding increase in fluorophore efflux, while at higher L-arabinose concentrations, increasing L-arabinose decreased fluorophore efflux activity. The WT S. Choleraesuis that lacks TolC (ΔtolC), an efflux protein associated with bacterial antibiotic resistance and virulence, was demonstrated to possess a significantly reduced ability to extrude L-arabinose. Further, due to the rapid export of L-arabinose, an efficient method for recombination-mediated gene knockout, the L-arabinose-inducible bacteriophage λ Red recombinase system, has a reduced recombination frequency (~ 12.5%) in clinically isolated MAR Salmonella strains. An increased recombination frequency (up to 60%) can be achieved using a higher concentration of L-arabinose (fivefold) for genetic manipulation and functional analysis for MAR Salmonella using the λ Red system. The study suggests that L-arabinose serves not only as an inducer of the TolC-dependent efflux system but also acts as a competitive substrate of the efflux system. In addition, understanding the TolC-dependent efflux of L-arabinose should facilitate the optimization of L-arabinose induction in strains with high efflux activity.

  19. A mixed diet supplemented with l-arabinose does not alter glycaemic or insulinaemic responses in healthy human subjects

    DEFF Research Database (Denmark)

    Halschou-Jensen, Kia; Knudsen, Knud E Bach; Nielsen, Soren

    2015-01-01

    of the present study showed that the peak plasma concentration, time to reach peak plasma concentration or AUC values of glucose, insulin and C-peptide were not altered after consumption of the test meals. Overall, it was not possible to reproduce the beneficial effects of L-arabinose added to sucrose drinks...... effects on postprandial blood glucose, insulin and C-peptide responses in humans. However, the effects of adding L-arabinose to mixed meals on the indices of glucose control are unknown. The purpose of the present study was to investigate whether the positive effects of L-arabinose added to a sugar drink...... could be reproduced in subjects consuming a mixed meal containing sucrose and/or starch from wheat flour. A total of seventeen healthy men participated in study 1, a randomised, double-blind, cross-over trial. In this study, the subjects consumed two different breakfast meals containing sucrose...

  20. Microbial production of xylitol from xylose and L-arabinose: conversion of L-arabitol to xylitol using bacterial oxidoreductases

    Science.gov (United States)

    Microbial production of xylitol, using hemicellulosic biomass such as agricultural residues, is becoming more attractive for reducing its manufacturing cost. L-arabitol is a particular problem to xylitol production from hemicellulosic hydrolyzates that contain both xylose and L-arabinose because it...

  1. ARA1 regulates not only l-arabinose but also d-galactose catabolism in Trichoderma reesei

    NARCIS (Netherlands)

    Benocci, Tiziano; Aguilar-Pontes, Maria Victoria; Kun, Roland Sándor; Seiboth, Bernhard; de Vries, Ronald P; Daly, Paul

    2017-01-01

    Trichoderma reesei is used to produce saccharifying enzyme cocktails for biofuels. There is limited understanding of the transcription factors (TFs) that regulate genes involved in release and catabolism of l-arabinose and d-galactose, as the main TF XYR1 is only partially involved. Here, the T.

  2. E-CAI: a novel server to estimate an expected value of Codon Adaptation Index (eCAI

    Directory of Open Access Journals (Sweden)

    Garcia-Vallvé Santiago

    2008-01-01

    Full Text Available Abstract Background The Codon Adaptation Index (CAI is a measure of the synonymous codon usage bias for a DNA or RNA sequence. It quantifies the similarity between the synonymous codon usage of a gene and the synonymous codon frequency of a reference set. Extreme values in the nucleotide or in the amino acid composition have a large impact on differential preference for synonymous codons. It is thence essential to define the limits for the expected value of CAI on the basis of sequence composition in order to properly interpret the CAI and provide statistical support to CAI analyses. Though several freely available programs calculate the CAI for a given DNA sequence, none of them corrects for compositional biases or provides confidence intervals for CAI values. Results The E-CAI server, available at http://genomes.urv.es/CAIcal/E-CAI, is a web-application that calculates an expected value of CAI for a set of query sequences by generating random sequences with G+C and amino acid content similar to those of the input. An executable file, a tutorial, a Frequently Asked Questions (FAQ section and several examples are also available. To exemplify the use of the E-CAI server, we have analysed the codon adaptation of human mitochondrial genes that codify a subunit of the mitochondrial respiratory chain (excluding those genes that lack a prokaryotic orthologue and are encoded in the nuclear genome. It is assumed that these genes were transferred from the proto-mitochondrial to the nuclear genome and that its codon usage was then ameliorated. Conclusion The E-CAI server provides a direct threshold value for discerning whether the differences in CAI are statistically significant or whether they are merely artifacts that arise from internal biases in the G+C composition and/or amino acid composition of the query sequences.

  3. Metabolome analysis-based design and engineering of a metabolic pathway in Corynebacterium glutamicum to match rates of simultaneous utilization of D-glucose and L-arabinose.

    Science.gov (United States)

    Kawaguchi, Hideo; Yoshihara, Kumiko; Hara, Kiyotaka Y; Hasunuma, Tomohisa; Ogino, Chiaki; Kondo, Akihiko

    2018-05-17

    L-Arabinose is the second most abundant component of hemicellulose in lignocellulosic biomass, next to D-xylose. However, few microorganisms are capable of utilizing pentoses, and catabolic genes and operons enabling bacterial utilization of pentoses are typically subject to carbon catabolite repression by more-preferred carbon sources, such as D-glucose, leading to a preferential utilization of D-glucose over pentoses. In order to simultaneously utilize both D-glucose and L-arabinose at the same rate, a modified metabolic pathway was rationally designed based on metabolome analysis. Corynebacterium glutamicum ATCC 31831 utilized D-glucose and L-arabinose simultaneously at a low concentration (3.6 g/L each) but preferentially utilized D-glucose over L-arabinose at a high concentration (15 g/L each), although L-arabinose and D-glucose were consumed at comparable rates in the absence of the second carbon source. Metabolome analysis revealed that phosphofructokinase and pyruvate kinase were major bottlenecks for D-glucose and L-arabinose metabolism, respectively. Based on the results of metabolome analysis, a metabolic pathway was engineered by overexpressing pyruvate kinase in combination with deletion of araR, which encodes a repressor of L-arabinose uptake and catabolism. The recombinant strain utilized high concentrations of D-glucose and L-arabinose (15 g/L each) at the same consumption rate. During simultaneous utilization of both carbon sources at high concentrations, intracellular levels of phosphoenolpyruvate declined and acetyl-CoA levels increased significantly as compared with the wild-type strain that preferentially utilized D-glucose. These results suggest that overexpression of pyruvate kinase in the araR deletion strain increased the specific consumption rate of L-arabinose and that citrate synthase activity becomes a new bottleneck in the engineered pathway during the simultaneous utilization of D-glucose and L-arabinose. Metabolome analysis

  4. Co-utilization of L-arabinose and D-xylose by laboratory and industrial Saccharomyces cerevisiae strains

    Directory of Open Access Journals (Sweden)

    Boles Eckhard

    2006-04-01

    Full Text Available Abstract Background Fermentation of lignocellulosic biomass is an attractive alternative for the production of bioethanol. Traditionally, the yeast Saccharomyces cerevisiae is used in industrial ethanol fermentations. However, S. cerevisiae is naturally not able to ferment the pentose sugars D-xylose and L-arabinose, which are present in high amounts in lignocellulosic raw materials. Results We describe the engineering of laboratory and industrial S. cerevisiae strains to co-ferment the pentose sugars D-xylose and L-arabinose. Introduction of a fungal xylose and a bacterial arabinose pathway resulted in strains able to grow on both pentose sugars. Introduction of a xylose pathway into an arabinose-fermenting laboratory strain resulted in nearly complete conversion of arabinose into arabitol due to the L-arabinose reductase activity of the xylose reductase. The industrial strain displayed lower arabitol yield and increased ethanol yield from xylose and arabinose. Conclusion Our work demonstrates simultaneous co-utilization of xylose and arabinose in recombinant strains of S. cerevisiae. In addition, the co-utilization of arabinose together with xylose significantly reduced formation of the by-product xylitol, which contributed to improved ethanol production.

  5. Sugar-metal ion interactions: The coordination behavior of cesium ion with lactose, D-arabinose and L-arabinose

    Science.gov (United States)

    Jiang, Ye; Xue, Junhui; Wen, Xiaodong; Zhai, Yanjun; Yang, Limin; Xu, Yizhuang; Zhao, Guozhong; Kou, Kuan; Liu, Kexin; Chen, Jia'er; Wu, Jinguang

    2016-04-01

    The novel cesium chloride-lactose complex (CsCl·C12H22O10 (Cs-Lac), cesium chloride-D-arabinose and L-arabinose complexes (CsCl·C5H10O5, Cs-D-Ara and Cs-L-Ara) have been synthesized and characterized using X-ray diffraction, FTIR, FIR, THz and Raman spectroscopies. Cs+ is 9-coordinated to two chloride ions and seven hydroxyl groups from five lactose molecules in Cs-Lac. In the structures of CsCl-D-arabinose and CsCl-L-arabinose complexes, two kinds of Cs+ ions coexist in the structures. Cs1 is 10-coordinated with two chloride ions and eight hydroxyl groups from five arabinose molecule; Cs2 is 9-coordinated to three chloride ions and six hydroxyl groups from five arabinose molecules. Two coordination modes of arabinose coexist in the structures. α-D-arabinopyranose and α-L-arabinopyranose appear in the structures of Cs-D-Ara and Cs-L-Ara complexes. FTIR and Raman results indicate variations of hydrogen bonds and the conformation of the ligands after complexation. FIR and THz spectra also confirm the formation of Cs-complexes. Crystal structure, FTIR, FIR, THz and Raman spectra provide detailed information on the structure and coordination of hydroxyl groups to metal ions in the cesium chloride-lactose, cesium chloride-D- and L-arabinose complexes.

  6. Coutilization of D-Glucose, D-Xylose, and L-Arabinose in Saccharomyces cerevisiae by Coexpressing the Metabolic Pathways and Evolutionary Engineering

    Directory of Open Access Journals (Sweden)

    Chengqiang Wang

    2017-01-01

    Full Text Available Efficient and cost-effective fuel ethanol production from lignocellulosic materials requires simultaneous cofermentation of all hydrolyzed sugars, mainly including D-glucose, D-xylose, and L-arabinose. Saccharomyces cerevisiae is a traditional D-glucose fermenting strain and could utilize D-xylose and L-arabinose after introducing the initial metabolic pathways. The efficiency and simultaneous coutilization of the two pentoses and D-glucose for ethanol production in S. cerevisiae still need to be optimized. Previously, we constructed an L-arabinose-utilizing S. cerevisiae BSW3AP. In this study, we further introduced the XI and XR-XDH metabolic pathways of D-xylose into BSW3AP to obtain D-glucose, D-xylose, and L-arabinose cofermenting strain. Benefits of evolutionary engineering: the resulting strain BSW4XA3 displayed a simultaneous coutilization of D-xylose and L-arabinose with similar consumption rates, and the D-glucose metabolic capacity was not decreased. After 120 h of fermentation on mixed D-glucose, D-xylose, and L-arabinose, BSW4XA3 consumed 24% more amounts of pentoses and the ethanol yield of mixed sugars was increased by 30% than that of BSW3AP. The resulting strain BSW4XA3 was a useful chassis for further enhancing the coutilization efficiency of mixed sugars for bioethanol production.

  7. Novel transporters from Kluyveromyces marxianus and Pichia guilliermondii expressed in Saccharomyces cerevisiae enable growth on L-arabinose and D-xylose.

    Science.gov (United States)

    Knoshaug, Eric P; Vidgren, Virve; Magalhães, Frederico; Jarvis, Eric E; Franden, Mary Ann; Zhang, Min; Singh, Arjun

    2015-10-01

    Genes encoding L-arabinose transporters in Kluyveromyces marxianus and Pichia guilliermondii were identified by functional complementation of Saccharomyces cerevisiae whose growth on L-arabinose was dependent on a functioning L-arabinose transporter, or by screening a differential display library, respectively. These transporters also transport D-xylose and were designated KmAXT1 (arabinose-xylose transporter) and PgAXT1, respectively. Transport assays using L-arabinose showed that KmAxt1p has K(m) 263 mM and V(max) 57 nM/mg/min, and PgAxt1p has K(m) 0.13 mM and V(max) 18 nM/mg/min. Glucose, galactose and xylose significantly inhibit L-arabinose transport by both transporters. Transport assays using D-xylose showed that KmAxt1p has K(m) 27 mM and V(max) 3.8 nM/mg/min, and PgAxt1p has K(m) 65 mM and V(max) 8.7 nM/mg/min. Neither transporter is capable of recovering growth on glucose or galactose in a S. cerevisiae strain deleted for hexose and galactose transporters. Transport kinetics of S. cerevisiae Gal2p showed K(m) 371 mM and V(max) 341 nM/mg/min for L-arabinose, and K(m) 25 mM and V(max) 76 nM/mg/min for galactose. Due to the ability of Gal2p and these two newly characterized transporters to transport both L-arabinose and D-xylose, one scenario for the complete usage of biomass-derived pentose sugars would require only the low-affinity, high-throughput transporter Gal2p and one additional high-affinity general pentose transporter, rather than dedicated D-xylose or L-arabinose transporters. Additionally, alignment of these transporters with other characterized pentose transporters provides potential targets for substrate recognition engineering. Copyright © 2015 John Wiley & Sons, Ltd.

  8. Bioproduction of D-Tagatose from D-Galactose Using Phosphoglucose Isomerase from Pseudomonas aeruginosa PAO1.

    Science.gov (United States)

    Patel, Manisha J; Patel, Arti T; Akhani, Rekha; Dedania, Samir; Patel, Darshan H

    2016-07-01

    Pseudomonas aeruginosa PAO1 phosphoglucose isomerase was purified as an active soluble form by a single-step purification using Ni-NTA chromatography that showed homogeneity on SDS-PAGE with molecular mass ∼62 kDa. The optimum temperature and pH for the maximum isomerization activity with D-galactose were 60 °C and 7.0, respectively. Generally, sugar phosphate isomerases show metal-independent activity but PA-PGI exhibited metal-dependent isomerization activity with aldosugars and optimally catalyzed the D-galactose isomerization in the presence of 1.0 mM MnCl2. The apparent Km and Vmax for D-galactose under standardized conditions were calculated to be 1029 mM (±31.30 with S.E.) and 5.95 U/mg (±0.9 with S.E.), respectively. Equilibrium reached after 180 min with production of 567.51 μM D-tagatose from 1000 mM of D-galactose. Though, the bioconversion ratio is low but it can be increased by immobilization and enzyme engineering. Although various L-arabinose isomerases have been characterized for bioproduction of D-tagatose, P. aeruginosa glucose phosphate isomerase is distinguished from the other L-arabinose isomerases by its optimal temperature (60 °C) for D-tagatose production being mesophilic bacteria, making it an alternate choice for bulk production.

  9. The Hypocrea jecorina (syn. Trichoderma reesei) lxr1 gene encodes a D-mannitol dehydrogenase and is not involved in L-arabinose catabolism

    NARCIS (Netherlands)

    Metz, Benjamin; de Vries, Ronald P; Polak, Stefan; Seidl, Verena; Seiboth, Bernhard

    2009-01-01

    The Hypocrea jecorina LXR1 was described as the first fungal L-xylulose reductase responsible for NADPH dependent reduction of L-xylulose to xylitol in L-arabinose catabolism. Phylogenetic analysis now reveals that LXR1 forms a clade with fungal D-mannitol 2-dehydrogenases. Lxr1 and the orthologous

  10. Targeted deletion of the ara operon of Salmonella typhimurium enhances L-arabinose accumulation and drives PBAD-promoted expression of anti-cancer toxins and imaging agents.

    Science.gov (United States)

    Hong, Hyun; Lim, Daejin; Kim, Geun-Joong; Park, Seung-Hwan; Sik Kim, Hyeon; Hong, Yeongjin; Choy, Hyon E; Min, Jung-Joon

    2014-01-01

    Tumor-specific expression of antitumor drugs can be achieved using attenuated Salmonella typhimurium harboring the PBAD promoter, which is induced by L-arabinose. However, L-arabinose does not accumulate because it is metabolized to D-xylulose-5-P by enzymes encoded by the ara operon in Salmonellae. To address this problem, we developed an engineered strain of S. typhimurium in which the ara operon is deleted. Linear DNA transformation was performed using λ red recombinase to exchange the ara operon with linear DNA carrying an antibiotic-resistance gene with homology to regions adjacent to the ara operon. The ara operon-deleted strain and its parental strain were transformed with a plasmid encoding Renilla luciferase variant 8 (RLuc8) or cytolysin A (clyA) under the control of the PBAD promoter. Luciferase assays demonstrated that RLuc8 expression was 49-fold higher in the ara operon-deleted S. typhimurium than in the parental strain after the addition of L-arabinose. In vivo bioluminescence imaging showed that the tumor tissue targeted by the ara operon-deleted Salmonella had a stronger imaging signal (~30-fold) than that targeted by the parental strain. Mice with murine colon cancer (CT26) that had been injected with the ara operon-deleted S. typhimurium expressing clyA showed significant tumor suppression. The present report demonstrates that deletion of the ara operon of S. typhimurium enhances L-arabinose accumulation and thereby drives PBAD-promoted expression of cytotoxic agents and imaging agents. This is a promising approach for tumor therapy and imaging.

  11. A combination of l-arabinose and chromium lowers circulating glucose and insulin levels after an acute oral sucrose challenge

    Directory of Open Access Journals (Sweden)

    Perricone Nicholas V

    2011-05-01

    Full Text Available Abstract Background A growing body of research suggests that elevated circulating levels of glucose and insulin accelerate risk factors for a wide range of disorders. Low-risk interventions that could suppress glucose without raising insulin levels could offer significant long-term health benefits. Methods To address this issue, we conducted two sequential studies, the first with two phases. In the first phase of Study 1, baseline fasting blood glucose was measured in 20 subjects who consumed 70 grams of sucrose in water and subsequently completed capillary glucose measurements at 30, 45, 60 and 90 minutes (Control. On day-2 the same procedure was followed, but with subjects simultaneously consuming a novel formula containing l-arabinose and a trivalent patented food source of chromium (LA-Cr (Treatment. The presence or absence of the LA-Cr was blinded to the subjects and testing technician. Comparisons of changes from baseline were made between Control and Treatment periods. In the second phase of Study 1, 10 subjects selected from the original 20 competed baseline measures of body composition (DXA, a 43-blood chemistry panel and a Quality of Life Inventory. These subjects subsequently took LA-Cr daily for 4 weeks completing daily tracking forms and repeating the baseline capillary tests at the end of each of the four weeks. In Study 2, the same procedures used in the first phase were repeated for 50 subjects, but with added circulating insulin measurements at 30 and 60 minutes from baseline. Results In both studies, as compared to Control, the Treatment group had significantly lower glucose responses for all four testing times (AUC = P P = Conclusions As compared to a placebo control, consumption of a LA-Cr formula after a 70-gram sucrose challenge was effective in safely lowering both circulating glucose and insulin levels. Trial Registration Clinical Trials.gov, NCT0110743

  12. Glucose (xylose) isomerase production from thermotolerant and ...

    African Journals Online (AJOL)

    Owner

    2012-11-13

    Nov 13, 2012 ... in the production of the high fructose corn syrup (HFCS) from corn starch. ... Key words: Glucose isomerase, xylose isomerase, enzyme activity, Klebsiella, ... Soil, water, and manure (five samples each) were collected from.

  13. The influence of Aspergillus niger transcription factors AraR and XlnR in the gene expression during growth in D-xylose, L-arabinose and steam-exploded sugarcane bagasse.

    Science.gov (United States)

    de Souza, Wagner Rodrigo; Maitan-Alfenas, Gabriela Piccolo; de Gouvêa, Paula Fagundes; Brown, Neil Andrew; Savoldi, Marcela; Battaglia, Evy; Goldman, Maria Helena S; de Vries, Ronald P; Goldman, Gustavo Henrique

    2013-11-01

    The interest in the conversion of plant biomass to renewable fuels such as bioethanol has led to an increased investigation into the processes regulating biomass saccharification. The filamentous fungus Aspergillus niger is an important microorganism capable of producing a wide variety of plant biomass degrading enzymes. In A. niger the transcriptional activator XlnR and its close homolog, AraR, controls the main (hemi-)cellulolytic system responsible for plant polysaccharide degradation. Sugarcane is used worldwide as a feedstock for sugar and ethanol production, while the lignocellulosic residual bagasse can be used in different industrial applications, including ethanol production. The use of pentose sugars from hemicelluloses represents an opportunity to further increase production efficiencies. In the present study, we describe a global gene expression analysis of A. niger XlnR- and AraR-deficient mutant strains, grown on a D-xylose/L-arabinose monosaccharide mixture and steam-exploded sugarcane bagasse. Different gene sets of CAZy enzymes and sugar transporters were shown to be individually or dually regulated by XlnR and AraR, with XlnR appearing to be the major regulator on complex polysaccharides. Our study contributes to understanding of the complex regulatory mechanisms responsible for plant polysaccharide-degrading gene expression, and opens new possibilities for the engineering of fungi able to produce more efficient enzymatic cocktails to be used in biofuel production. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Neurological findings in triosephosphate isomerase deficiency

    NARCIS (Netherlands)

    Poll-The, B. T.; Aicardi, J.; Girot, R.; Rosa, R.

    1985-01-01

    Two siblings with hemolytic anemia caused by triosephosphate isomerase deficiency developed a progressive neurological syndrome featuring dystonic movements, tremor, pyramidal tract signs, and evidence of spinal motor neuron involvement. Intelligence was unaffected. The findings in these patients

  15. Thermoinactivation Mechanism of Glucose Isomerase

    Science.gov (United States)

    Lim, Leng Hong; Saville, Bradley A.

    In this article, the mechanisms of thermoinactivation of glucose isomerase (GI) from Streptomyces rubiginosus (in soluble and immobilized forms) were investigated, particularly the contributions of thiol oxidation of the enzyme's cysteine residue and a "Maillard-like" reaction between the enzyme and sugars in high fructose corn syrup (HFCS). Soluble GI (SGI) was successfully immobilized on silica gel (13.5 μm particle size), with an activity yield between 20 and 40%. The immobilized GI (IGI) has high enzyme retention on the support during the glucose isomerization process. In batch reactors, SGI (half-life =145 h) was more stable than IGI (half-life=27 h) at 60°C in HFCS, whereas at 80°C, IGI (half-life=12 h) was more stable than SGI (half-life=5.2 h). IGI was subject to thiol oxidation at 60°C, which contributed to the enzyme's deactivation. IGI was subject to thiol oxidation at 80°C, but this did not contribute to the deactivation of the enzyme. SGI did not undergo thiol oxidation at 60°C, but at 80°C SGI underwent severe precipitation and thiol oxidation, which caused the enzyme to deactivate. Experimental results show that immobilization suppresses the destablizing effect of thiol oxidation on GI. A "Maillard-like" reaction between SGI and the sugars also caused SGI thermoinactivation at 60, 70, and 80°C, but had minimal effect on IGI. At 60 and 80°C, IGI had higher thermostability in continuous reactors than in batch reactors, possibily because of reduced contact with deleterious compounds in HFCS.

  16. Functional differences in yeast protein disulfide isomerases

    DEFF Research Database (Denmark)

    Nørgaard, P; Westphal, V; Tachibana, C

    2001-01-01

    PDI1 is the essential gene encoding protein disulfide isomerase in yeast. The Saccharomyces cerevisiae genome, however, contains four other nonessential genes with homology to PDI1: MPD1, MPD2, EUG1, and EPS1. We have investigated the effects of simultaneous deletions of these genes. In several...

  17. 21 CFR 862.1570 - Phosphohexose isomerase test system.

    Science.gov (United States)

    2010-04-01

    .... Measurements of phosphohexose isomerase are used in the diagnosis and treatment of muscle diseases such as muscular dystrophy, liver diseases such as hepatitis or cirrhosis, and metastatic carcinoma. (b...

  18. Compact conformations of human protein disulfide isomerase.

    Directory of Open Access Journals (Sweden)

    Shang Yang

    Full Text Available Protein disulfide isomerase (PDI composed of four thioredoxin-like domains a, b, b', and a', is a key enzyme catalyzing oxidative protein folding in the endoplasmic reticulum. Large scale molecular dynamics simulations starting from the crystal structures of human PDI (hPDI in the oxidized and reduced states were performed. The results indicate that hPDI adopts more compact conformations in solution than in the crystal structures, which are stabilized primarily by inter-domain interactions, including the salt bridges between domains a and b' observed for the first time. A prominent feature of the compact conformations is that the two catalytic domains a and a' can locate close enough for intra-molecular electron transfer, which was confirmed by the characterization of an intermediate with a disulfide between the two domains. Mutations, which disrupt the inter-domain interactions, lead to decreased reductase activity of hPDI. Our molecular dynamics simulations and biochemical experiments reveal the intrinsic conformational dynamics of hPDI and its biological impact.

  19. The human protein disulfide isomerase gene family

    Directory of Open Access Journals (Sweden)

    Galligan James J

    2012-07-01

    Full Text Available Abstract Enzyme-mediated disulfide bond formation is a highly conserved process affecting over one-third of all eukaryotic proteins. The enzymes primarily responsible for facilitating thiol-disulfide exchange are members of an expanding family of proteins known as protein disulfide isomerases (PDIs. These proteins are part of a larger superfamily of proteins known as the thioredoxin protein family (TRX. As members of the PDI family of proteins, all proteins contain a TRX-like structural domain and are predominantly expressed in the endoplasmic reticulum. Subcellular localization and the presence of a TRX domain, however, comprise the short list of distinguishing features required for gene family classification. To date, the PDI gene family contains 21 members, varying in domain composition, molecular weight, tissue expression, and cellular processing. Given their vital role in protein-folding, loss of PDI activity has been associated with the pathogenesis of numerous disease states, most commonly related to the unfolded protein response (UPR. Over the past decade, UPR has become a very attractive therapeutic target for multiple pathologies including Alzheimer disease, Parkinson disease, alcoholic and non-alcoholic liver disease, and type-2 diabetes. Understanding the mechanisms of protein-folding, specifically thiol-disulfide exchange, may lead to development of a novel class of therapeutics that would help alleviate a wide range of diseases by targeting the UPR.

  20. Purification, crystallization and preliminary crytallographic analysis of phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus

    NARCIS (Netherlands)

    Akerboom, A.P.; Turnbull, A.P.; Hargreaves, D.; Fischer, M.; Geus, de D.; Sedelnikova, S.E.; Berrisford, J.M.; Baker, P.J.; Verhees, C.H.; Oost, van der J.; Rice, D.W.

    2003-01-01

    The glycolytic enzyme phosphoglucose isomerase catalyses the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate. The phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus, which shows no sequence similarity to any known bacterial or eukaryotic

  1. Cloning and characterization of peptidylprolyl isomerase B in the ...

    African Journals Online (AJOL)

    Peptidylprolyl isomerases (PPIases) play essential roles in protein folding and are implicated in immune response and cell cycle control. Our previous proteomic analysis indicated that Bombyx mori PPIases may be involved in anti- Bombyx mori nucleopolyhedrovirus (BmNPV) response. To help investigate this mechanism, ...

  2. Characteristics of chalcone isomerase promoter in crabapple leaves ...

    African Journals Online (AJOL)

    Anthocyanins are secondary metabolites found in higher plants that contribute to the colors of plants and chalcone isomerase (CHI) is one of the key enzymes in anthocyanin biosynthetic pathway. What characteristic is CHI promoter known as the regulation sequence of CHI gene, has been rarely investigated. We isolated A ...

  3. [Deficiency of triosephosphate isomerase. Apropos of 2 new cases].

    Science.gov (United States)

    Delso Martínez, M C; Uriel Miñana, P; Pérez Lugmus, G; Giménez Mas, J A; Baldellou Vázquez, A

    1983-08-01

    Two siblings, born of a no consanguineous couple, a female and a male, affected by a severe and progressive neurological disease and chronic hemolytic anemia are presented. Their clinical, hematological, biochemical and pathological studies are discussed. One of the patients showed a triosephosphate isomerase deficiency and the carrier condition of their parents was tested. Commentaries about physiopathology of this disease are made.

  4. Purification and characterization of the d-xylose isomerase gene from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Ho, N W.Y.; Rosenfeld, S; Stevis, P; Tsao, G T

    1983-11-01

    A DNA fragment containing both the Escherichia coli D-xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) gene and the D-xylulokinase (ATP: D-xylulose 5-phosphotransferase, EC 2.7.1.17) gene has been cloned on an E. coli plasmid. The D-xylose isomerase gene was separated from the D-xylulokinase gene by the construction of a new deletion plasmid, pLX7. The D-xylose isomerase gene cloned on pLX7 was found still to be an intact gene. The precise location of the D-xylose isomerase gene on the plasmid pLX7 was further determined by the construction of two more plasmids, pLX8 and pLX9. This is believed to be the first D-xylose isomerase gene that has been isolated and extensively purified from any organism. D-Xylose isomerase, the enzyme product of the D-xylose isomerase gene, is responsible for the conversion of D-xylose to D-xylulose, as well as D-glucose to D-fructose. It is widely believed that yeast cannot ferment D-xylose to ethanol primarily because of the lack of D-xylose isomerase in yeast. D-Xylose isomerase (also known as D-glucose isomerase) is also used for the commercial production of high-fructose syrups. The purification of the D-xylose isomerase gene may lead to the following industrial applications: (1) cloning and expression of the gene in yeast to make the latter organism capable of directly fermenting D-xylose to ethanol, and (2) cloning of the gene on a high-copy-number plasmid in a proper host to overproduce the enzyme, which should have a profound impact on the high-fructose syrup technology. 14 references.

  5. Methods of measuring Protein Disulfide Isomerase activity: a critical overview

    Science.gov (United States)

    Watanabe, Monica; Laurindo, Francisco; Fernandes, Denise

    2014-09-01

    Protein disulfide isomerase is an essential redox chaperone from the endoplasmic reticulum (ER) and is responsible for correct disulfide bond formation in nascent proteins. PDI is also found in other cellular locations in the cell, particularly the cell surface. Overall, PDI contributes to ER and global cell redox homeostasis and signaling. The knowledge about PDI structure and function progressed substantially based on in vitro studies using recombinant PDI and chimeric proteins. In these experimental scenarios, PDI reductase and chaperone activities are readily approachable. In contrast, assays to measure PDI isomerase activity, the hallmark of PDI family, are more complex. Assessment of PDI roles in cells and tissues mainly relies on gain- or loss-of-function studies. However, there is limited information regarding correlation of experimental readouts with the distinct types of PDI activities. In this mini-review, we evaluate the main methods described for measuring the different kinds of PDI activity: thiol reductase, thiol oxidase, thiol isomerase and chaperone. We emphasize the need to use appropriate controls and the role of critical interferents (e.g., detergent, presence of reducing agents). We also discuss the translation of results from in vitro studies with purified recombinant PDI to cellular and tissue samples, with critical comments on the interpretation of results.

  6. Nucleotide sequence of the triosephosphate isomerase gene from Macaca mulatta

    Energy Technology Data Exchange (ETDEWEB)

    Old, S.E.; Mohrenweiser, H.W. (Univ. of Michigan, Ann Arbor (USA))

    1988-09-26

    The triosephosphate isomerase gene from a rhesus monkey, Macaca mulatta, charon 34 library was sequenced. The human and chimpanzee enzymes differ from the rhesus enzyme at ASN 20 and GLU 198. The nucleotide sequence identity between rhesus and human is 97% in the coding region and >94% in the flanking regions. Comparison of the rhesus and chimp genes, including the intron and flanking sequences, does not suggest a mechanism for generating the two TPI peptides of proliferating cells from hominoids and a single peptide from the rhesus gene.

  7. Arabidopsis Phosphomannose Isomerase 1, but Not Phosphomannose Isomerase 2, Is Essential for Ascorbic Acid Biosynthesis*S⃞

    OpenAIRE

    Maruta, Takanori; Yonemitsu, Miki; Yabuta, Yukinori; Tamoi, Masahiro; Ishikawa, Takahiro; Shigeoka, Shigeru

    2008-01-01

    We studied molecular and functional properties of Arabidopsis phosphomannose isomerase isoenzymes (PMI1 and PMI2) that catalyze reversible isomerization between d-fructose 6-phosphate and d-mannose 6-phosphate (Man-6P). The apparent Km and Vmax values for Man-6P of purified recombinant PMI1 were 41.3 ± 4.2 μm and 1.89 μmol/min/mg protein, respectively, whereas those of purified recombinant PMI2 were 372 ± 13 μm and 22.5 μmol/min/mg protein, respectively. Both PMI1 ...

  8. Studies on the production of glucose isomerase by Bacillus licheniformis

    Directory of Open Access Journals (Sweden)

    Nwokoro Ogbonnaya

    2015-09-01

    Full Text Available This work reports the effects of some culture conditions on the production of glucose isomerase by Bacillus licheniformis. The bacterium was selected based on the release of 3.62 mg/mL fructose from the fermentation of glucose. Enzyme was produced using a variety of carbon substrates but the highest enzyme activity was detected in a medium containing 0.5% xylose and 1% glycerol (specific activity = 6.88 U/mg protein. Media containing only xylose or glucose gave lower enzyme productivies (specific activities= 4.60 and 2.35 U/mg protein respectively. The effects of nitrogen substrates on glucose isomerase production showed that yeast extract supported maximum enzyme activity (specific activity = 5.24 U/mg protein. Lowest enzyme activity was observed with sodium trioxonitrate (specific activity = 2.44 U/mg protein. In general, organic nitrogen substrates supported higher enzyme productivity than inorganic nitrogen substrates. Best enzyme activity was observed in the presence of Mg2+ (specific activity = 6.85 U/mg protein while Hg2+ was inhibitory (specific activity = 1.02 U/mg protein. The optimum pH for best enzyme activity was 6.0 while optimum temperature for enzyme production was 50ºC.

  9. Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases

    DEFF Research Database (Denmark)

    Spetzler, J C; Westphal, V; Winther, Jakob R.

    1998-01-01

    Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish...... the quenching chromophore (Tyr(NO2)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the interchain disulphide bonds in the library were monitored under a fluorescence microscope. Substrates to investigate the properties of protein disulphide isomerase in solution were...

  10. Overexpression, purification, crystallization and preliminary X-ray crystal analysis of Bacillus pallidusd-arabinose isomerase

    International Nuclear Information System (INIS)

    Takeda, Kosei; Yoshida, Hiromi; Takada, Goro; Izumori, Ken; Kamitori, Shigehiro

    2008-01-01

    Recombinant B. pallidusd-arabinose isomerase was crystallized and diffraction data were collected to 2.3 Å resolution. d-Arabinose isomerase catalyzes the isomerization of d-arabinose to d-ribulose. Bacillus pallidusd-arabinose isomerase has broad substrate specificity and can catalyze the isomerization of d-arabinose, l-fucose, l-xylose, l-galactose and d-altrose. Recombinant B. pallidusd-arabinose isomerase was overexpressed, purified and crystallized. A crystal of the enzyme was obtained by the sitting-drop method at room temperature and belonged to the orthorhombic space group P2 1 2 1 2, with unit-cell parameters a = 144.9, b = 127.9, c = 109.5 Å. Diffraction data were collected to 2.3 Å resolution

  11. Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118

    International Nuclear Information System (INIS)

    Lobley, Carina M. C.; Aller, Pierre; Douangamath, Alice; Reddivari, Yamini; Bumann, Mario; Bird, Louise E.; Nettleship, Joanne E.; Brandao-Neto, Jose; Owens, Raymond J.; O’Toole, Paul W.; Walsh, Martin A.

    2012-01-01

    The crystal structure of ribose 5-phosphate isomerase has been determined to 1.72 Å resolution and is presented with a brief comparison to other known ribose 5-phosphate isomerase A structures. The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Å resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and β d-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography

  12. Triosephosphate isomerase is a common crystallization contaminant of soluble His-tagged proteins produced in Escherichia coli

    International Nuclear Information System (INIS)

    Kozlov, Guennadi; Vinaik, Roohi; Gehring, Kalle

    2013-01-01

    Crystals of E. coli triosephosphate isomerase were obtained as a contaminant and its structure was determined to 1.85 Å resolution. Attempts to crystallize several mammalian proteins overexpressed in Escherichia coli revealed a common contaminant, triosephosphate isomerase, a protein involved in glucose metabolism. Even with triosephosphate isomerase present in very small amounts, similarly shaped crystals appeared in the crystallization drops in a number of polyethylene glycol-containing conditions. All of the target proteins were His-tagged and their purification involved immobilized metal-affinity chromatography (IMAC), a step that was likely to lead to triosephosphate isomerase contamination. Analysis of the triosephosphate isomerase crystals led to the structure of E. coli triosephosphate isomerase at 1.85 Å resolution, which is a significant improvement over the previous structure

  13. Glucose isomerization in simulated moving bed reactor by Glucose isomerase

    Directory of Open Access Journals (Sweden)

    Eduardo Alberto Borges da Silva

    2006-05-01

    Full Text Available Studies were carried out on the production of high-fructose syrup by Simulated Moving Bed (SMB technology. A mathematical model and numerical methodology were used to predict the behavior and performance of the simulated moving bed reactors and to verify some important aspects for application of this technology in the isomerization process. The developed algorithm used the strategy that considered equivalences between simulated moving bed reactors and true moving bed reactors. The kinetic parameters of the enzymatic reaction were obtained experimentally using discontinuous reactors by the Lineweaver-Burk technique. Mass transfer effects in the reaction conversion using the immobilized enzyme glucose isomerase were investigated. In the SMB reactive system, the operational variable flow rate of feed stream was evaluated to determine its influence on system performance. Results showed that there were some flow rate values at which greater purities could be obtained.Neste trabalho a tecnologia de Leito Móvel Simulado (LMS reativo é aplicada no processo de isomerização da glicose visando à produção de xarope concentrado de frutose. É apresentada a modelagem matemática e uma metodologia numérica para predizer o comportamento e o desempenho de unidades reativas de leito móvel simulado para verificar alguns aspectos importantes para o emprego desta tecnologia no processo de isomerização. O algoritmo desenvolvido utiliza a abordagem que considera as equivalências entre as unidades reativas de leito móvel simulado e leito móvel verdadeiro. Parâmetros cinéticos da reação enzimática são obtidos experimentalmente usando reatores em batelada pela técnica Lineweaver-Burk. Efeitos da transferência de massa na conversão de reação usando a enzima imobilizada glicose isomerase são verificados. No sistema reativo de LMS, a variável operacional vazão da corrente de alimentação é avaliada para conhecer o efeito de sua influência no

  14. Roles of Prolyl Isomerases in RNA-Mediated Gene Expression

    Directory of Open Access Journals (Sweden)

    Roopa Thapar

    2015-05-01

    Full Text Available The peptidyl-prolyl cis-trans isomerases (PPIases that include immunophilins (cyclophilins and FKBPs and parvulins (Pin1, Par14, Par17 participate in cell signaling, transcription, pre-mRNA processing and mRNA decay. The human genome encodes 19 cyclophilins, 18 FKBPs and three parvulins. Immunophilins are receptors for the immunosuppressive drugs cyclosporin A, FK506, and rapamycin that are used in organ transplantation. Pin1 has also been targeted in the treatment of Alzheimer’s disease, asthma, and a number of cancers. While these PPIases are characterized as molecular chaperones, they also act in a nonchaperone manner to promote protein-protein interactions using surfaces outside their active sites. The immunosuppressive drugs act by a gain-of-function mechanism by promoting protein-protein interactions in vivo. Several immunophilins have been identified as components of the spliceosome and are essential for alternative splicing. Pin1 plays roles in transcription and RNA processing by catalyzing conformational changes in the RNA Pol II C-terminal domain. Pin1 also binds several RNA binding proteins such as AUF1, KSRP, HuR, and SLBP that regulate mRNA decay by remodeling mRNP complexes. The functions of ribonucleoprotein associated PPIases are largely unknown. This review highlights PPIases that play roles in RNA-mediated gene expression, providing insight into their structures, functions and mechanisms of action in mRNP remodeling in vivo.

  15. Protein Disulfide Isomerase and Host-Pathogen Interaction

    Directory of Open Access Journals (Sweden)

    Beatriz S. Stolf

    2011-01-01

    Full Text Available Reactive oxygen species (ROS production by immunological cells is known to cause damage to pathogens. Increasing evidence accumulated in the last decade has shown, however, that ROS (and redox signals functionally regulate different cellular pathways in the host-pathogen interaction. These especially affect (i pathogen entry through protein redox switches and redox modification (i.e., intra- and interdisulfide and cysteine oxidation and (ii phagocytic ROS production via Nox family NADPH oxidase enzyme and the control of phagolysosome function with key implications for antigen processing. The protein disulfide isomerase (PDI family of redox chaperones is closely involved in both processes and is also implicated in protein unfolding and trafficking across the endoplasmic reticulum (ER and towards the cytosol, a thiol-based redox locus for antigen processing. Here, we summarise examples of the cellular association of host PDI with different pathogens and explore the possible roles of pathogen PDIs in infection. A better understanding of these complex regulatory steps will provide insightful information on the redox role and coevolutional biological process, and assist the development of more specific therapeutic strategies in pathogen-mediated infections.

  16. Immobilization of Recombinant Glucose Isomerase for Efficient Production of High Fructose Corn Syrup.

    Science.gov (United States)

    Jin, Li-Qun; Xu, Qi; Liu, Zhi-Qiang; Jia, Dong-Xu; Liao, Cheng-Jun; Chen, De-Shui; Zheng, Yu-Guo

    2017-09-01

    Glucose isomerase is the important enzyme for the production of high fructose corn syrup (HFCS). One-step production of HFCS containing more than 55% fructose (HFCS-55) is receiving much attention for its industrial applications. In this work, the Escherichia coli harboring glucose isomerase mutant TEGI-W139F/V186T was immobilized for efficient production of HFCS-55. The immobilization conditions were optimized, and the maximum enzyme activity recovery of 92% was obtained. The immobilized glucose isomerase showed higher pH, temperature, and operational stabilities with a K m value of 272 mM and maximum reaction rate of 23.8 mM min -1 . The fructose concentration still retained above 55% after the immobilized glucose isomerase was reused for 10 cycles, and more than 85% of its initial activity was reserved even after 15 recycles of usage at temperature of 90 °C. The results highlighted the immobilized glucose isomerase as a potential biocatalyst for HFCS-55 production.

  17. Molecular identification, immunolocalization, and characterization of Clonorchis sinensis triosephosphate isomerase.

    Science.gov (United States)

    Zhou, Juanjuan; Liao, Hua; Li, Shan; Zhou, Chenhui; Huang, Yan; Li, Xuerong; Liang, Chi; Yu, Xinbing

    2015-08-01

    Clonorchis sinensis triosephosphate isomerase (CsTIM) is a key regulatory enzyme of glycolysis and gluconeogenesis, which catalyzes the interconversion of glyceraldehyde 3-phosphate to dihydroxyacetone phosphate. In this study, the biochemical characterizations of CsTIM have been examined. A full-length complementary DNA (cDNA; Cs105350) sequence encoding CsTIM was obtained from our C. sinensis cDNA library. The open reading frame of CsTIM contains 759 bp which encodes 252 amino acids. The amino acid sequence of CsTIM shares 60-65% identity with other species. Western blot analysis displayed that recombinant CsTIM (rCsTIM) can be probed by anti-rCsTIM rat serum and anti-C. sinensis excretory/secretory products (anti-CsESPs) rat serum. Quantitative reverse transcription (RT)-PCR and western blotting analysis revealed that CsTIM messenger RNA (mRNA) and protein were differentially expressed in development cycle stages of the parasite, including adult worm, metacercaria, excysted metacercaria, and egg. In addition, immunolocalization assay showed that CsTIM was located in the seminal vesicle, eggs, and testicle. Moreover, rCsTIM exhibited active enzyme activity in catalytic reactions. The Michaelis constant (K m) of rCsTIM was 0.33 mM, when using glyceraldehyde 3-phosphate as the substrate. The optimal temperature and pH of CsTIM were 37 °C and 7.5-9.5, respectively. Collectively, these results suggest that CsTIM is an important protein involved in glycometabolism, and CsTIM possibly take part in many biological functions in the growth and development of C. sinensis.

  18. Crystallization and preliminary X-ray characterization of phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv

    International Nuclear Information System (INIS)

    Mathur, Divya; Anand, Kanchan; Mathur, Deepika; Jagadish, Nirmala; Suri, Anil; Garg, Lalit C.

    2007-01-01

    The phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv was crystallized and diffraction data were collected to 2.8 Å resolution. Phosphoglucose isomerase is a ubiquitous enzyme that catalyzes the isomerization of d-glucopyranose-6-phosphate to d-fructofuranose-6-phosphate. The present investigation reports the expression, purification, crystallization and preliminary crystallographic studies of the phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv, which shares 46% sequence identity with that of its human host. The recombinant protein, which was prepared using an Escherichia coli expression system, was crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.8 Å and belonged to the orthorhombic space group I2 1 2 1 2 1 , with unit-cell parameters a = 109.0, b = 119.8, c = 138.9 Å

  19. Directed evolution of xylose isomerase for improved xylose catabolism and fermentation in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S

    2012-08-01

    The heterologous expression of a highly functional xylose isomerase pathway in Saccharomyces cerevisiae would have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways in S. cerevisiae suffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of the Piromyces sp. xylose isomerase (encoded by xylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing a gre3 knockout and tal1 and XKS1 overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.

  20. Escherichia coli rpiA gene encoding ribose phosphate isomerase A

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Maigaard, Marianne

    1993-01-01

    The rpiA gene encoding ribose phosphate isomerase A was cloned from phage 1A2(471) of the Kohara gene library. Subcloning, restriction, and complementation analyses revealed an 1,800-bp SspI-generated DNA fragment that contained the entire control and coding sequences. This DNA fragment was seque......The rpiA gene encoding ribose phosphate isomerase A was cloned from phage 1A2(471) of the Kohara gene library. Subcloning, restriction, and complementation analyses revealed an 1,800-bp SspI-generated DNA fragment that contained the entire control and coding sequences. This DNA fragment...

  1. Human triose-phosphate isomerase deficiency: a single amino acid substitution results in a thermolabile enzyme.

    OpenAIRE

    Daar, I O; Artymiuk, P J; Phillips, D C; Maquat, L E

    1986-01-01

    Triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) deficiency is a recessive disorder that results in hemolytic anemia and neuromuscular dysfunction. To determine the molecular basis of this disorder, a TPI allele from two unrelated patients homozygous for TPI deficiency was compared with an allele from a normal individual. Each disease-associated sequence harbors a G X C----C X G transversion in the codon for amino acid-104 and specifies a structurally...

  2. Obtaining mutants of Streptomyces griseoflavus strain 1339, producers of glucose isomerase, following gamma irradiation

    International Nuclear Information System (INIS)

    Dzhedzheva, G.; Stoeva, N.; Stojchev, M.

    1990-01-01

    A water suspension of Streptomyces griseoflavus strain 1339 spores of a density of 8.7.10 6 spores/cm 3 is gamma irradiated ( 60 Co, RHM-γ-20, 30.3 Gy/min). The survival of Streptomyces griseoflavus strain 1339 spores was determined depending on radiation doses, exposure times and incubation temperature. Five major morphological types of colonies were isolated, characterized by different levels of glucose isomerase activity. Maximum specific glucose isomerase activity (GIU/g) was attained after the third gamma irradiation step using a dose of 3000 Gy. 2 tabs., 3 figs., 7 refs

  3. Xylose isomerase improves growth and ethanol production rates from biomass sugars for both Saccharomyces pastorianus and Saccharomyces cerevisiae.

    Science.gov (United States)

    Miller, Kristen P; Gowtham, Yogender Kumar; Henson, J Michael; Harcum, Sarah W

    2012-01-01

    The demand for biofuel ethanol made from clean, renewable nonfood sources is growing. Cellulosic biomass, such as switch grass (Panicum virgatum L.), is an alternative feedstock for ethanol production; however, cellulosic feedstock hydrolysates contain high levels of xylose, which needs to be converted to ethanol to meet economic feasibility. In this study, the effects of xylose isomerase on cell growth and ethanol production from biomass sugars representative of switch grass were investigated using low cell density cultures. The lager yeast species Saccharomyces pastorianus was grown with immobilized xylose isomerase in the fermentation step to determine the impact of the glucose and xylose concentrations on the ethanol production rates. Ethanol production rates were improved due to xylose isomerase; however, the positive effect was not due solely to the conversion of xylose to xylulose. Xylose isomerase also has glucose isomerase activity, so to better understand the impact of the xylose isomerase on S. pastorianus, growth and ethanol production were examined in cultures provided fructose as the sole carbon. It was observed that growth and ethanol production rates were higher for the fructose cultures with xylose isomerase even in the absence of xylose. To determine whether the positive effects of xylose isomerase extended to other yeast species, a side-by-side comparison of S. pastorianus and Saccharomyces cerevisiae was conducted. These comparisons demonstrated that the xylose isomerase increased ethanol productivity for both the yeast species by increasing the glucose consumption rate. These results suggest that xylose isomerase can contribute to improved ethanol productivity, even without significant xylose conversion. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

  4. Microbial Production of Xylitol from L-arabinose by Metabolically Engineered Escherichia coli

    Science.gov (United States)

    Xylitol is used commercially as a natural sweetener in some food products such as chewing gum, soft drinks, and confectionery. It is currently produced by chemical reduction of D-xylose derived from plant materials, mainly hemicellulosic hydrolysates from birch trees. Expanding the substrate range...

  5. Effect of pH on simultaneous saccharification and isomerization by glucoamylase and glucose isomerase.

    Science.gov (United States)

    Mishra, Abha; Debnath Das, Meera

    2002-01-01

    pH and temperature play critical roles in multistep enzymatic conversions. In such conversions, the optimal pH for individual steps differs greatly. In this article, we describe the production of glucoamylase (from Aspergillus oryzae MTCC152 in solid-state fermentation) and glucose isomerase (from Streptomyces griseus NCIM2020 in submerged fermentation), used in industries for producing high-fructose syrup. Optimum pH for glucoamylase was found to be 5.0. For glucose isomerase, the optimum pH ranged between 7.0 and 8.5, depending on the type of buffer used. Optimum temperature for glucoamylase and glucose isomerase was 50 and 60 degrees C, respectively. When both the enzymatic conversions were performed simultaneously at a compromised pH of 6.5, both the enzymes showed lowered activity. We also studied the kinetics at different pHs, which allows the two-step reaction to take place simultaneously. This was done by separating two steps by a thin layer of urease. Ammonia generated by the hydrolysis of urea consumed the hydrogen ions, thereby allowing optimal activity of glucose isomerase at an acidic pH of 5.0.

  6. Crystal structure of Pyrococcus furiosus phosphoglucose isomerase: Implications for substrate binding and catalysis

    NARCIS (Netherlands)

    Berrisford, J.M.; Akerboom, A.P.; Turnbull, A.P.; Geus, de D.; Sedelnikova, S.E.; Staton, I.; McLeod, C.W.; Verhees, C.H.; Oost, van der J.; Rice, D.W.; Baker, P.J.

    2003-01-01

    Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization between D-fructose 6-phosphate and D-glucose 6-phosphate as part of the glycolytic pathway. PGI from the Archaea Pyrococcus furiosus (Pfu) was crystallized, and its structure was determined by x-ray diffraction to a 2-Angstrom

  7. Inhibiting prolyl isomerase activity by hybrid organic-inorganic molecules containing rhodium(II) fragments.

    Science.gov (United States)

    Coughlin, Jane M; Kundu, Rituparna; Cooper, Julian C; Ball, Zachary T

    2014-11-15

    A small molecule containing a rhodium(II) tetracarboxylate fragment is shown to be a potent inhibitor of the prolyl isomerase FKBP12. The use of small molecules conjugates of rhodium(II) is presented as a general strategy for developing new protein inhibitors based on distinct structural and sequence features of the enzyme active site. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. A library of fluorescent peptides for exploring the substrate specificities of prolyl isomerases

    NARCIS (Netherlands)

    Zoldak, G.; Aumuller, T.; Lucke, C.; Hritz, J.; Oostenbrink, C.; Fischer, G.; Schmid, F.X.

    2009-01-01

    To fully explore the substrate specificities of prolyl isomerases, we synthesized a library of 20 tetrapeptides that are labeled with a 2-aminobenzoyl (Abz) group at the amino terminus and a p-nitroanilide (pNA) group at the carboxy terminus. In this peptide library of the general formula

  9. Structural and Biochemical Characterization of the Human Cyclophilin Family of Peptidyl-Prolyl Isomerases

    Energy Technology Data Exchange (ETDEWEB)

    Davis, Tara L.; Walker, John R.; Campagna-Slater, Valérie; Finerty, Jr., Patrick J.; Paramanathan, Ragika; Bernstein, Galina; MacKenzie, Farrell; Tempel, Wolfram; Ouyang, Hui; Lee, Wen Hwa; Eisenmesser, Elan Z.; Dhe-Paganon, Sirano (Toronto); (Colorado)

    2011-12-14

    Peptidyl-prolyl isomerases catalyze the conversion between cis and trans isomers of proline. The cyclophilin family of peptidyl-prolyl isomerases is well known for being the target of the immunosuppressive drug cyclosporin, used to combat organ transplant rejection. There is great interest in both the substrate specificity of these enzymes and the design of isoform-selective ligands for them. However, the dearth of available data for individual family members inhibits attempts to design drug specificity; additionally, in order to define physiological functions for the cyclophilins, definitive isoform characterization is required. In the current study, enzymatic activity was assayed for 15 of the 17 human cyclophilin isomerase domains, and binding to the cyclosporin scaffold was tested. In order to rationalize the observed isoform diversity, the high-resolution crystallographic structures of seven cyclophilin domains were determined. These models, combined with seven previously solved cyclophilin isoforms, provide the basis for a family-wide structure:function analysis. Detailed structural analysis of the human cyclophilin isomerase explains why cyclophilin activity against short peptides is correlated with an ability to ligate cyclosporin and why certain isoforms are not competent for either activity. In addition, we find that regions of the isomerase domain outside the proline-binding surface impart isoform specificity for both in vivo substrates and drug design. We hypothesize that there is a well-defined molecular surface corresponding to the substrate-binding S2 position that is a site of diversity in the cyclophilin family. Computational simulations of substrate binding in this region support our observations. Our data indicate that unique isoform determinants exist that may be exploited for development of selective ligands and suggest that the currently available small-molecule and peptide-based ligands for this class of enzyme are insufficient for isoform

  10. Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

    Directory of Open Access Journals (Sweden)

    Hahn-Hägerdal Bärbel

    2008-10-01

    Full Text Available Abstract Background Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose. Results The specific aerobic arabinose growth rate was identical, 0.03 h-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells-1 h-1 than the xylose isomerase strain, which only reached 0.03 h-1 and 0.02 g (g cells-1h-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g-1 compared with 0.18 g g-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g-1 compared with 0.32 g g-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells-1 h-1 compared with 0.01 g (g cells-1 h-1

  11. Genomic analysis of a xylose operon and characterization of novel xylose isomerase and xylulokinase from Bacillus coagulans NL01.

    Science.gov (United States)

    Zheng, Zhaojuan; Lin, Xi; Jiang, Ting; Ye, Weihua; Ouyang, Jia

    2016-08-01

    To investigate the xylose operon and properties of xylose isomerase and xylulokinase in Bacillus coagulans that can effectively ferment xylose to lactic acid. The xylose operon is widely present in B. coagulans. It is composed of four putative ORFs. Novel xylA and xylB from B. coagulans NL01 were cloned and expressed in Escherichia coli. Sequence of xylose isomerase was more conserved than that of xylulokinase. Both the enzymes exhibited maximum activities at pH 7-8 but with a high temperature maximum of 80-85 °C, divalent metal ion was prerequisite for their activation. Xylose isomerase and xylulokinase were most effectively activated by Ni(2+) and Co(2+), respectively. Genomic analysis of xylose operon has contributed to understanding xylose metabolism in B. coagulans and the novel xylose isomerase and xylulokinase might provide new alternatives for metabolic engineering of other strains to improve their fermentation performance on xylose.

  12. Open reading frame 176 in the photosynthesis gene cluster of Rhodobacter capsulatus encodes idi, a gene for isopentenyl diphosphate isomerase.

    OpenAIRE

    Hahn, F M; Baker, J A; Poulter, C D

    1996-01-01

    Isopentenyl diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway. A database search based on probes from the highly conserved regions in three eukaryotic IPP isomerases revealed substantial similarity with ORF176 in the photosynthesis gene cluster in Rhodobacter capsulatus. The open reading frame was cloned into an Escherichia coli expression vector. The encoded 20-kDa protein, which was purified in two steps by ion exchange and hydrophobic...

  13. Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118.

    Science.gov (United States)

    Lobley, Carina M C; Aller, Pierre; Douangamath, Alice; Reddivari, Yamini; Bumann, Mario; Bird, Louise E; Nettleship, Joanne E; Brandao-Neto, Jose; Owens, Raymond J; O'Toole, Paul W; Walsh, Martin A

    2012-12-01

    The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Å resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and β D-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography.

  14. A Protein Disulfide Isomerase Gene Fusion Expression System That Increases the Extracellular Productivity of Bacillus brevis

    Science.gov (United States)

    Kajino, Tsutomu; Ohto, Chikara; Muramatsu, Masayoshi; Obata, Shusei; Udaka, Shigezo; Yamada, Yukio; Takahashi, Haruo

    2000-01-01

    We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system. PMID:10653729

  15. Molecular Characterization and Analysis of a Novel Protein Disulfide Isomerase-Like Protein of Eimeria tenella

    OpenAIRE

    Han, Hongyu; Dong, Hui; Zhu, Shunhai; Zhao, Qiping; Jiang, Lianlian; Wang, Yange; Li, Liujia; Wu, Youlin; Huang, Bing

    2014-01-01

    Protein disulfide isomerase (PDI) and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE) according to the expressed sequence tag (EST). The EtPDI...

  16. Interaction of p53 with prolyl isomerases: Healthy and unhealthy relationships.

    Science.gov (United States)

    Mantovani, Fiamma; Zannini, Alessandro; Rustighi, Alessandra; Del Sal, Giannino

    2015-10-01

    The p53 protein family, comprising p53, p63 and p73, is primarily involved in preserving genome integrity and preventing tumor onset, and also affects a range of physiological processes. Signal-dependent modifications of its members and of other pathway components provide cells with a sophisticated code to transduce a variety of stress signaling into appropriate responses. TP53 mutations are highly frequent in cancer and lead to the expression of mutant p53 proteins that are endowed with oncogenic activities and sensitive to stress signaling. p53 family proteins have unique structural and functional plasticity, and here we discuss the relevance of prolyl-isomerization to actively shape these features. The anti-proliferative functions of the p53 family are carefully activated upon severe stress and this involves the interaction with prolyl-isomerases. In particular, stress-induced stabilization of p53, activation of its transcriptional control over arrest- and cell death-related target genes and of its mitochondrial apoptotic function, as well as certain p63 and p73 functions, all require phosphorylation of specific S/T-P motifs and their subsequent isomerization by the prolyl-isomerase Pin1. While these functions of p53 counteract tumorigenesis, under some circumstances their activation by prolyl-isomerases may have negative repercussions (e.g. tissue damage induced by anticancer therapies and ischemia-reperfusion, neurodegeneration). Moreover, elevated Pin1 levels in tumor cells may transduce deregulated phosphorylation signaling into activation of mutant p53 oncogenic functions. The complex repertoire of biological outcomes induced by p53 finds mechanistic explanations, at least in part, in the association between prolyl-isomerases and the p53 pathway. This article is part of a Special Issue entitled Proline-directed foldases: Cell signaling catalysts and drug targets. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Genome sequence of carboxylesterase, carboxylase and xylose isomerase producing alkaliphilic haloarchaeon Haloterrigena turkmenica WANU15

    Directory of Open Access Journals (Sweden)

    Samy Selim

    2016-03-01

    Full Text Available We report draft genome sequence of Haloterrigena turkmenica strain WANU15, isolated from Soda Lake. The draft genome size is 2,950,899 bp with a G + C content of 64% and contains 49 RNA sequence. The genome sequence can be accessed at DDBJ/EMBL/GenBank under the accession no. LKCV00000000. Keywords: Soda Lake, Haloterrigena turkmenica, Carboxylesterase, Carboxylase, Xylose isomerase, Whole genome sequencing

  18. The crystal structure of a multifunctional protein: Phosphoglucose isomerase/autocrine motility factor/neuroleukin

    OpenAIRE

    Sun, Yuh-Ju; Chou, Chia-Cheng; Chen, Wei-Shone; Wu, Rong-Tsun; Meng, Menghsiao; Hsiao, Chwan-Deng

    1999-01-01

    Phosphoglucose isomerase (PGI) plays a central role in both the glycolysis and the gluconeogenesis pathways. We present here the complete crystal structure of PGI from Bacillus stearothermophilus at 2.3-Å resolution. We show that PGI has cell-motility-stimulating activity on mouse colon cancer cells similar to that of endogenous autocrine motility factor (AMF). PGI can also enhance neurite outgrowth on neuronal progenitor cells similar to that observed for neuroleukin. The results confirm tha...

  19. Domain architecture of protein-disulfide isomerase facilitates its dual role as an oxidase and an isomerase in Ero1p-mediated disulfide formation

    DEFF Research Database (Denmark)

    Kulp, M. S.; Frickel, E. M.; Ellgaard, Lars

    2006-01-01

    reduction/rearrangement of non-native disulfides is poorly understood. We analyzed the role of individual PDI domains in disulfide bond formation in a reaction driven by their natural oxidant, Ero1p. We found that Ero1p oxidizes the isolated PDI catalytic thioredoxin domains, A and A' at the same rate......Native disulfide bond formation in eukaryotes is dependent on protein-disulfide isomerase (PDI) and its homologs, which contain varying combinations of catalytically active and inactive thioredoxin domains. However, the specific contribution of PDI to the formation of new disulfides versus...... catalytic (A) domain. The specific order of thioredoxin domains in PDI is important in establishing the asymmetry in the rate of oxidation of the two active sites thus allowing A and A', two thioredoxin domains that are similar in sequence and structure, to serve opposing functional roles as a disulfide...

  20. Triosephosphate isomerase: energetics of the reaction catalyzed by the yeast enzyme expressed in Escherichia coli

    International Nuclear Information System (INIS)

    Nickbarg, E.B.; Knowles, J.R.

    1988-01-01

    Triosephosphate isomerase from bakers' yeast, expressed in Escherichia coli strain DF502(p12), has been purified to homogeneity. The kinetics of the reaction in each direction have been determined at pH 7.5 and 30 degrees C. Deuterium substitution at the C-2 position of substrate (R)-glyceraldehyde phosphate and at the 1-pro-R position of substrate dihydroxyacetone phosphate results in kinetic isotope effects on kcat of 1.6 and 3.4, respectively. The extent of transfer of tritium from [1(R)- 3 H]dihydroxyacetone phosphate to product (R)-glyceraldehyde phosphate during the catalyzed reaction is only 3% after 66% conversion to product, indicating that the enzymic base that mediates proton transfer is in rapid exchange with solvent protons. When the isomerase-catalyzed reaction is run in tritiated water in each direction, radioactivity is incorporated both into the remaining substrate and into the product. In the exchange-conversion experiment with dihydroxyacetone phosphate as substrate, the specific radioactivity of remaining dihydroxyacetone phosphate rises as a function of the extent of reaction with a slope of about 0.3, while the specific radioactivity of the products is 54% that of the solvent. In the reverse direction with (R)-glyceraldehyde phosphate as substrate, the specific radioactivity of the product formed is only 11% that of the solvent, while the radioactivity incorporated into the remaining substrate (R)-glyceraldehyde phosphate also rises as a function of the extent of reaction with a slope of 0.3. These results have been analyzed according to the protocol described earlier to yield the free energy profile of the reaction catalyzed by the yeast isomerase

  1. Affinity labeling and characterization of the active site histidine of glucosephosphate isomerase

    International Nuclear Information System (INIS)

    Gibson, D.R.; Gracy, R.W.; Hartman, F.C.

    1980-01-01

    N-bromoacetylethanolamine phosphate was found to act as a specific affinity label for the active center of glucosephosphate isomerase. The inactivation process followed pseudo-first order kinetics, was irreversible, and exhibited rate saturation kinetics with minimal half-lives of inactivation of 4.5 and 6.3 min for the enzyme isolated from human placenta and rabbit muscle, respectively. The pH dependence of the inactivation process closely paralleled the pH dependence of the overall catalytic process with pK/sub a/ values at pH 6.4 and 9.0. The stoichiometry of labeling of either enzyme, as determined with N-bromo[ 14 C 2 ]acetylethanolamine phosphate, was 1 eq of the affinity label/subunit of enzyme. After acid hydrolysis and amino acid analysis of the radioactive affinity-labeled human enzyme, only radioactive 3-carboxymethyl histidine was found. In the case of the rabbit enzyme, the only radioactive derivative obtained was 1-carboxymethyl histidine. Active site tryptic peptides were isolated by solvent extraction, thin layer peptide fingerprinting, and ion exchange chromatography before and after removal of the phosphate from the active site peptide. Amino acid analysis of the labeled peptides from the two species were very similar. Using high sensitivity methods for sequence analysis, the primary structure of the active site was established as Val-Leu-His-Ala-Glu-Asn-Val-Asp (Gly,Thr,Ser) Glu-Ile (Thr-Gly-His-Lys-Glx)-Tyr-Phe. Apparent sequence homology between the catalytic center of glucosephosphate isomerase and triosephosphate isomerase suggest that the two enzymes may have evolved from a common ancestral gene

  2. Glucose(xylose isomerase production by Streptomyces sp. CH7 grown on agricultural residues

    Directory of Open Access Journals (Sweden)

    Kankiya Chanitnun

    2012-09-01

    Full Text Available Streptomyces sp. CH7 was found to efficiently produce glucose(xylose isomerase when grown on either xylan or agricultural residues. This strain produced a glucose(xylose isomerase activity of roughly 1.8 U/mg of protein when it was grown in medium containing 1% xylose as a carbon source. Maximal enzymatic activities of about 5 and 3 U/mg were obtained when 1% xylan and 2.5% corn husks were used, respectively. The enzyme was purified from a mycelial extract to 16-fold purity with only two consecutive column chromatography steps using Macro-prep DEAE and Sephacryl-300, respectively. The approximate molecular weight of the purified enzyme is 170 kDa, and it has four identical subunits of 43.6 kDa as estimated by SDS-PAGE. Its Km values for glucose and xylose were found to be 258.96 and 82.77 mM, respectively, and its Vmax values are 32.42 and 63.64 μM/min/mg, respectively. The purified enzyme is optimally active at 85ºC and pH 7.0. It is stable at pH 5.5-8.5 and at temperatures up to 60ºC after 30 min. These findings indicate that glucose(xylose isomerase from Streptomyces sp. CH7 has the potential for industrial applications, especially for high-fructose syrup production and bioethanol fermentation from hemicellulosic hydrolysates by Saccharomyces cerevisiae.

  3. Rapid expansion of the protein disulfide isomerase gene family facilitates the folding of venom peptides

    DEFF Research Database (Denmark)

    Safavi-Hemami, Helena; Li, Qing; Jackson, Ronneshia L.

    2016-01-01

    Formation of correct disulfide bonds in the endoplasmic reticulum is a crucial step for folding proteins destined for secretion. Protein disulfide isomerases (PDIs) play a central role in this process. We report a previously unidentified, hypervariable family of PDIs that represents the most...... diverse gene family of oxidoreductases described in a single genus to date. These enzymes are highly expressed specifically in the venom glands of predatory cone snails, animals that synthesize a remarkably diverse set of cysteine-rich peptide toxins (conotoxins). Enzymes in this PDI family, termed...

  4. Crystal Structure and Substrate Specificity of D-Galactose-6-Phosphate Isomerase Complexed with Substrates

    Science.gov (United States)

    Lee, Jung-Kul; Pan, Cheol-Ho

    2013-01-01

    D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26), which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD), catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi). Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays. PMID:24015281

  5. Polimorfisme Enzim Glucose-6-Phosphate Isomerase pada Tiga Populasi Tuna Sirip Kuning (Thunnus albacares)

    OpenAIRE

    Permana, Gusti Ngurah; Hutapea, Jhon H.; Moria, Sari Budi; Haryanti, Haryanti

    2006-01-01

    Samples of yellowfin tuna (Thunnus albacares) were taken from three locations Bali, North Sulawesi and North Maluku. The glucose-6-phosphate isomerase (GPI) was analyzed from liver using allozyme electrophoresis method. Polymorphism of GPI enzyme was observed and four alleles (A, B ,C, D) were found in Bali population, three alleles (A,B,C) were found in North Maluku and North Sulawesi populations. Heterozygosity values, from Bali, North Maluku and North Sulawesi were 0.419; 0.417; 0.143 resp...

  6. Crystal structure and substrate specificity of D-galactose-6-phosphate isomerase complexed with substrates.

    Directory of Open Access Journals (Sweden)

    Woo-Suk Jung

    Full Text Available D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26, which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD, catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi. Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays.

  7. Effect of gamma irradiation on whole-cell glucose isomerase. Pt.1

    International Nuclear Information System (INIS)

    Bachman, S.; Gebicka, L.

    1984-01-01

    Gamma-rays induced inactivation of Actinoplanes missouriensis and Streptomyces olivaceus glucose isomerase has been studied. This enzyme exhibits high resistance against ionizing radiation. The D 37 value was found to be equal to 131 kGy for Actinoplanes missouriensis cells and 88 kGy for Streptomyces olivaceus cells when irradiated in the dry state in the presence of air. Mg 2+ ions do not affect the radiosensitivity of the enzyme in cells, while the addition of Co 2+ ions to the cell suspension increases its stability against ionizing radiation. (orig.) [de

  8. Control analysis of the role of triosephosphate isomerase in glucose metabolism in Lactococcus lactis

    DEFF Research Database (Denmark)

    Solem, Christian; Købmann, Brian Jensen; Jensen, Peter Ruhdal

    2008-01-01

    Triosephosphate isomerase (TPI), which catalyses the conversion of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (G3P), was studied for its control on glycolysis and mixed acid production in L. lactis subspecies lactis IL1403 and L. lactis subspecies cremoris MG1363. Strains...... metabolites glucose-6-phosphate, fructose-1,6-bisphosphate and DHAP in the IL1403 derivatives were essentially unchanged for TPI activities from 26% to 225%. At a TPI activity of 3%, the level of DHAP increased four times. The finding that an increased level of DHAP coincides with an increase in formate...

  9. MTH1745, a protein disulfide isomerase-like protein from thermophilic archaea, Methanothermobacter thermoautotrophicum involving in stress response.

    Science.gov (United States)

    Ding, Xia; Lv, Zhen-Mei; Zhao, Yang; Min, Hang; Yang, Wei-Jun

    2008-01-01

    MTH1745 is a putative protein disulfide isomerase characterized with 151 amino acid residues and a CPAC active-site from the anaerobic archaea Methanothermobacter thermoautotrophicum. The potential functions of MTH1745 are not clear. In the present study, we show a crucial role of MTH1745 in protecting cells against stress which may be related to its functions as a disulfide isomerase and its chaperone properties. Using real-time polymerase chain reaction analyses, the level of MTH1745 messenger RNA (mRNA) in the thermophilic archaea M. thermoautotrophicum was found to be stress-induced in that it was significantly higher under low (50 degrees C) and high (70 degrees C) growth temperatures than under the optimal growth temperature for the organism (65 degrees C). Additionally, the expression of MTH1745 mRNA was up-regulated by cold shock (4 degrees C). Furthermore, the survival of MTH1745 expressing Escherichia coli cells was markedly higher than that of control cells in response to heat shock (51.0 degrees C). These results indicated that MTH1745 plays an important role in the resistance of stress. By assay of enzyme activities in vitro, MTH1745 also exhibited a chaperone function by promoting the functional folding of citrate synthase after thermodenaturation. On the other hand, MTH1745 was also shown to function as a disulfide isomerase on the refolding of denatured and reduced ribonuclease A. On the basis of its single thioredoxin domain, function as a disulfide isomerase, and its chaperone activity, we suggest that MTH1745 may be an ancient protein disulfide isomerase. These studies may provide clues to the understanding of the function of protein disulfide isomerase in archaea.

  10. SAXS-WAXS studies of the low-resolution structure in solution of xylose/glucose isomerase from Streptomyces rubiginosus

    Science.gov (United States)

    Kozak, Maciej; Taube, Michał

    2009-10-01

    The structure and conformation of molecule of xylose/glucose isomerase from Streptomyces rubiginosus in solution (at pH 6 and 7.6; with and without the substrate) has been studied by small- and wide-angle scattering of synchrotron radiation (SAXS-WAXS). On the basis of the SAXS-WAXS data, the low-resolution structure in solution has been reconstructed using ab inito methods. A comparison of the models of glucose isomerase shows only small differences between the model in solution and the crystal structure.

  11. Theileria parasites secrete a prolyl isomerase to maintain host leukocyte transformation.

    Science.gov (United States)

    Marsolier, J; Perichon, M; DeBarry, J D; Villoutreix, B O; Chluba, J; Lopez, T; Garrido, C; Zhou, X Z; Lu, K P; Fritsch, L; Ait-Si-Ali, S; Mhadhbi, M; Medjkane, S; Weitzman, J B

    2015-04-16

    Infectious agents develop intricate mechanisms to interact with host cell pathways and hijack their genetic and epigenetic machinery to change host cell phenotypic states. Among the Apicomplexa phylum of obligate intracellular parasites, which cause veterinary and human diseases, Theileria is the only genus that transforms its mammalian host cells. Theileria infection of bovine leukocytes induces proliferative and invasive phenotypes associated with activated signalling pathways, notably JNK and AP-1 (ref. 2). The transformed phenotypes are reversed by treatment with the theilericidal drug buparvaquone. We used comparative genomics to identify a homologue of the peptidyl-prolyl isomerase PIN1 in T. annulata (TaPIN1) that is secreted into the host cell and modulates oncogenic signalling pathways. Here we show that TaPIN1 is a bona fide prolyl isomerase and that it interacts with the host ubiquitin ligase FBW7, leading to its degradation and subsequent stabilization of c-JUN, which promotes transformation. We performed in vitro and in silico analysis and in vivo zebrafish xenograft experiments to demonstrate that TaPIN1 is directly inhibited by the anti-parasite drug buparvaquone (and other known PIN1 inhibitors) and is mutated in a drug-resistant strain. Prolyl isomerization is thus a conserved mechanism that is important in cancer and is used by Theileria parasites to manipulate host oncogenic signalling.

  12. Crystallization and preliminary X-ray diffraction studies of l-rhamnose isomerase from Pseudomonas stutzeri

    International Nuclear Information System (INIS)

    Yoshida, Hiromi; Wayoon, Poonperm; Takada, Goro; Izumori, Ken; Kamitori, Shigehiro

    2006-01-01

    Recombinant l-rhamnose isomerase from P. stutzeri has been crystallized. Diffraction data have been collected to 2.0 Å resolution. l-Rhamnose isomerase from Pseudomonas stutzeri (P. stutzeril-RhI) catalyzes not only the reversible isomerization of l-rhamnose to l-rhamnulose, but also isomerization between various rare aldoses and ketoses. Purified His-tagged P. stutzeril-RhI was crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group P2 1 , with unit-cell parameters a = 74.3, b = 104.0, c = 107.0 Å, β = 106.8°. Diffraction data have been collected to 2.0 Å resolution. The molecular weight of the purified P. stutzeril-RhI with a His tag at the C-terminus was confirmed to be 47.7 kDa by MALDI–TOF mass-spectrometric analysis and the asymmetric unit is expected to contain four molecules

  13. In-house SIRAS phasing of the polyunsaturated fatty-acid isomerase from Propionibacterium acnes

    International Nuclear Information System (INIS)

    Liavonchanka, Alena; Hornung, Ellen; Feussner, Ivo; Rudolph, Markus

    2006-01-01

    Low iodide concentrations were sufficient to allow SAD and SIRAS phasing of cubic crystals of a novel fatty acid isomerase using Cu Kα radiation. The polyenoic fatty-acid isomerase from Propionibacterium acnes (PAI) catalyzes the double-bond isomerization of linoleic acid to conjugated linoleic acid, which is a dairy- or meat-derived fatty acid in the human diet. PAI was overproduced in Escherichia coli and purified to homogeneity as a yellow-coloured protein. The nature of the bound cofactor was analyzed by absorption and fluorescence spectroscopy. Single crystals of PAI were obtained in two crystal forms. Cubic shaped crystals belong to space group I2 1 3, with a unit-cell parameter of 160.4 Å, and plate-like crystals belong to the monoclinic space group C2, with unit-cell parameters a = 133.7, b = 60.8, c = 72.2 Å, β = 115.8°. Both crystal forms contain one molecule per asymmetric unit and diffract to a resolution of better than 2.0 Å. Initial phases were obtained by SIRAS from in-house data from a cubic crystal that was soaked with an unusually low KI concentration of 0.25 M

  14. Ethanol production from lignocellulosic hydrolysates using engineered Saccharomyces cerevisiae harboring xylose isomerase-based pathway.

    Science.gov (United States)

    Ko, Ja Kyong; Um, Youngsoon; Woo, Han Min; Kim, Kyoung Heon; Lee, Sun-Mi

    2016-06-01

    The efficient co-fermentation of glucose and xylose is necessary for the economically feasible bioethanol production from lignocellulosic biomass. Even with xylose utilizing Saccharomyces cerevisiae, the efficiency of the lignocellulosic ethanol production remains suboptimal mainly due to the low conversion yield of xylose to ethanol. In this study, we evaluated the co-fermentation performances of SXA-R2P-E, a recently engineered isomerase-based xylose utilizing strain, in mixed sugars and in lignocellulosic hydrolysates. In a high-sugar fermentation with 70g/L of glucose and 40g/L of xylose, SXA-R2P-E produced 50g/L of ethanol with an yield of 0.43gethanol/gsugars at 72h. From dilute acid-pretreated hydrolysates of rice straw and hardwood (oak), the strain produced 18-21g/L of ethanol with among the highest yield of 0.43-0.46gethanol/gsugars ever reported. This study shows a highly promising potential of a xylose isomerase-expressing strain as an industrially relevant ethanol producer from lignocellulosic hydrolysates. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Characterization of a monoclonal antibody that specifically inhibits triosephosphate isomerase activity of Taenia solium.

    Science.gov (United States)

    Víctor, Sanabria-Ayala; Yolanda, Medina-Flores; Araceli, Zavala-Carballo; Lucía, Jiménez; Abraham, Landa

    2013-08-01

    In the present study, we obtained and characterized partially a monoclonal antibody (4H11D10B11 mAb) against triosephosphate isomerase from Taenia solium (TTPI). This antibody recognized the enzyme by both ELISA and western blot and was able to inhibit its enzymatic activity in 74%. Moreover, the antigen-binding fragments (Fabs), products of digestion of the monoclonal antibody with papain, retained almost the same inhibitory effect. We determined the binding site by ELISA; synthetic peptides containing sequences from different non-conserved regions of the TTPI were confronted to the 4H11D10B11 mAb. The epitope recognized by the monoclonal antibody was located on peptide TTPI-56 (ATPAQAQEVHKVVRDWIRKHVDAGIADKARI), and an analysis of mimotopes, obtained with the 4H11D10B11 mAb, suggests that the epitope spans the sequence WIRKHVDAGIAD, residues 193-204 of the enzyme. This epitope is located within helix 6, next to loop 6, an essential active loop during catalysis. The antibody did not recognize triosephosphate isomerase from man and pig, definitive and intermediary hosts of T. solium, respectively. Furthermore, it did not bind to the catalytic site, since kinetic analysis demonstrated that inhibition had a non-competitive profile. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Overexpression, purification, crystallization and preliminary diffraction studies of the Protaminobacter rubrum sucrose isomerase SmuA

    International Nuclear Information System (INIS)

    Ravaud, Stéphanie; Watzlawick, Hildegard; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

    2005-01-01

    The P. rubrum sucrose isomerase SmuA, a key enzyme in the industrial production of isomaltulose, was crystallized and diffraction data were collected to 1.95 Å resolution. Palatinose (isomaltulose, α-d-glucosylpyranosyl-1,6-d-fructofuranose), a nutritional and acariogenic reducing sugar, is industrially obtained from sucrose by using immobilized cells of Protaminobacter rubrum that produce the sucrose isomerase SmuA. The isomerization of sucrose catalyzed by this enzyme also results in the formation of trehalulose (α-d-glucosylpyranosyl-1,1-d-fructofuranose) in smaller amounts and glucose, fructose and eventually isomaltose as by-products, which lower the yield of the reaction and complicate the recovery of palatinose. The determination of the three-dimensional structure of SmuA will provide a basis for rational protein-engineering studies in order to optimize the industrial production of palatinose. A recombinant form of the 67.3 kDa SmuA enzyme has been crystallized in the native state by the vapour-diffusion method. Crystals belong to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 61.6, b = 81.4, c = 135.6 Å, and diffract to 1.95 Å resolution on a synchrotron-radiation source

  17. Preliminary crystallographic analysis of two hypothetical ribose-5-phosphate isomerases from Streptococcus mutans

    International Nuclear Information System (INIS)

    Wang, Chen; Fan, Xuexin; Cao, Xiaofang; Liu, Xiang; Li, Lanfen; Su, Xiaodong

    2012-01-01

    Two hypothetical ribose-5-phosphate isomerases from S. mutans have been produced in E. coli and crystallized. The crystals diffracted to high resolutions suitable for crystallographic analyses. Study of the enzymes from sugar metabolic pathways may provide a better understanding of the pathogenesis of the human oral pathogen Streptococcus mutans. Bioinformatics, biochemical and crystallization methods were used to characterize and understand the function of two putative ribose-5-phosphate isomerases: SMU1234 and SMU2142. The proteins were cloned and constructed with N-terminal His tags. Protein purification was performed by Ni 2+ -chelating and size-exclusion chromatography. The crystals of SUM1234 diffracted to 1.9 Å resolution and belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 48.97, b = 98.27, c = 101.09 Å, α = β = γ = 90°. The optimized SMU2142 crystals diffracted to 2.7 Å resolution and belonged to space group P1, with unit-cell parameters a = 53.7, b = 54.1, c = 86.5 Å, α = 74.2, β = 73.5, γ = 83.7°. Initial phasing of both proteins was attempted by molecular replacement; the structure of SMU1234 could easily be solved, but no useful results were obtained for SMU2142. Therefore, SeMet-labelled SMU2142 will be prepared for phasing

  18. Human triose-phosphate isomerase deficiency: a single amino acid substitution results in a thermolabile enzyme.

    Science.gov (United States)

    Daar, I O; Artymiuk, P J; Phillips, D C; Maquat, L E

    1986-10-01

    Triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) deficiency is a recessive disorder that results in hemolytic anemia and neuromuscular dysfunction. To determine the molecular basis of this disorder, a TPI allele from two unrelated patients homozygous for TPI deficiency was compared with an allele from a normal individual. Each disease-associated sequence harbors a G X C----C X G transversion in the codon for amino acid-104 and specifies a structurally altered protein in which a glutamate residue is replaced by an aspartate residue. The importance of glutamate-104 to enzyme structure and function is implicated by its conservation in the TPI protein of all species that have been characterized to date. The glutamate-to-aspartate substitution results in a thermolabile enzyme as demonstrated by assays of TPI activity in cultured fibroblasts of each patient and cultured Chinese hamster ovary (CHO) cells that were stably transformed with the mutant alleles. Although this substitution conserves the overall charge of amino acid-104, the x-ray crystal structure of chicken TPI indicates that the loss of a side-chain methylene group (-CH2CH2COO- ---- -CH2COO-) is sufficient to disrupt the counterbalancing of charges that normally exists within a hydrophobic pocket of the native enzyme.

  19. Structural insights from a novel invertebrate triosephosphate isomerase from Litopenaeus vannamei

    Science.gov (United States)

    Lopez-Zavala, Alonso A.; Carrasco-Miranda, Jesus S.; Ramirez-Aguirre, Claudia D.; López-Hidalgo, Marisol; Benitez-Cardoza, Claudia G.; Ochoa-Leyva, Adrian; Cardona-Felix, Cesar S.; Diaz-Quezada, Corina; Rudiño-Piñera, Enrique; Sotelo-Mundo, Rogerio R.; Brieba, Luis G.

    2016-01-01

    Triosephosphate isomerase (TIM; EC 5.3.1.1) is a key enzyme involved in glycolysis and gluconeogenesis. Glycolysis is one of the most regulated metabolic pathways, however little is known about the structural mechanisms for its regulation in non-model organisms, like crustaceans. To understand the structure and function of this enzyme in invertebrates, we obtained the crystal structure of triosephosphate isomerase from the marine Pacific whiteleg shrimp (Litopenaeus vannamei, LvTIM) in complex with its inhibitor 2-phosphogyceric acid (2-PG) at 1.7 Å resolution. LvTIM assembles as a homodimer with residues 166-176 covering the active site and residue Glu166 interacting with the inhibitor. We found that LvTIM is the least stable TIM characterized to date, with the lowest range of melting temperatures, and with the lowest activation enthalpy associated with the thermal unfolding process reported. In TIMs dimer stabilization is maintained by an interaction of loop 3 by a set of hydrophobic contacts between subunits. Within these contacts, the side chain of a hydrophobic residue of one subunit fits into a cavity created by a set of hydrophobic residues in the neighboring subunit, via a "ball and socket" interaction. LvTIM presents a Cys47 at the "ball" inter-subunit contact indicating that the character of this residue is responsible for the decrease in dimer stability. Mutational studies show that this residue plays a role in dimer stability but is not a solely determinant for dimer formation. PMID:27614148

  20. A preliminary X-ray study of sedoheptulose-7-phosphate isomerase from Burkholderia pseudomallei

    International Nuclear Information System (INIS)

    Kim, Mi-Sun; Shin, Dong Hae

    2009-01-01

    Sedoheptulose-7-phosphate isomerase (GmhA) from B. pseudomallei is one of the targets of antibiotic adjuvants for melioidosis. In this study, GmhA has been cloned, expressed, purified and crystallized. Sedoheptulose-7-phosphate isomerase (GmhA) converts d-sedoheptulose 7-phosphate to d,d-heptose 7-phosphate. This is the first step in the biosynthesis pathway of NDP-heptose, which is responsible for the pleiotropic phenotype. This biosynthesis pathway is the target of inhibitors to increase the membrane permeability of Gram-negative pathogens or of adjuvants working synergistically with known antibiotics. Burkholderia pseudomallei is the causative agent of melioidosis, a seriously invasive disease in animals and humans in tropical and subtropical areas. GmhA from B. pseudomallei is one of the targets of antibiotic adjuvants for melioidosis. In this study, GmhA has been cloned, expressed, purified and crystallized. Synchrotron X-ray data were also collected to 1.9 Å resolution. The crystal belonged to the primitive orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 61.3, b = 84.2, c = 142.3 Å. A full structural determination is under way in order to provide insights into the structure–function relationships of this protein

  1. Enhanced pest resistance and increased phenolic production in maize callus transgenically expressing a maize chalcone isomerase -3 like gene

    Science.gov (United States)

    Significant losses in maize production are due to damage by insects and ear rot fungi. A gene designated as chalcone-isomerase-like, located in a quantitative trait locus for resistance to Fusarium ear rot fungi, was cloned from a Fusarium ear rot resistant inbred and transgenically expressed in mai...

  2. Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors.

    Science.gov (United States)

    Yamamoto, Kenta; Wang, Jiguang; Sprinzen, Lisa; Xu, Jun; Haddock, Christopher J; Li, Chen; Lee, Brian J; Loredan, Denis G; Jiang, Wenxia; Vindigni, Alessandro; Wang, Dong; Rabadan, Raul; Zha, Shan

    2016-06-15

    Missense mutations in ATM kinase, a master regulator of DNA damage responses, are found in many cancers, but their impact on ATM function and implications for cancer therapy are largely unknown. Here we report that 72% of cancer-associated ATM mutations are missense mutations that are enriched around the kinase domain. Expression of kinase-dead ATM (Atm(KD/-)) is more oncogenic than loss of ATM (Atm(-/-)) in mouse models, leading to earlier and more frequent lymphomas with Pten deletions. Kinase-dead ATM protein (Atm-KD), but not loss of ATM (Atm-null), prevents replication-dependent removal of Topo-isomerase I-DNA adducts at the step of strand cleavage, leading to severe genomic instability and hypersensitivity to Topo-isomerase I inhibitors. Correspondingly, Topo-isomerase I inhibitors effectively and preferentially eliminate Atm(KD/-), but not Atm-proficientor Atm(-/-) leukemia in animal models. These findings identify ATM kinase-domain missense mutations as a potent oncogenic event and a biomarker for Topo-isomerase I inhibitor based therapy.

  3. Physiological and Pathogenic Roles of Prolyl Isomerase Pin1 in Metabolic Regulations via Multiple Signal Transduction Pathway Modulations

    Directory of Open Access Journals (Sweden)

    Yusuke Nakatsu

    2016-09-01

    Full Text Available Prolyl isomerases are divided into three groups, the FKBP family, Cyclophilin and the Parvulin family (Pin1 and Par14. Among these isomerases, Pin1 is a unique prolyl isomerase binding to the motif including pSer/pThr-Pro that is phosphorylated by kinases. Once bound, Pin1 modulates the enzymatic activity, protein stability or subcellular localization of target proteins by changing the cis- and trans-formations of proline. Several studies have examined the roles of Pin1 in the pathogenesis of cancers and Alzheimer’s disease. On the other hand, recent studies have newly demonstrated Pin1 to be involved in regulating glucose and lipid metabolism. Interestingly, while Pin1 expression is markedly increased by high-fat diet feeding, Pin1 KO mice are resistant to diet-induced obesity, non-alcoholic steatohepatitis and diabetic vascular dysfunction. These phenomena result from the binding of Pin1 to several key factors regulating metabolic functions, which include insulin receptor substrate-1, AMPK, Crtc2 and NF-κB p65. In this review, we focus on recent advances in elucidating the physiological roles of Pin1 as well as the pathogenesis of disorders involving this isomerase, from the viewpoint of the relationships between signal transductions and metabolic functions.

  4. L-Rhamnose isomerase and its use for biotechnological production of rare sugars.

    Science.gov (United States)

    Xu, Wei; Zhang, Wenli; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

    2016-04-01

    L-Rhamnose isomerase (L-RI, EC 5.3.1.14), catalyzing the isomerization between L-rhamnose and L-rhamnulose, plays an important role in microbial L-rhamnose metabolism and thus occurs in a wide range of microorganisms. It attracts more and more attention because of its broad substrate specificity and its great potential in enzymatic production of various rare sugars. In this article, the enzymatic properties of various reported L-RIs were compared in detail, and their applications in the production of L-rhamnulose and various rare sugars including D-allose, D-gulose, L-lyxose, L-mannose, L-talose, and L-galactose were also reviewed.

  5. Mechanisms of Neuroprotection by Protein Disulphide Isomerase in Amyotrophic Lateral Sclerosis

    Directory of Open Access Journals (Sweden)

    Adam K. Walker

    2011-01-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a devastating neurodegenerative disease characterised by the progressive loss of motor neurons, leading to paralysis and death within several years of onset. Although protein misfolding is a key feature of ALS, the upstream triggers of disease remain elusive. Recently, endoplasmic reticulum (ER stress was identified as an early and central feature in ALS disease models as well as in human patient tissues, indicating that ER stress could be an important process in disease pathogenesis. One important chaperone induced by ER stress is protein disulphide isomerase (PDI, which is both upregulated and posttranslationally inhibited by S-nitrosylation in ALS. In this paper, we present evidence from studies of genetics, model organisms, and patient tissues which indicate an active role for PDI and ER stress in ALS disease processes.

  6. In silico cloning and B/T cell epitope prediction of triosephosphate isomerase from Echinococcus granulosus.

    Science.gov (United States)

    Wang, Fen; Ye, Bin

    2016-10-01

    Cystic echinococcosis is a worldwide zoonosis caused by Echinococcus granulosus. Because the methods of diagnosis and treatment for cystic echinococcosis were limited, it is still necessary to screen target proteins for the development of new anti-hydatidosis vaccine. In this study, the triosephosphate isomerase gene of E. granulosus was in silico cloned. The B cell and T cell epitopes were predicted by bioinformatics methods. The cDNA sequence of EgTIM was composition of 1094 base pairs, with an open reading frame of 753 base pairs. The deduced amino acid sequences were composed of 250 amino acids. Five cross-reactive epitopes, locating on 21aa-35aa, 43aa-57aa, 94aa-107aa, 115-129aa, and 164aa-183aa, could be expected to serve as candidate epitopes in the development of vaccine against E. granulosus. These results could provide bases for gene cloning, recombinant expression, and the designation of anti-hydatidosis vaccine.

  7. Effects of peptidyl-prolyl isomerase 1 depletion in animal models of prion diseases.

    Science.gov (United States)

    Legname, Giuseppe; Virgilio, Tommaso; Bistaffa, Edoardo; De Luca, Chiara Maria Giulia; Catania, Marcella; Zago, Paola; Isopi, Elisa; Campagnani, Ilaria; Tagliavini, Fabrizio; Giaccone, Giorgio; Moda, Fabio

    2018-04-20

    Pin1 is a peptidyl-prolyl isomerase that induces the cis-trans conversion of specific Ser/Thr-Pro peptide bonds in phosphorylated proteins, leading to conformational changes through which Pin1 regulates protein stability and activity. Since down-regulation of Pin1 has been described in several neurodegenerative disorders, including Alzheimer's Disease (AD), Parkinson's Disease (PD) and Huntington's Disease (HD), we investigated its potential role in prion diseases. Animals generated on wild-type (Pin1 +/+ ), hemizygous (Pin1 +/- ) or knock-out (Pin1 -/- ) background for Pin1 were experimentally infected with RML prions. The study indicates that, neither the total depletion nor reduced levels of Pin1 significantly altered the clinical and neuropathological features of the disease.

  8. Helicobacter pylori Peptidyl Prolyl Isomerase Expression Is Associated with the Severity of Gastritis.

    Science.gov (United States)

    Oghalaie, Akbar; Saberi, Samaneh; Esmaeili, Maryam; Ebrahimzadeh, Fatemeh; Barkhordari, Farzaneh; Ghamarian, Abdolreza; Tashakoripoor, Mohammad; Abdirad, Afshin; Eshagh Hosseini, Mahmoud; Khalaj, Vahid; Mohammadi, Marjan

    2016-12-01

    Helicobacter pylori secretory peptidyl prolyl isomerase, HP0175, is progressively identified as a pro-inflammatory and pro-carcinogenic protein, which serves to link H. pylori infection to its more severe clinical outcomes. Here, we have analyzed host HP0175-specific antibody responses in relation to the severity of gastritis. The HP0175 gene fragment was PCR-amplified, cloned, expressed and purified by Ni-NTA affinity chromatography. Serum antigen-specific antibody responses of non-ulcer dyspeptic patients (N = 176) against recombinant HP0175 were detected by western blotting. The infection status of these subjects was determined by rapid urease test, culture, histology, and serology. The grade of inflammation and stage of atrophy were scored blindly according to the OLGA staging system. The recombinant HP0175 (rHP0175) was expressed as a ~35 kDa protein and its identity was confirmed by western blotting using anti-6X His tag antibody and pooled H. pylori-positive sera. Serum IgG antibodies against rHP0175 segregated our patients into two similar-sized groups of sero-positives (90/176, 51.1 %) and sero-negatives (86/176, 48.9 %). The former presented with higher grades of gastric inflammation (OR = 4.4, 95 % CI = 1.9-9.9, P = 0.001) and stages of gastric atrophy (OR = 18.3, 95 %CI = 1.4-246.6, P = 0.028). Our findings lend further support to the pro-inflammatory nature of H. pylori peptidyl prolyl isomerase (HP0175) and recommends this antigen as a non-invasive serum biomarker of the severity of H. pylori-associated gastritis.

  9. Cyclophilin40 isomerase activity is regulated by a temperature-dependent allosteric interaction with Hsp90.

    Science.gov (United States)

    Blackburn, Elizabeth A; Wear, Martin A; Landré, Vivian; Narayan, Vikram; Ning, Jia; Erman, Burak; Ball, Kathryn L; Walkinshaw, Malcolm D

    2015-09-01

    Cyclophilin 40 (Cyp40) comprises an N-terminal cyclophilin domain with peptidyl-prolyl isomerase (PPIase) activity and a C-terminal tetratricopeptide repeat (TPR) domain that binds to the C-terminal-EEVD sequence common to both heat shock protein 70 (Hsp70) and Hsp90. We show in the present study that binding of peptides containing the MEEVD motif reduces the PPIase activity by ∼30%. CD and fluorescence assays show that the TPR domain is less stable than the cyclophilin domain and is stabilized by peptide binding. Isothermal titration calorimetry (ITC) shows that the affinity for the-MEEVD peptide is temperature sensitive in the physiological temperature range. Results from these biophysical studies fit with the MD simulations of the apo and holo (peptide-bound) structures which show a significant reduction in root mean square (RMS) fluctuation in both TPR and cyclophilin domains when-MEEVD is bound. The MD simulations of the apo-protein also highlight strong anti-correlated motions between residues around the PPIase-active site and a band of residues running across four of the seven helices in the TPR domain. Peptide binding leads to a distortion in the shape of the active site and a significant reduction in these strongly anti-correlated motions, providing an explanation for the allosteric effect of ligand binding and loss of PPIase activity. Together the experimental and MD results suggest that on heat shock, dissociation of Cyp40 from complexes mediated by the TPR domain leads to an increased pool of free Cyp40 capable of acting as an isomerase/chaperone in conditions of cellular stress. © 2015 Authors.

  10. Mannose Phosphate Isomerase Isoenzymes in Plutella xylostella Support Common Genetic Bases of Resistance to Bacillus thuringiensis Toxins in Lepidopteran Species

    OpenAIRE

    Herrero, Salvador; Ferré, Juan; Escriche, Baltasar

    2001-01-01

    A strong correlation between two mannose phosphate isomerase (MPI) isoenzymes and resistance to Cry1A toxins from Bacillus thuringiensis has been found in a Plutella xylostella population. MPI linkage to Cry1A resistance had previously been reported for a Heliothis virescens population. The fact that the two populations share similar biochemical, genetic, and cross-resistance profiles of resistance suggests the occurrence of homologous resistance loci in both species.

  11. Revisiting the mechanistic basis of the French Paradox: red wine inhibits the activity of protein disulfide isomerase in vitro

    Science.gov (United States)

    Galinski, Christine N.; Zwicker, Jeffrey I.; Kennedy, Daniel R.

    2015-01-01

    Introduction Although epidemiologic evidence points to cardioprotective activity of red wine, the mechanistic basis for antithrombotic activity has not been established. Quercetin and related flavonoids are present in high concentrations in red but not white wine. Quercetin-glycosides were recently shown to prevent thrombosis in animal models through the inhibition of extracellular protein disulfide isomerase (PDI). We evaluated whether red or white wine inhibited PDI activity in vitro. Methods Quercetin levels in red and white wines were measured by HPLC analysis. Inhibition of PDI activity by red and white wines was assessed by an insulin reduction turbidity assay at various concentrations of wine. PDI inhibition was confirmed using a reduced peptide that contained a disulfide containing peptide as a substrate. The inhibition of PDI related thiol isomerases ERp5 and ERp57 was also assessed. Results We observed a dose-dependent decrease of PDI activity for a variety of red but not white wines. Red wine diluted to 3% final concentration resulted in over 80% inhibition of PDI activity by insulin reductase assay for all varieties tested. This inhibition was also observed in the peptide based assay. Red grape juice yielded similar results but ethanol alone did not affect PDI activity. Interestingly, red wine also inhibited the PDI related thiol isomerases ERp5 and ERp57, albeit to a lesser degree than PDI. Conclusions PDI activity is inhibited by red wine and grape juice, identifying a potentially novel mechanism underlying the cardiovascular benefits attributed to wine consumption. PMID:26585763

  12. A Role of a Newly Identified Isomerase From Yarrowia lipolytica in Erythritol Catabolism

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    Aleksandra M. Mirończuk

    2018-05-01

    Full Text Available Erythritol is a natural sweetener produced by microorganisms as an osmoprotectant. It belongs to the group of polyols and it can be utilized by the oleaginous yeast Yarrowia lipolytica. Despite the recent identification of the transcription factor of erythritol utilization (EUF1, the metabolic pathway of erythritol catabolism remains unknown. In this study we identified a new gene, YALI0F01628g, involved in erythritol assimilation. In silico analysis showed that YALI0F01628g is a putative isomerase and it is localized in the same region as EUF1. qRT-PCR analysis of Y. lipolytica showed a significant increase in YALI0F01628g expression during growth on erythritol and after overexpression of EUF1. Moreover, the deletion strain ΔF01628 showed significantly impaired erythritol assimilation, whereas synthesis of erythritol remained unchanged. The results showed that YALI0F1628g is involved in erythritol assimilation; thus we named the gene EYI1. Moreover, we suggest the metabolic pathway of erythritol assimilation in yeast Y. lipolytica.

  13. Crystal structure and enzymatic properties of chalcone isomerase from the Antarctic vascular plant Deschampsia antarctica Desv.

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    Sun-Ha Park

    Full Text Available Chalcone isomerase (CHI is an important enzyme for flavonoid biosynthesis that catalyzes the intramolecular cyclization of chalcones into (S-flavanones. CHIs have been classified into two types based on their substrate specificity. Type I CHIs use naringenin chalcone as a substrate and are found in most of plants besides legumes, whereas type II CHIs in leguminous plants can also utilize isoliquiritigenin. In this study, we found that the CHI from the Antarctic plant Deschampsia antarctica (DaCHI1 is of type I based on sequence homology but can use type II CHI substrates. To clarify the enzymatic mechanism of DaCHI1 at the molecular level, the crystal structures of unliganded DaCHI1 and isoliquiritigenin-bound DaCHI1 were determined at 2.7 and 2.1 Å resolutions, respectively. The structures revealed that isoliquiritigenin binds to the active site of DaCHI1 and induces conformational changes. Additionally, the activity assay showed that while DaCHI1 exhibits substrate preference for naringenin chalcone, it can also utilize isoliquiritigenin although the catalytic activity was relatively low. Based on these results, we propose that DaCHI1 uses various substrates to produce antioxidant flavonoids as an adaptation to oxidative stresses associated with harsh environmental conditions.

  14. Crystal structure and enzymatic properties of chalcone isomerase from the Antarctic vascular plant Deschampsia antarctica Desv.

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    Park, Sun-Ha; Lee, Chang Woo; Cho, Sung Mi; Lee, Hyoungseok; Park, Hyun; Lee, Jungeun; Lee, Jun Hyuck

    2018-01-01

    Chalcone isomerase (CHI) is an important enzyme for flavonoid biosynthesis that catalyzes the intramolecular cyclization of chalcones into (S)-flavanones. CHIs have been classified into two types based on their substrate specificity. Type I CHIs use naringenin chalcone as a substrate and are found in most of plants besides legumes, whereas type II CHIs in leguminous plants can also utilize isoliquiritigenin. In this study, we found that the CHI from the Antarctic plant Deschampsia antarctica (DaCHI1) is of type I based on sequence homology but can use type II CHI substrates. To clarify the enzymatic mechanism of DaCHI1 at the molecular level, the crystal structures of unliganded DaCHI1 and isoliquiritigenin-bound DaCHI1 were determined at 2.7 and 2.1 Å resolutions, respectively. The structures revealed that isoliquiritigenin binds to the active site of DaCHI1 and induces conformational changes. Additionally, the activity assay showed that while DaCHI1 exhibits substrate preference for naringenin chalcone, it can also utilize isoliquiritigenin although the catalytic activity was relatively low. Based on these results, we propose that DaCHI1 uses various substrates to produce antioxidant flavonoids as an adaptation to oxidative stresses associated with harsh environmental conditions.

  15. Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α.

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    Hyeong-Jun Han

    Full Text Available Peptidyl prolyl isomerase (PIN1 regulates the functional activity of a subset of phosphoproteins through binding to phosphorylated Ser/Thr-Pro motifs and subsequently isomerization of the phosphorylated bonds. Interestingly, PIN1 is overexpressed in many types of malignancies including breast, prostate, lung and colon cancers. However, its oncogenic functions have not been fully elucidated. Here, we report that PIN1 directly interacts with hypoxia-inducible factor (HIF-1α in human colon cancer (HCT116 cells. PIN1 binding to HIF-1α occurred in a phosphorylation-dependent manner. We also found that PIN1 interacted with HIF-1α at both exogenous and endogenous levels. Notably, PIN1 binding stabilized the HIF-1α protein, given that their levels were significantly increased under hypoxic conditions. The stabilization of HIF-1α resulted in increased transcriptional activity, consequently upregulating expression of vascular endothelial growth factor, a major contributor to angiogenesis. Silencing of PIN1 or pharmacologic inhibition of its activity abrogated the angiogenesis. By utilizing a bioluminescence imaging technique, we were able to demonstrate that PIN1 inhibition dramatically reduced the tumor volume in a subcutaneous mouse xenograft model and angiogenesis as well as hypoxia-induced transcriptional activity of HIF-1α. These results suggest that PIN1 interacting with HIF-1α is a potential cancer chemopreventive and therapeutic target.

  16. Identification of triosephosphate isomerase as a novel allergen in Octopus fangsiao.

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    Yang, Yang; Chen, Zhong-Wei; Hurlburt, Barry K; Li, Gui-Ling; Zhang, Yong-Xia; Fei, Dan-Xia; Shen, Hai-Wang; Cao, Min-Jie; Liu, Guang-Ming

    2017-05-01

    Octopus is an important mollusk in human dietary for its nutritional value, however it also causes allergic reactions in humans. Major allergens from octopus have been identified, while the knowledge of novel allergens remains poor. In the present study, a novel allergen with molecular weight of 28kDa protein was purified from octopus (Octopus fangsiao) and identified as triosephosphate isomerase (TIM) by mass spectrometry. TIM aggregated beyond 45°C, and its IgE-binding activity was affected under extreme pH conditions due to the altered secondary structure. In simulated gastric fluid digestion, TIM can be degraded into small fragments, while retaining over 80% of the IgE-binding activity. The full-length cDNA of O. fangsiao TIM (1140bp) was cloned, which encodes 247 amino acid residues, and the entire recombinant TIM was successfully expressed in Escherichia coli BL21, which showed similar immunoreactivity to the native TIM. Different intensity of cross-reactivity among TIM from related species revealed the complexity of its epitopes. Eight linear epitopes of TIM were predicted following bioinformatic analysis. Furthermore, a conformational epitope (A 71 G 74 S 69 D 75 T 73 F 72 V 67 ) was confirmed by the phage display technology. The results revealed the physicochemical and immunological characteristics of TIM, which is significant in the development of hyposensitivity food and allergy diagnosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Mapping Soluble Guanylyl Cyclase and Protein Disulfide Isomerase Regions of Interaction.

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    Erin J Heckler

    Full Text Available Soluble guanylyl cyclase (sGC is a heterodimeric nitric oxide (NO receptor that produces cyclic GMP. This signaling mechanism is a key component in the cardiovascular system. NO binds to heme in the β subunit and stimulates the catalytic conversion of GTP to cGMP several hundred fold. Several endogenous factors have been identified that modulate sGC function in vitro and in vivo. In previous work, we determined that protein disulfide isomerase (PDI interacts with sGC in a redox-dependent manner in vitro and that PDI inhibited NO-stimulated activity in cells. To our knowledge, this was the first report of a physical interaction between sGC and a thiol-redox protein. To characterize this interaction between sGC and PDI, we first identified peptide linkages between sGC and PDI, using a lysine cross-linking reagent and recently developed mass spectrometry analysis. Together with Flag-immunoprecipitation using sGC domain deletions, wild-type (WT and mutated PDI, regions of sGC involved in this interaction were identified. The observed data were further explored with computational modeling to gain insight into the interaction mechanism between sGC and oxidized PDI. Our results indicate that PDI interacts preferentially with the catalytic domain of sGC, thus providing a mechanism for PDI inhibition of sGC. A model in which PDI interacts with either the α or the β catalytic domain is proposed.

  18. Comparison between serum levels of carcinoembryonic antigen, sialic acid and phosphohexose isomerase in lung cancer

    International Nuclear Information System (INIS)

    Patel, P.S.; Raval, G.N.; Rawal, R.M.; Balar, D.B.; Patel, G.H.; Shah, P.M.; Patel, D.D.

    1995-01-01

    The identification and application of quantifiable tumor markers as adjuncts to clinical care is a story of both success and failure. The present study compared serum levels of carcinoembryogenic antigen (CEA) with total sialic acid/total protein (TSA/TP) ration and phosphohexose isomerase (PHI) in 192 untreated lung cancer patients as well as 80 age and sex matched controls (44 non-smokers). CEA values were significantly raised (p < 0.001) in smokers as compared to the non-smokers; whereas, TSA/TP and PHI values were comparable between the groups of the groups of the controls. All the bio-markers were significantly elevated (p < 0.00.1) in untreated lung cancer patients as compared to the controls. Receiver operating characteristic curve analysis revealed higher sensitivities of TSA/TP and PHI as compared to CEA at different specificity levels between 60% and 95%. Mean values of CEA, TSA/TP and PHI were higher in non-responders compared to the responders. The results indicate that TSA/TP and PHI are superior tumor markers than CEA for lung cancer patients. (author)

  19. Functional Role of the Disulfide Isomerase ERp57 in Axonal Regeneration.

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    Valentina Castillo

    Full Text Available ERp57 (also known as grp58 and PDIA3 is a protein disulfide isomerase that catalyzes disulfide bonds formation of glycoproteins as part of the calnexin and calreticulin cycle. ERp57 is markedly upregulated in most common neurodegenerative diseases downstream of the endoplasmic reticulum (ER stress response. Despite accumulating correlative evidence supporting a neuroprotective role of ERp57, the contribution of this foldase to the physiology of the nervous system remains unknown. Here we developed a transgenic mouse model that overexpresses ERp57 in the nervous system under the control of the prion promoter. We analyzed the susceptibility of ERp57 transgenic mice to undergo neurodegeneration. Unexpectedly, ERp57 overexpression did not affect dopaminergic neuron loss and striatal denervation after injection of a Parkinson's disease-inducing neurotoxin. In sharp contrast, ERp57 transgenic animals presented enhanced locomotor recovery after mechanical injury to the sciatic nerve. These protective effects were associated with enhanced myelin removal, macrophage infiltration and axonal regeneration. Our results suggest that ERp57 specifically contributes to peripheral nerve regeneration, whereas its activity is dispensable for the survival of a specific neuronal population of the central nervous system. These results demonstrate for the first time a functional role of a component of the ER proteostasis network in peripheral nerve regeneration.

  20. The unfolded protein response and the role of protein disulphide isomerase in neurodegeneration.

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    Emma ePerri

    2016-01-01

    Full Text Available The maintenance and regulation of proteostasis is a critical function for post-mitotic neurons and dysregulation of proteostasis is increasingly implicated in neurodegenerative diseases. Despite having different clinical manifestations, these disorders share similar pathology; an accumulation of misfolded proteins in neurons and subsequent disruption to cellular proteostasis. The endoplasmic reticulum (ER is an important component of proteostasis, and when the accumulation of misfolded proteins occurs within the ER, this disturbs ER homeostasis, giving rise to ER stress. This triggers the unfolded protein response (UPR, distinct signalling pathways that whilst initially protective, are pro-apoptotic if ER stress is prolonged. ER stress is increasingly implicated in neurodegenerative diseases, and emerging evidence highlights the complexity of the UPR in these disorders, with both protective and detrimental components being described. Protein Disulphide Isomerase (PDI is an ER chaperone induced during ER stress that is responsible for the formation of disulphide bonds in proteins. Whilst initially considered to be protective, recent studies have revealed unconventional roles for PDI in neurodegenerative diseases, distinct from its normal function in the UPR and the ER, although these mechanisms remain poorly defined. However specific aspects of PDI function may offer the potential to be exploited therapeutically in the future. This review will focus on the evidence linking ER stress and the UPR to neurodegenerative diseases, with particular emphasis on the emerging functions ascribed to PDI in these conditions.

  1. Styrene Oxide Isomerase of Rhodococcus opacus 1CP, a Highly Stable and Considerably Active Enzyme

    Science.gov (United States)

    Gröning, Janosch A. D.; Tischler, Dirk; Kaschabek, Stefan R.; Schlömann, Michael

    2012-01-01

    Styrene oxide isomerase (SOI) is involved in peripheral styrene catabolism of bacteria and converts styrene oxide to phenylacetaldehyde. Here, we report on the identification, enrichment, and biochemical characterization of a novel representative from the actinobacterium Rhodococcus opacus 1CP. The enzyme, which is strongly induced during growth on styrene, was shown to be membrane integrated, and a convenient procedure was developed to highly enrich the protein in active form from the wild-type host. A specific activity of about 370 U mg−1 represents the highest activity reported for this enzyme class so far. This, in combination with a wide pH and temperature tolerance, the independence from cofactors, and the ability to convert a spectrum of substituted styrene oxides, makes a biocatalytic application imaginable. First, semipreparative conversions were performed from which up to 760 μmol of the pure phenylacetaldehyde could be obtained from 130 U of enriched SOI. Product concentrations of up to 76 mM were achieved. However, due to the high chemical reactivity of the aldehyde function, SOI was shown to be the subject of an irreversible product inhibition. A half-life of 15 min was determined at a phenylacetaldehyde concentration of about 55 mM, indicating substantial limitations of applicability and the need to modify the process. PMID:22504818

  2. The crystal structure of a multifunctional protein: phosphoglucose isomerase/autocrine motility factor/neuroleukin.

    Science.gov (United States)

    Sun, Y J; Chou, C C; Chen, W S; Wu, R T; Meng, M; Hsiao, C D

    1999-05-11

    Phosphoglucose isomerase (PGI) plays a central role in both the glycolysis and the gluconeogenesis pathways. We present here the complete crystal structure of PGI from Bacillus stearothermophilus at 2.3-A resolution. We show that PGI has cell-motility-stimulating activity on mouse colon cancer cells similar to that of endogenous autocrine motility factor (AMF). PGI can also enhance neurite outgrowth on neuronal progenitor cells similar to that observed for neuroleukin. The results confirm that PGI is neuroleukin and AMF. PGI has an open twisted alpha/beta structural motif consisting of two globular domains and two protruding parts. Based on this substrate-free structure, together with the previously published biological, biochemical, and modeling results, we postulate a possible substrate-binding site that is located within the domains' interface for PGI and AMF. In addition, the structure provides evidence suggesting that the top part of the large domain together with one of the protruding loops might participate in inducing the neurotrophic activity.

  3. Perturbation of the dimer interface of triosephosphate isomerase and its effect on Trypanosoma cruzi.

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    Vanesa Olivares-Illana

    2007-10-01

    Full Text Available Chagas disease affects around 18 million people in the American continent. Unfortunately, there is no satisfactory treatment for the disease. The drugs currently used are not specific and exert serious toxic effects. Thus, there is an urgent need for drugs that are effective. Looking for molecules to eliminate the parasite, we have targeted a central enzyme of the glycolytic pathway: triosephosphate isomerase (TIM. The homodimeric enzyme is catalytically active only as a dimer. Because there are significant differences in the interface of the enzymes from the parasite and humans, we searched for small molecules that specifically disrupt contact between the two subunits of the enzyme from Trypanosoma cruzi but not those of TIM from Homo sapiens (HTIM, and tested if they kill the parasite.Dithiodianiline (DTDA at nanomolar concentrations completely inactivates recombinant TIM of T. cruzi (TcTIM. It also inactivated HTIM, but at concentrations around 400 times higher. DTDA was also tested on four TcTIM mutants with each of its four cysteines replaced with either valine or alanine. The sensitivity of the mutants to DTDA was markedly similar to that of the wild type. The crystal structure of the TcTIM soaked in DTDA at 2.15 A resolution, and the data on the mutants showed that inactivation resulted from alterations of the dimer interface. DTDA also prevented the growth of Escherichia coli cells transformed with TcTIM, had no effect on normal E. coli, and also killed T. cruzi epimastigotes in culture.By targeting on the dimer interface of oligomeric enzymes from parasites, it is possible to discover small molecules that selectively thwart the life of the parasite. Also, the conformational changes that DTDA induces in the dimer interface of the trypanosomal enzyme are unique and identify a region of the interface that could be targeted for drug discovery.

  4. Phycoerythrin-specific bilin lyase-isomerase controls blue-green chromatic acclimation in marine Synechococcus.

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    Shukla, Animesh; Biswas, Avijit; Blot, Nicolas; Partensky, Frédéric; Karty, Jonathan A; Hammad, Loubna A; Garczarek, Laurence; Gutu, Andrian; Schluchter, Wendy M; Kehoe, David M

    2012-12-04

    The marine cyanobacterium Synechococcus is the second most abundant phytoplanktonic organism in the world's oceans. The ubiquity of this genus is in large part due to its use of a diverse set of photosynthetic light-harvesting pigments called phycobiliproteins, which allow it to efficiently exploit a wide range of light colors. Here we uncover a pivotal molecular mechanism underpinning a widespread response among marine Synechococcus cells known as "type IV chromatic acclimation" (CA4). During this process, the pigmentation of the two main phycobiliproteins of this organism, phycoerythrins I and II, is reversibly modified to match changes in the ambient light color so as to maximize photon capture for photosynthesis. CA4 involves the replacement of three molecules of the green light-absorbing chromophore phycoerythrobilin with an equivalent number of the blue light-absorbing chromophore phycourobilin when cells are shifted from green to blue light, and the reverse after a shift from blue to green light. We have identified and characterized MpeZ, an enzyme critical for CA4 in marine Synechococcus. MpeZ attaches phycoerythrobilin to cysteine-83 of the α-subunit of phycoerythrin II and isomerizes it to phycourobilin. mpeZ RNA is six times more abundant in blue light, suggesting that its proper regulation is critical for CA4. Furthermore, mpeZ mutants fail to normally acclimate in blue light. These findings provide insights into the molecular mechanisms controlling an ecologically important photosynthetic process and identify a unique class of phycoerythrin lyase/isomerases, which will further expand the already widespread use of phycoerythrin in biotechnology and cell biology applications.

  5. A chalcone isomerase-like protein enhances flavonoid production and flower pigmentation.

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    Morita, Yasumasa; Takagi, Kyoko; Fukuchi-Mizutani, Masako; Ishiguro, Kanako; Tanaka, Yoshikazu; Nitasaka, Eiji; Nakayama, Masayoshi; Saito, Norio; Kagami, Takashi; Hoshino, Atsushi; Iida, Shigeru

    2014-04-01

    Flavonoids are major pigments in plants, and their biosynthetic pathway is one of the best-studied metabolic pathways. Here we have identified three mutations within a gene that result in pale-colored flowers in the Japanese morning glory (Ipomoea nil). As the mutations lead to a reduction of the colorless flavonoid compound flavonol as well as of anthocyanins in the flower petal, the identified gene was designated enhancer of flavonoid production (EFP). EFP encodes a chalcone isomerase (CHI)-related protein classified as a type IV CHI protein. CHI is the second committed enzyme of the flavonoid biosynthetic pathway, but type IV CHI proteins are thought to lack CHI enzymatic activity, and their functions remain unknown. The spatio-temporal expression of EFP and structural genes encoding enzymes that produce flavonoids is very similar. Expression of both EFP and the structural genes is coordinately promoted by genes encoding R2R3-MYB and WD40 family proteins. The EFP gene is widely distributed in land plants, and RNAi knockdown mutants of the EFP homologs in petunia (Petunia hybrida) and torenia (Torenia hybrida) had pale-colored flowers and low amounts of anthocyanins. The flavonol and flavone contents in the knockdown petunia and torenia flowers, respectively, were also significantly decreased, suggesting that the EFP protein contributes in early step(s) of the flavonoid biosynthetic pathway to ensure production of flavonoid compounds. From these results, we conclude that EFP is an enhancer of flavonoid production and flower pigmentation, and its function is conserved among diverse land plant species. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  6. Structural effects of protein aging: terminal marking by deamidation in human triosephosphate isomerase.

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    Ignacio de la Mora-de la Mora

    Full Text Available Deamidation, the loss of the ammonium group of asparagine and glutamine to form aspartic and glutamic acid, is one of the most commonly occurring post-translational modifications in proteins. Since deamidation rates are encoded in the protein structure, it has been proposed that they can serve as molecular clocks for the timing of biological processes such as protein turnover, development and aging. Despite the importance of this process, there is a lack of detailed structural information explaining the effects of deamidation on the structure of proteins. Here, we studied the effects of deamidation on human triosephosphate isomerase (HsTIM, an enzyme for which deamidation of N15 and N71 has been long recognized as the signal for terminal marking of the protein. Deamidation was mimicked by site directed mutagenesis; thus, three mutants of HsTIM (N15D, N71D and N15D/N71D were characterized. The results show that the N71D mutant resembles, structurally and functionally, the wild type enzyme. In contrast, the N15D mutant displays all the detrimental effects related to deamidation. The N15D/N71D mutant shows only minor additional effects when compared with the N15D mutation, supporting that deamidation of N71 induces negligible effects. The crystal structures show that, in contrast to the N71D mutant, where minimal alterations are observed, the N15D mutation forms new interactions that perturb the structure of loop 1 and loop 3, both critical components of the catalytic site and the interface of HsTIM. Based on a phylogenetic analysis of TIM sequences, we propose the conservation of this mechanism for mammalian TIMs.

  7. Protein disulfide isomerases in the endoplasmic reticulum promote anchorage-independent growth of breast cancer cells.

    Science.gov (United States)

    Wise, Randi; Duhachek-Muggy, Sara; Qi, Yue; Zolkiewski, Michal; Zolkiewska, Anna

    2016-06-01

    Metastatic breast cancer cells are exposed to stress of detachment from the extracellular matrix (ECM). Cultured breast cancer cells that survive this stress and are capable of anchorage-independent proliferation form mammospheres. The purpose of this study was to explore a link between mammosphere growth, ECM gene expression, and the protein quality control system in the endoplasmic reticulum (ER). We compared the mRNA and protein levels of ER folding factors in SUM159PT and MCF10DCIS.com breast cancer cells grown as mammospheres versus adherent conditions. Publicly available gene expression data for mammospheres formed by primary breast cancer cells and for circulating tumor cells (CTCs) were analyzed to assess the status of ECM/ER folding factor genes in clinically relevant samples. Knock-down of selected protein disulfide isomerase (PDI) family members was performed to examine their roles in SUM159PT mammosphere growth. We found that cells grown as mammospheres had elevated expression of ECM genes and ER folding quality control genes. CTC gene expression data for an index patient indicated that upregulation of ECM and ER folding factor genes occurred at the time of acquired therapy resistance and disease progression. Knock-down of PDI, ERp44, or ERp57, three members of the PDI family with elevated protein levels in mammospheres, in SUM159PT cells partially inhibited the mammosphere growth. Thus, breast cancer cell survival and growth under detachment conditions require enhanced assistance of the ER protein folding machinery. Targeting ER folding factors, in particular members of the PDI family, may improve the therapeutic outcomes in metastatic breast cancer.

  8. Crystal structure of glucose isomerase in complex with xylitol inhibitor in one metal binding mode.

    Science.gov (United States)

    Bae, Ji-Eun; Kim, In Jung; Nam, Ki Hyun

    2017-11-04

    Glucose isomerase (GI) is an intramolecular oxidoreductase that interconverts aldoses and ketoses. These characteristics are widely used in the food, detergent, and pharmaceutical industries. In order to obtain an efficient GI, identification of novel GI genes and substrate binding/inhibition have been studied. Xylitol is a well-known inhibitor of GI. In Streptomyces rubiginosus, two crystal structures have been reported for GI in complex with xylitol inhibitor. However, a structural comparison showed that xylitol can have variable conformation at the substrate binding site, e.g., a nonspecific binding mode. In this study, we report the crystal structure of S. rubiginosus GI in a complex with xylitol and glycerol. Our crystal structure showed one metal binding mode in GI, which we presumed to represent the inactive form of the GI. The metal ion was found only at the M1 site, which was involved in substrate binding, and was not present at the M2 site, which was involved in catalytic function. The O 2 and O 4 atoms of xylitol molecules contributed to the stable octahedral coordination of the metal in M1. Although there was no metal at the M2 site, no large conformational change was observed for the conserved residues coordinating M2. Our structural analysis showed that the metal at the M2 site was not important when a xylitol inhibitor was bound to the M1 site in GI. Thus, these findings provided important information for elucidation or engineering of GI functions. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. The disulfide isomerase ERp57 is required for fibrin deposition in vivo.

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    Zhou, J; Wu, Y; Wang, L; Rauova, L; Hayes, V M; Poncz, M; Essex, D W

    2014-11-01

    ERp57 is required for platelet function; however, whether ERp57 contributes to fibrin generation is unknown. Using an inhibitory anti-ERp57 antibody (mAb1), Pf4-Cre/ERp57(fl/fl) mice, Tie2-Cre/ERp57(fl/fl) mice, and mutants of ERp57, we analyzed the function of ERp57 in laser-induced thrombosis. Fibrin deposition was decreased in Pf4-Cre/ERp57(fl/fl) mice, consistent with a role for platelet ERp57 in fibrin generation. Fibrin deposition was further decreased with infusion of mAb1 and in Tie2-Cre/ERp57(fl/fl) mice, consistent with endothelial cells also contributing to fibrin deposition. Infusion of eptibifatide inhibited platelet and fibrin deposition, confirming a role for platelets in fibrin deposition. Infusion of recombinant ERp57 corrected the defect in fibrin deposition but not platelet accumulation, suggesting a direct effect of ERp57 on coagulation. mAb1 inhibited thrombin generation in vitro, consistent with a requirement for ERp57 in coagulation. Platelet accumulation was decreased to similar extents in Pf4-Cre/ERp57(fl/fl) mice, Tie2-Cre/ERp57(fl/fl) mice and normal mice infused with mAb1. Infusion of completely inactivated ERp57 or ERp57 with a non-functional second active site inhibited fibrin deposition and platelet accumulation, indicating that the isomerase activity of the second active site is required for these processes. ERp57 regulates thrombosis via multiple targets. © 2014 International Society on Thrombosis and Haemostasis.

  10. Attachment and entry of Chlamydia have distinct requirements for host protein disulfide isomerase.

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    Stephanie Abromaitis

    2009-04-01

    Full Text Available Chlamydia is an obligate intracellular pathogen that causes a wide range of diseases in humans. Attachment and entry are key processes in infectivity and subsequent pathogenesis of Chlamydia, yet the mechanisms governing these interactions are unknown. It was recently shown that a cell line, CHO6, that is resistant to attachment, and thus infectivity, of multiple Chlamydia species has a defect in protein disulfide isomerase (PDI N-terminal signal sequence processing. Ectopic expression of PDI in CHO6 cells led to restoration of Chlamydia attachment and infectivity; however, the mechanism leading to this recovery was not ascertained. To advance our understanding of the role of PDI in Chlamydia infection, we used RNA interference to establish that cellular PDI is essential for bacterial attachment to cells, making PDI the only host protein identified as necessary for attachment of multiple species of Chlamydia. Genetic complementation and PDI-specific inhibitors were used to determine that cell surface PDI enzymatic activity is required for bacterial entry into cells, but enzymatic function was not required for bacterial attachment. We further determined that it is a PDI-mediated reduction at the cell surface that triggers bacterial uptake. While PDI is necessary for Chlamydia attachment to cells, the bacteria do not appear to utilize plasma membrane-associated PDI as a receptor, suggesting that Chlamydia binds a cell surface protein that requires structural association with PDI. Our findings demonstrate that PDI has two essential and independent roles in the process of chlamydial infectivity: it is structurally required for chlamydial attachment, and the thiol-mediated oxido-reductive function of PDI is necessary for entry.

  11. Role of Loop-Clamping Side Chains in Catalysis by Triosephosphate Isomerase.

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    Zhai, Xiang; Amyes, Tina L; Richard, John P

    2015-12-09

    The side chains of Y208 and S211 from loop 7 of triosephosphate isomerase (TIM) form hydrogen bonds to backbone amides and carbonyls from loop 6 to stabilize the caged enzyme-substrate complex. The effect of seven mutations [Y208T, Y208S, Y208A, Y208F, S211G, S211A, Y208T/S211G] on the kinetic parameters for TIM catalyzed reactions of the whole substrates dihydroxyacetone phosphate and d-glyceraldehyde 3-phosphate [(k(cat)/K(m))(GAP) and (k(cat)/K(m))DHAP] and of the substrate pieces glycolaldehyde and phosphite dianion (k(cat)/K(HPi)K(GA)) are reported. The linear logarithmic correlation between these kinetic parameters, with slope of 1.04 ± 0.03, shows that most mutations of TIM result in an identical change in the activation barriers for the catalyzed reactions of whole substrate and substrate pieces, so that the transition states for these reactions are stabilized by similar interactions with the protein catalyst. The second linear logarithmic correlation [slope = 0.53 ± 0.16] between k(cat) for isomerization of GAP and K(d)(⧧) for phosphite dianion binding to the transition state for wildtype and many mutant TIM-catalyzed reactions of substrate pieces shows that ca. 50% of the wildtype TIM dianion binding energy, eliminated by these mutations, is expressed at the wildtype Michaelis complex, and ca. 50% is only expressed at the wildtype transition state. Negative deviations from this correlation are observed when the mutation results in a decrease in enzyme reactivity at the catalytic site. The main effect of Y208T, Y208S, and Y208A mutations is to cause a reduction in the total intrinsic dianion binding energy, but the effect of Y208F extends to the catalytic site.

  12. Substrate-Induced Dimerization of Engineered Monomeric Variants of Triosephosphate Isomerase from Trichomonas vaginalis.

    Directory of Open Access Journals (Sweden)

    Samuel Lara-Gonzalez

    Full Text Available The dimeric nature of triosephosphate isomerases (TIMs is maintained by an extensive surface area interface of more than 1600 Å2. TIMs from Trichomonas vaginalis (TvTIM are held in their dimeric state by two mechanisms: a ball and socket interaction of residue 45 of one subunit that fits into the hydrophobic pocket of the complementary subunit and by swapping of loop 3 between subunits. TvTIMs differ from other TIMs in their unfolding energetics. In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer. Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model. In contrast to other monomeric TIMs, monomeric variants of TvTIM1 are stable and unexpectedly one of them (I45A is only 29-fold less active than wild-type TvTIM1. The high enzymatic activity of monomeric TvTIMs contrast with the marginal catalytic activity of diverse monomeric TIMs variants. The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.

  13. Isolation of xylose isomerases by sequence- and function-based screening from a soil metagenomic library

    Directory of Open Access Journals (Sweden)

    Parachin Nádia

    2011-05-01

    Full Text Available Abstract Background Xylose isomerase (XI catalyses the isomerisation of xylose to xylulose in bacteria and some fungi. Currently, only a limited number of XI genes have been functionally expressed in Saccharomyces cerevisiae, the microorganism of choice for lignocellulosic ethanol production. The objective of the present study was to search for novel XI genes in the vastly diverse microbial habitat present in soil. As the exploitation of microbial diversity is impaired by the ability to cultivate soil microorganisms under standard laboratory conditions, a metagenomic approach, consisting of total DNA extraction from a given environment followed by cloning of DNA into suitable vectors, was undertaken. Results A soil metagenomic library was constructed and two screening methods based on protein sequence similarity and enzyme activity were investigated to isolate novel XI encoding genes. These two screening approaches identified the xym1 and xym2 genes, respectively. Sequence and phylogenetic analyses revealed that the genes shared 67% similarity and belonged to different bacterial groups. When xym1 and xym2 were overexpressed in a xylA-deficient Escherichia coli strain, similar growth rates to those in which the Piromyces XI gene was expressed were obtained. However, expression in S. cerevisiae resulted in only one-fourth the growth rate of that obtained for the strain expressing the Piromyces XI gene. Conclusions For the first time, the screening of a soil metagenomic library in E. coli resulted in the successful isolation of two active XIs. However, the discrepancy between XI enzyme performance in E. coli and S. cerevisiae suggests that future screening for XI activity from soil should be pursued directly using yeast as a host.

  14. A dual inhibitor against prolyl isomerase Pin1 and cyclophilin discovered by a novel real-time fluorescence detection method

    International Nuclear Information System (INIS)

    Mori, Tadashi; Hidaka, Masafumi; Lin, Yi-Chin; Yoshizawa, Ibuki; Okabe, Takayoshi; Egashira, Shinichiro; Kojima, Hirotatsu; Nagano, Tetsuo; Koketsu, Mamoru; Takamiya, Mari; Uchida, Takafumi

    2011-01-01

    Research highlights: → A Pin1 (prolyl isomerase) inhibitor, TME-001, has been discovered by using a new established high-throughput screening method. → The TME-001 showed a cell-active inhibition with lower cytotoxic effect than known Pin1 inhibitors. → Kinetic analyses revealed that the TME-001 is the first compound that exhibits dual inhibition of Pin1 and another type of prolyl isomerase, cyclophilin. → Thus, similarities of structure and reaction mechanism between Pin1 and cyclophilin are proposed. -- Abstract: Pin1, a peptidyl prolyl cis/trans isomerase (PPIase), is a potential target molecule for cancer, infectious disease, and Alzheimer's disease. We established a high-throughput screening method for Pin1 inhibitors, which employs a real-time fluorescence detector. This screening method identified 66 compounds that inhibit Pin1 out of 9756 compounds from structurally diverse chemical libraries. Further evaluations of surface plasmon resonance methods and a cell proliferation assay were performed. We discovered a cell-active inhibitor, TME-001 (2-(3-chloro-4-fluoro-phenyl)-isothiazol-3-one). Surprisingly, kinetic analyses revealed that TME-001 is the first compound that exhibits dual inhibition of Pin1 (IC 50 = 6.1 μM) and cyclophilin, another type of PPIase, (IC 50 = 13.7 μM). This compound does not inhibit FKBP. This finding suggests the existence of similarities of structure and reaction mechanism between Pin1 and cyclophilin, and may lead to a more complete understanding of the active sites of PPIases.

  15. Kinetic analysis of the mechanism and specificity of protein-disulfide isomerase using fluorescence-quenched peptides

    DEFF Research Database (Denmark)

    Westphal, V; Spetzler, J C; Meldal, M

    1998-01-01

    Protein-disulfide isomerase (PDI) is an abundant folding catalyst in the endoplasmic reticulum of eukaryotic cells. PDI introduces disulfide bonds into newly synthesized proteins and catalyzes disulfide bond isomerizations. We have synthesized a library of disulfide-linked fluorescence......-quenched peptides, individually linked to resin beads, for two purposes: 1) to probe PDI specificity, and 2) to identify simple, sensitive peptide substrates of PDI. Using this library, beads that became rapidly fluorescent by reduction by human PDI were selected. Amino acid sequencing of the bead-linked peptides...

  16. The Expression of Millettia pinnata Chalcone Isomerase in Saccharomyces cerevisiae Salt-Sensitive Mutants Enhances Salt-Tolerance

    OpenAIRE

    Wang, Hui; Hu, Tangjin; Huang, Jianzi; Lu, Xiang; Huang, Baiqu; Zheng, Yizhi

    2013-01-01

    The present study demonstrates a new Millettia pinnata chalcone isomerase (MpCHI) whose transcription level in leaf was confirmed to be enhanced after being treated by seawater or NaCl (500 mM) via transcriptome sequencing and Real-Time Quantitative Reverse Transcription PCR (QRT-PCR) analyses. Its full length cDNA (666 bp) was obtained by 3'-end and 5'-end Rapid Amplification of cDNA Ends (RACE). The analysis via NCBI BLAST indicates that both aminoacid sequence and nucleotide sequ...

  17. Direct production of D-arabinose from D-xylose by a coupling reaction using D-xylose isomerase, D-tagatose 3-epimerase and D-arabinose isomerase.

    Science.gov (United States)

    Sultana, Ishrat; Mizanur, Rahman Md; Takeshita, Kei; Takada, Goro; Izumori, Ken

    2003-01-01

    Klebsiella pneumoniae 40bXX, a mutant strain that constitutively produces D-arabinose isomerase (D-AI), was isolated through a series of repeated subcultures from the parent strain on a mineral salt medium supplemented with L-Xylose as the sole carbon source. D-AI could be efficiently immobilized on chitopearl beads. The optimum temperature for the activity of the immobilized enzyme was 40 degrees C and the enzyme was stable up to 50 degrees C. The D-Al was active at pH 10.0 and was stable in the range of pH 6.0-11.0. The enzyme required manganese ions for maximum activity. Three immobilized enzymes, D-xylose isomerase (D-XI), D-tagatose 3-epimerase (D-TE and D-AI were used for the preparation of D-arabinose from D-xylose in a coupling reaction. After completion of the reaction, degradation of D-xylulose was carried out by Saccharomyces cerevisiae. The reaction mixture containing D-Xylose, D-ribulose and the product was then separated by ion exchange column chromatography. After crystallization, the product was checked by HPLC, IR spectroscopy, NMR spectroscopy and optical rotation measurements. Finally, 2.0 g of D-arabinose could be obtained from 5 g of the substrate.

  18. Transmutation of human glutathione transferase A2-2 with peroxidase activity into an efficient steroid isomerase.

    Science.gov (United States)

    Pettersson, Par L; Johansson, Ann-Sofie; Mannervik, Bengt

    2002-08-16

    A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.

  19. Characterization of the triple-component linoleic acid isomerase in Lactobacillus plantarum ZS2058 by genetic manipulation.

    Science.gov (United States)

    Yang, B; Qi, H; Gu, Z; Zhang, H; Chen, W; Chen, H; Chen, Y Q

    2017-11-01

    To assess the mechanism for conjugated linoleic acid (CLA) production in Lactobacillus plantarum ZS2058. CLA has attracted great interests for decades due to its health-associated benefits including anticancer, anti-atherogenic, anti-obesity and modulation of the immune system. A number of microbial CLA producers were widely reported including lactic acid bacteria. Lactobacillus plantarum ZS2058, an isolate from Chinese traditional fermented food, could convert LA to CLA with various intermediates. To characterize the genetic determinants for generating CLA, a cre-lox-based system was utilized to delete the genes encoding myosin cross-reactive antigen (MCRA), short-chain dehydrogenase/oxidoreductase (DH) and acetoacetate decarboxylase (DC) in Lact. plantarum ZS2058, respectively. Neither intermediate was detected in the corresponding gene deletion mutant. Meanwhile all those mutants could recover the ability to convert linoleic acid to CLA when the corresponding gene was completed. The results indicated that CLA production was a multiple-step reaction catalysed by triple-component linoleate isomerase system encoded by mcra, dh and dc. Multicomponent linoleic acid isomerase provided important results for illustration unique mechanism for CLA production in Lact. plantarum ZS2058. Lactobacilli with CLA production ability offer novel opportunities for functional food development. © 2017 The Society for Applied Microbiology.

  20. Construction of phosphomannose isomerase (PMI) transformation vectors and evaluation of the effectiveness of vectors in tobacco (Nicotiana tabacum L).

    Science.gov (United States)

    Bahariah, Bohari; Parveez, Ghulam Kadir Ahmad; Masani, Mat Yunus Abdul; Khalid, Norzulaani

    2012-01-01

    Phosphomannose isomerase (pmi) gene isolated from Escherichia coli allows transgenic plants carrying it to convert mannose-6- phosphate (from mannose), a carbon source that could not be naturally utilized by plants into fructose-6-phosphate which can be utilized by plants as a carbon source. This conversion ability provides energy source to allow the transformed cells to survive on the medium containing mannose. In this study, four transformation vectors carrying the pmi gene alone or in combination with the β-glucuronidase (gusA) gene were constructed and driven by either the maize ubiquitin (Ubi1) or the cauliflower mosaic virus (CaMV35S) promoter. Restriction digestion, PCR amplification and sequencing were carried out to ensure sequence integrity and orientation. Tobacco was used as a model system to study the effectiveness of the constructs and selection system. PMI11G and pMI3G, which carry gusA gene, were used to study the gene transient expression in tobacco. PMI3 construct, which only carries the pmi gene driven by CaMV35S promoter, was stably transformed into tobacco using biolistics after selection on 30 g 1(-1) mannose without sucrose. Transgenic plants were verified using PCR analysis. PMI/pmi - Phosphomannose isomerase, Ubi1 - Maize ubiquitin promoter, CaMV35S - Cauliflower mosaic virus 35S promoter, gusA - β-glucuronidase GUS reporter gene.

  1. Human cyclophilin B: A second cyclophilin gene encodes a peptidyl-prolyl isomerase with a signal sequence

    International Nuclear Information System (INIS)

    Price, E.R.; Zydowsky, L.D.; Jin, Mingjie; Baker, C.H.; McKeon, F.D.; Walsh, C.T.

    1991-01-01

    The authors report the cloning and characterization of a cDNA encoding a second human cyclosporin A-binding protein (hCyPB). Homology analyses reveal that hCyPB is a member of the cyclophilin B (CyPB) family, which includes yeast CyPB, Drosophila nina A, and rat cyclophilin-like protein. This family is distinguished from the cyclophilin A (CyPA) family by the presence of endoplasmic reticulum (ER)-directed signal sequences. hCyPB has a hydrophobic leader sequence not found in hCyPA, and its first 25 amino acids are removed upon expression in Escherichia coli. Moreover, they show that hCyPB is a peptidyl-prolyl cis-trans isomerase which can be inhibited by cyclosporin A. These observations suggest that other members of the CyPB family will have similar enzymatic properties. Sequence comparisons of the CyPB proteins show a central, 165-amino acid peptidyl-prolyl isomerase and cyclosprorin A-binding domain, flanked by variable N-terminal and C-terminal domains. These two variable regions may impart compartmental specificity and regulation to this family of cyclophilin proteins containing the conserved core domain. Northern blot analyses show that hCyPB mRNA is expressed in the Jurkat T-cell line, consistent with its possible target role in cyclosporin A-mediated immunosuppression

  2. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of the mannose 6-phosphate isomerase from Salmonella typhimurium

    Energy Technology Data Exchange (ETDEWEB)

    Gowda, Giri; Sagurthi, Someswar Rao [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 (India); Savithri, H. S. [Department of Biochemistry, Indian Institute of Science, Bangalore 560 012 (India); Murthy, M. R. N., E-mail: mrn@mbu.iisc.ernet.in [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 (India)

    2008-02-01

    The cloning, expression, purification, crystallization and preliminary X-ray crystallographic studies of mannose 6-phosphate isomerase from S. typhimurium are reported. Mannose 6-phosphate isomerase (MPI; EC 5.3.1.8) catalyzes the reversible isomerization of d-mannose 6-phosphate (M6P) and d-fructose 6-phosphate (F6P). In the eukaryotes and prokaryotes investigated to date, the enzyme has been reported to play a crucial role in d-mannose metabolism and supply of the activated mannose donor guanosine diphosphate d-mannose (GDP-d-mannose). In the present study, MPI was cloned from Salmonella typhimurium, overexpressed in Escherichia coli and purified using Ni–NTA affinity column chromatography. Purified MPI crystallized in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 36.03, b = 92.2, c = 111.01 Å. A data set extending to 1.66 Å resolution was collected with 98.8% completeness using an image-plate detector system mounted on a rotating-anode X-ray generator. The asymmetric unit of the crystal cell was compatible with the presence of a monomer of MPI. A preliminary structure solution of the enzyme has been obtained by molecular replacement using Candida albicans MPI as the phasing model and the program Phaser. Further refinement and model building are in progress.

  3. Insights into evolution in Andean Polystichum (Dryopteridaceae) from expanded understanding of the cytosolic phosphoglucose isomerase gene.

    Science.gov (United States)

    Lyons, Brendan M; McHenry, Monique A; Barrington, David S

    2017-07-01

    Cytosolic phosphoglucose isomerase (pgiC) is an enzyme essential to glycolysis found universally in eukaryotes, but broad understanding of variation in the gene coding for pgiC is lacking for ferns. We used a substantially expanded representation of the gene for Andean species of the fern genus Polystichum to characterize pgiC in ferns relative to angiosperms, insects, and an amoebozoan; assess the impact of selection versus neutral evolutionary processes on pgiC; and explore evolutionary relationships of selected Andean species. The dataset of complete sequences comprised nine accessions representing seven species and one hybrid from the Andes and Serra do Mar. The aligned sequences of the full data set comprised 3376 base pairs (70% of the entire gene) including 17 exons and 15 introns from two central areas of the gene. The exons are highly conserved relative to angiosperms and retain substantial homology to insect pgiC, but intron length and structure are unique to the ferns. Average intron size is similar to angiosperms; intron number and location in insects are unlike those of the plants we considered. The introns included an array of indels and, in intron 7, an extensive microsatellite array with potential utility in analyzing population-level histories. Bayesian and maximum-parsimony analysis of 129 variable nucleotides in the Andean polystichums revealed that 59 (1.7% of the 3376 total) were phylogenetically informative; most of these united sister accessions. The phylogenetic trees for the Andean polystichums were incongruent with previously published cpDNA trees for the same taxa, likely the result of rapid evolutionary change in the introns and contrasting stability in the exons. The exons code a total of seven amino-acid substitutions. Comparison of non-synonymous to synonymous substitutions did not suggest that the pgiC gene is under selection in the Andes. Variation in pgiC including two additional accessions represented by incomplete sequences

  4. Molecular characterization and analysis of a novel protein disulfide isomerase-like protein of Eimeria tenella.

    Science.gov (United States)

    Han, Hongyu; Dong, Hui; Zhu, Shunhai; Zhao, Qiping; Jiang, Lianlian; Wang, Yange; Li, Liujia; Wu, Youlin; Huang, Bing

    2014-01-01

    Protein disulfide isomerase (PDI) and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE) according to the expressed sequence tag (EST). The EtPDIL cDNA contained 1129 nucleotides encoding 216 amino acids. The deduced EtPDIL protein belonged to thioredoxin-like superfamily and had a single predicted thioredoxin domain with a non-classical thioredoxin-like motif (SXXC). BLAST analysis showed that the EtPDIL protein was 55-59% identical to PDI-like proteins of other apicomplexan parasites. The transcript and protein levels of EtPDIL at different development stages were investigated by real-time quantitative PCR and western blot. The messenger RNA and protein levels of EtPDIL were higher in sporulated oocysts than in unsporulated oocysts, sporozoites or merozoites. Protein expression was barely detectable in unsporulated oocysts. Western blots showed that rabbit antiserum against recombinant EtPDIL recognized only a native 24 kDa protein from parasites. Immunolocalization with EtPDIL antibody showed that EtPDIL had a disperse distribution in the cytoplasm of whole sporozoites and merozoites. After sporozoites were incubated in complete medium, EtPDIL protein concentrated at the anterior of the sporozoites and appeared on the surface of parasites. Specific staining was more intense and mainly located on the parasite surface after merozoites released from mature schizonts invaded DF-1 cells. After development of parasites in DF-1 cells, staining intensified in trophozoites, immature schizonts and mature schizonts. Antibody inhibition of EtPDIL function reduced the ability of E. tenella to invade DF-1 cells. These results

  5. Structural Basis for Redox Regulation of Cytoplasmic and Chloroplastic Triosephosphate Isomerases from Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Laura Margarita López-Castillo

    2016-12-01

    Full Text Available In plants triosephosphate isomerase (TPI interconverts glyceraldehyde 3-phosphate (G3P and dihydroxyacetone phosphate (DHAP during glycolysis, gluconeogenesis, and the Calvin-Benson cycle. The nuclear genome of land plants encodes two tpi genes, one gene product is located in the cytoplasm and the other is imported into the chloroplast. Herein we report the crystal structures of the TPIs from the vascular plant Arabidopsis thaliana (AtTPIs and address their enzymatic modulation by redox agents. Cytoplasmic TPI (cTPI and chloroplast TPI (pdTPI share more than 60% amino acid identity and assemble as (β-α8 dimers with high structural homology. cTPI and pdTPI harbor two and one accessible thiol groups per monomer respectively. cTPI and pdTPI present a cysteine at an equivalent structural position (C13 and C15 respectively and cTPI also contains a specific solvent accessible cysteine at residue 218 (cTPI-C218. Site directed mutagenesis of residues pdTPI-C15, cTPI-C13 and cTPI-C218 to serine substantially decreases enzymatic activity, indicating that the structural integrity of these cysteines is necessary for catalysis. AtTPIs exhibit differential responses to oxidative agents, cTPI is susceptible to oxidative agents such as diamide and H2O2, whereas pdTPI is resistant to inhibition. Incubation of AtTPIs with the sulfhydryl conjugating reagents methylmethane thiosulfonate (MMTS and glutathione inhibits enzymatic activity. However, the concentration necessary to inhibit pdTPI is at least two orders of magnitude higher than the concentration needed to inhibit cTPI. Western-blot analysis indicates that residues cTPI-C13, cTPI-C218, and pdTPI-C15 conjugate with glutathione. In summary, our data indicate that AtTPIs could be redox regulated by the derivatization of specific AtTPI cysteines (cTPI-C13 and pdTPI-C15 and cTPI-C218. Since AtTPIs have evolved by gene duplication, the higher resistance of pdTPI to redox agents may be an adaptive consequence to

  6. Molecular characterization and analysis of a novel protein disulfide isomerase-like protein of Eimeria tenella.

    Directory of Open Access Journals (Sweden)

    Hongyu Han

    Full Text Available Protein disulfide isomerase (PDI and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE according to the expressed sequence tag (EST. The EtPDIL cDNA contained 1129 nucleotides encoding 216 amino acids. The deduced EtPDIL protein belonged to thioredoxin-like superfamily and had a single predicted thioredoxin domain with a non-classical thioredoxin-like motif (SXXC. BLAST analysis showed that the EtPDIL protein was 55-59% identical to PDI-like proteins of other apicomplexan parasites. The transcript and protein levels of EtPDIL at different development stages were investigated by real-time quantitative PCR and western blot. The messenger RNA and protein levels of EtPDIL were higher in sporulated oocysts than in unsporulated oocysts, sporozoites or merozoites. Protein expression was barely detectable in unsporulated oocysts. Western blots showed that rabbit antiserum against recombinant EtPDIL recognized only a native 24 kDa protein from parasites. Immunolocalization with EtPDIL antibody showed that EtPDIL had a disperse distribution in the cytoplasm of whole sporozoites and merozoites. After sporozoites were incubated in complete medium, EtPDIL protein concentrated at the anterior of the sporozoites and appeared on the surface of parasites. Specific staining was more intense and mainly located on the parasite surface after merozoites released from mature schizonts invaded DF-1 cells. After development of parasites in DF-1 cells, staining intensified in trophozoites, immature schizonts and mature schizonts. Antibody inhibition of EtPDIL function reduced the ability of E. tenella to invade DF-1 cells

  7. Role of hydrogen bonds in the reaction mechanism of chalcone isomerase.

    Science.gov (United States)

    Jez, Joseph M; Bowman, Marianne E; Noel, Joseph P

    2002-04-23

    In flavonoid, isoflavonoid, and anthocyanin biosynthesis, chalcone isomerase (CHI) catalyzes the intramolecular cyclization of chalcones into (S)-flavanones with a second-order rate constant that approaches the diffusion-controlled limit. The three-dimensional structures of alfalfa CHI complexed with different flavanones indicate that two sets of hydrogen bonds may possess critical roles in catalysis. The first set of interactions includes two conserved amino acids (Thr48 and Tyr106) that mediate a hydrogen bond network with two active site water molecules. The second set of hydrogen bonds occurs between the flavanone 7-hydroxyl group and two active site residues (Asn113 and Thr190). Comparison of the steady-state kinetic parameters of wild-type and mutant CHIs demonstrates that efficient cyclization of various chalcones into their respective flavanones requires both sets of contacts. For example, the T48A, T48S, Y106F, N113A, and T190A mutants exhibit 1550-, 3-, 30-, 7-, and 6-fold reductions in k(cat) and 2-3-fold changes in K(m) with 4,2',4'-trihydroxychalcone as a substrate. Kinetic comparisons of the pH-dependence of the reactions catalyzed by wild-type and mutant enzymes indicate that the active site hydrogen bonds contributed by these four residues do not significantly alter the pK(a) of the intramolecular cyclization reaction. Determinations of solvent kinetic isotope and solvent viscosity effects for wild-type and mutant enzymes reveal a change from a diffusion-controlled reaction to one limited by chemistry in the T48A and Y106F mutants. The X-ray crystal structures of the T48A and Y106F mutants support the assertion that the observed kinetic effects result from the loss of key hydrogen bonds at the CHI active site. Our results are consistent with a reaction mechanism for CHI in which Thr48 polarizes the ketone of the substrate and Tyr106 stabilizes a key catalytic water molecule. Hydrogen bonds contributed by Asn113 and Thr190 provide additional

  8. Identification and comparative analysis of sixteen fungal peptidyl-prolyl cis/trans isomerase repertoires

    Directory of Open Access Journals (Sweden)

    Pemberton Trevor J

    2006-09-01

    Full Text Available Abstract Background The peptidyl-prolyl cis/trans isomerase (PPIase class of proteins is present in all known eukaryotes, prokaryotes, and archaea, and it is comprised of three member families that share the ability to catalyze the cis/trans isomerisation of a prolyl bond. Some fungi have been used as model systems to investigate the role of PPIases within the cell, however how representative these repertoires are of other fungi or humans has not been fully investigated. Results PPIase numbers within these fungal repertoires appears associated with genome size and orthology between repertoires was found to be low. Phylogenetic analysis showed the single-domain FKBPs to evolve prior to the multi-domain FKBPs, whereas the multi-domain cyclophilins appear to evolve throughout cyclophilin evolution. A comparison of their known functions has identified, besides a common role within protein folding, multiple roles for the cyclophilins within pre-mRNA splicing and cellular signalling, and within transcription and cell cycle regulation for the parvulins. However, no such commonality was found with the FKBPs. Twelve of the 17 human cyclophilins and both human parvulins, but only one of the 13 human FKBPs, identified orthologues within these fungi. hPar14 orthologues were restricted to the Pezizomycotina fungi, and R. oryzae is unique in the known fungi in possessing an hCyp33 orthologue and a TPR-containing FKBP. The repertoires of Cryptococcus neoformans, Aspergillus fumigatus, and Aspergillus nidulans were found to exhibit the highest orthology to the human repertoire, and Saccharomyces cerevisiae one of the lowest. Conclusion Given this data, we would hypothesize that: (i the evolution of the fungal PPIases is driven, at least in part, by the size of the proteome, (ii evolutionary pressures differ both between the different PPIase families and the different fungi, and (iii whilst the cyclophilins and parvulins have evolved to perform conserved

  9. Protein disulfide isomerase interacts with tau protein and inhibits its fibrillization.

    Directory of Open Access Journals (Sweden)

    Li-Rong Xu

    Full Text Available BACKGROUND: Tau protein is implicated in the pathogenesis of neurodegenerative disorders such as tauopathies including Alzheimer disease, and Tau fibrillization is thought to be related to neuronal toxicity. Physiological inhibitors of Tau fibrillization hold promise for developing new strategies for treatment of Alzheimer disease. Because protein disulfide isomerase (PDI is both an enzyme and a chaperone, and implicated in neuroprotection against Alzheimer disease, we want to know whether PDI can prevent Tau fibrillization. In this study, we have investigated the interaction between PDI and Tau protein and the effect of PDI on Tau fibrillization. METHODOLOGY/PRINCIPAL FINDINGS: As evidenced by co-immunoprecipitation and confocal laser scanning microscopy, human PDI interacts and co-locates with some endogenous human Tau on the endoplasmic reticulum of undifferentiated SH-SY5Y neuroblastoma cells. The results from isothermal titration calorimetry show that one full-length human PDI binds to one full-length human Tau (or human Tau fragment Tau244-372 monomer with moderate, micromolar affinity at physiological pH and near physiological ionic strength. As revealed by thioflavin T binding assays, Sarkosyl-insoluble SDS-PAGE, and transmission electron microscopy, full-length human PDI remarkably inhibits both steps of nucleation and elongation of Tau244-372 fibrillization in a concentration-dependent manner. Furthermore, we find that two molecules of the a-domain of human PDI interact with one Tau244-372 molecule with sub-micromolar affinity, and inhibit both steps of nucleation and elongation of Tau244-372 fibrillization more strongly than full-length human PDI. CONCLUSIONS/SIGNIFICANCE: We demonstrate for the first time that human PDI binds to Tau protein mainly through its thioredoxin-like catalytic domain a, forming a 1∶1 complex and preventing Tau misfolding. Our findings suggest that PDI could act as a physiological inhibitor of Tau

  10. The peptidyl prolyl cis/trans isomerase Pin1/Ess1 inhibits phosphorylation and toxicity of tau in a yeast model for Alzheimer’s disease

    Directory of Open Access Journals (Sweden)

    Ann De Vos

    2015-04-01

    Full Text Available Since hyperphosphorylation of protein tau is a crucial event in Alzheimer’s disease, additional mechanisms besides the interplay of kinase and phosphatase activities are investigated, such as the effect of the peptidyl prolyl cis/trans isomerase Pin1. This isomerase was shown to bind and isomerize phosphorylated protein tau, thereby restoring the microtubule associated protein function of tau as well as promoting the dephosphorylation of the protein by the trans-dependent phosphatase PP2A. In this study we used models based on Saccharomyces cerevisiae to further elucidate the influence of Pin1 and its yeast ortholog Ess1 on tau phosphorylation and self-assembly. We could demonstrate that in yeast, a lack of Pin1 isomerase activity leads to an increase in phosphorylation of tau at Thr231, comparable to AD brain and consistent with earlier findings in other model organisms. However, we could also distinguish an effect by Pin1 on other residues of tau, i.e. Ser235 and Ser198/199/202. Furthermore, depletion of Pin1 isomerase activity results in reduced growth of the yeast cells, which is enhanced upon expression of tau. This suggests that the accumulation of hyperphosphorylated and aggregation-prone tau causes cytotoxicity in yeast. This study introduces yeast as a valuable model organism to characterize in detail the effect of Pin1 on the biochemical characteristics of protein tau, more specifically its phosphorylation and aggregation.

  11. Effects of polybrominated diphenyl ethers (PBDEs) and their derivatives on protein disulfide isomerase activity and growth hormone release of GH3 cells.

    Science.gov (United States)

    Hashimoto, Shoko; Yoshimura, Hiromi; Okada, Kazushi; Uramaru, Naoto; Sugihara, Kazumi; Kitamura, Shigeyuki; Imaoka, Susumu

    2012-03-19

    Polybrominated diphenyl ethers (PBDEs) have been used in a variety of consumer products such as flame retardants and recently have been known to be widespread environmental pollutants, which probably affect biological functions of mammalian cells. However, the risk posed by PBDE metabolites has not been clarified. Our previous study suggested that bisphenol A (BPA), an endocrine-disrupting chemical, binds to protein disulfide isomerase (PDI) and inhibits its activity. PDI is an isomerase enzyme in the endoplasmic reticulum and facilitates the formation or cleavage of disulfide bonds. PDI consists of a, b, b', and a' domains and the c region, with the a and a' domains having isomerase active sites. In the present study, we tested the effects of 10 kinds of PBDE compounds and their metabolites on PDI. OH-PBDEs specifically inhibited the isomerase activity of PDI, with 4'-OH-PBDE more effective than 2' (or 2)-OH-PBDEs. 4'-OH-PBDE inhibited the isomerase activity of the b'a'c fragment but not that of ab and a'c, suggesting that the b' domain of PDI is essential for the inhibition by 4'-OH-PBDE. We also investigated the effects of these chemicals on the production of growth hormone (GH) in GH3 cells. In GH3 cells, levels of mRNA and protein of GH stimulated by T(3) were reduced by 4'-OH-PBDE and 4'-MeO-PBDE. The reduction in GH expression caused by these compounds was not changed by the overexpression or knockdown of PDI in GH3 cells, while these manipulations of PDI levels significantly suppressed the expression of GH. These results suggest that the biological effects of PBDEs differed depending on their brominated and hydroxylated positions. © 2011 American Chemical Society

  12. The multidrug resistance IncA/C transferable plasmid encodes a novel domain-swapped dimeric protein-disulfide isomerase.

    Science.gov (United States)

    Premkumar, Lakshmanane; Kurth, Fabian; Neyer, Simon; Schembri, Mark A; Martin, Jennifer L

    2014-01-31

    The multidrug resistance-encoding IncA/C conjugative plasmids disseminate antibiotic resistance genes among clinically relevant enteric bacteria. A plasmid-encoded disulfide isomerase is associated with conjugation. Sequence analysis of several IncA/C plasmids and IncA/C-related integrative and conjugative elements (ICE) from commensal and pathogenic bacteria identified a conserved DsbC/DsbG homolog (DsbP). The crystal structure of DsbP reveals an N-terminal domain, a linker region, and a C-terminal catalytic domain. A DsbP homodimer is formed through domain swapping of two DsbP N-terminal domains. The catalytic domain incorporates a thioredoxin-fold with characteristic CXXC and cis-Pro motifs. Overall, the structure and redox properties of DsbP diverge from the Escherichia coli DsbC and DsbG disulfide isomerases. Specifically, the V-shaped dimer of DsbP is inverted compared with EcDsbC and EcDsbG. In addition, the redox potential of DsbP (-161 mV) is more reducing than EcDsbC (-130 mV) and EcDsbG (-126 mV). Other catalytic properties of DsbP more closely resemble those of EcDsbG than EcDsbC. These catalytic differences are in part a consequence of the unusual active site motif of DsbP (CAVC); substitution to the EcDsbC-like (CGYC) motif converts the catalytic properties to those of EcDsbC. Structural comparison of the 12 independent subunit structures of DsbP that we determined revealed that conformational changes in the linker region contribute to mobility of the catalytic domain, providing mechanistic insight into DsbP function. In summary, our data reveal that the conserved plasmid-encoded DsbP protein is a bona fide disulfide isomerase and suggest that a dedicated oxidative folding enzyme is important for conjugative plasmid transfer.

  13. Progranulin, a glycoprotein deficient in frontotemporal dementia, is a novel substrate of several protein disulfide isomerase family proteins.

    Directory of Open Access Journals (Sweden)

    Sandra Almeida

    Full Text Available The reduced production or activity of the cysteine-rich glycoprotein progranulin is responsible for about 20% of cases of familial frontotemporal dementia. However, little is known about the molecular mechanisms that govern the level and secretion of progranulin. Here we show that progranulin is expressed in mouse cortical neurons and more prominently in mouse microglia in culture and is abundant in the endoplasmic reticulum (ER and Golgi. Using chemical crosslinking, immunoprecipitation, and mass spectrometry, we found that progranulin is bound to a network of ER Ca(2+-binding chaperones including BiP, calreticulin, GRP94, and four members of the protein disulfide isomerase (PDI family. Loss of ERp57 inhibits progranulin secretion. Thus, progranulin is a novel substrate of several PDI family proteins and modulation of the ER chaperone network may be a therapeutic target for controlling progranulin secretion.

  14. Inhibition of d-xylose isomerase by polyols: atomic details by joint X-ray/neutron crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Kovalevsky, Andrey, E-mail: ayk@lanl.gov [Los Alamos National Laboratory, PO Box 1663, MS M888, Los Alamos, NM 87545 (United States); Hanson, B. Leif [University of Toledo, 2801 West Bancroft Street, Toledo, OH 43606 (United States); Mason, Sax A. [Institut Laue–Langevin, 6 Rue Jules Horowitz, 38042 Grenoble (France); Forsyth, V. Trevor [Institut Laue–Langevin, 6 Rue Jules Horowitz, 38042 Grenoble (France); Keele University, Staffordshire (United Kingdom); Fisher, Zoe [Los Alamos National Laboratory, PO Box 1663, MS M888, Los Alamos, NM 87545 (United States); Mustyakimov, Marat [Los Alamos National Laboratory, PO Box 1663, MS M888, Los Alamos, NM 87545 (United States); Oak Ridge National Laboratory, PO Box 2008, MS 6475, Oak Ridge, TN 37831 (United States); Blakeley, Matthew P. [Institut Laue–Langevin, 6 Rue Jules Horowitz, 38042 Grenoble (France); Keen, David A. [Harwell Science and Innovation Campus, Didcot, Oxon OX11 0QX (United Kingdom); Langan, Paul [Oak Ridge National Laboratory, PO Box 2008, MS 6475, Oak Ridge, TN 37831 (United States); Los Alamos National Laboratory, PO Box 1663, MS M888, Los Alamos, NM 87545 (United States)

    2012-09-01

    A joint X-ray/neutron structure of d-xylose isomerase in complex with the inhibitor sorbitol was determined at room temperature at an acidic pH of 5.9. Protonation of the O5 O atom of the sugar was directly observed in the nuclear density maps. Under acidic conditions sorbitol gains a water-mediated interaction with the enzyme active site, which may explain the increased potency of the inhibitor at low pH. d-Xylose isomerase (XI) converts the aldo-sugars xylose and glucose to their keto analogs xylulose and fructose, but is strongly inhibited by the polyols xylitol and sorbitol, especially at acidic pH. In order to understand the atomic details of polyol binding to the XI active site, a 2.0 Å resolution room-temperature joint X-ray/neutron structure of XI in complex with Ni{sup 2+} cofactors and sorbitol inhibitor at pH 5.9 and a room-temperature X-ray structure of XI containing Mg{sup 2+} ions and xylitol at the physiological pH of 7.7 were obtained. The protonation of oxygen O5 of the inhibitor, which was found to be deprotonated and negatively charged in previous structures of XI complexed with linear glucose and xylulose, was directly observed. The Ni{sup 2+} ions occupying the catalytic metal site (M2) were found at two locations, while Mg{sup 2+} in M2 is very mobile and has a high B factor. Under acidic conditions sorbitol gains a water-mediated interaction that connects its O1 hydroxyl to Asp257. This contact is not found in structures at basic pH. The new interaction that is formed may improve the binding of the inhibitor, providing an explanation for the increased affinity of the polyols for XI at low pH.

  15. Phospho-carboxyl-terminal domain binding and the role of a prolyl isomerase in pre-mRNA 3'-End formation.

    Science.gov (United States)

    Morris, D P; Phatnani, H P; Greenleaf, A L

    1999-10-29

    A phospho-carboxyl-terminal domain (CTD) affinity column created with yeast CTD kinase I and the CTD of RNA polymerase II was used to identify Ess1/Pin1 as a phospho-CTD-binding protein. Ess1/Pin1 is a peptidyl prolyl isomerase involved in both mitotic regulation and pre-mRNA 3'-end formation. Like native Ess1, a GSTEss1 fusion protein associates specifically with the phosphorylated but not with the unphosphorylated CTD. Further, hyperphosphorylated RNA polymerase II appears to be the dominant Ess1 binding protein in total yeast extracts. We demonstrate that phospho-CTD binding is mediated by the small WW domain of Ess1 rather than the isomerase domain. These findings suggest a mechanism in which the WW domain binds the phosphorylated CTD of elongating RNA polymerase II and the isomerase domain reconfigures the CTD though isomerization of proline residues perhaps by a processive mechanism. This process may be linked to a variety of pre-mRNA maturation events that use the phosphorylated CTD, including the coupled processes of pre-mRNA 3'-end formation and transcription termination.

  16. 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

  17. The basic tilted helix bundle domain of the prolyl isomerase FKBP25 is a novel double-stranded RNA binding module

    Science.gov (United States)

    Dilworth, David; Bonnafous, Pierre; Edoo, Amiirah Bibi; Bourbigot, Sarah; Pesek-Jardim, Francy; Gudavicius, Geoff; Serpa, Jason J.; Petrotchenko, Evgeniy V.; Borchers, Christoph H.

    2017-01-01

    Abstract Prolyl isomerases are defined by a catalytic domain that facilitates the cis–trans interconversion of proline residues. In most cases, additional domains in these enzymes add important biological function, including recruitment to a set of protein substrates. Here, we report that the N-terminal basic tilted helix bundle (BTHB) domain of the human prolyl isomerase FKBP25 confers specific binding to double-stranded RNA (dsRNA). This binding is selective over DNA as well as single-stranded oligonucleotides. We find that FKBP25 RNA-association is required for its nucleolar localization and for the vast majority of its protein interactions, including those with 60S pre-ribosome and early ribosome biogenesis factors. An independent mobility of the BTHB and FKBP catalytic domains supports a model by which the N-terminus of FKBP25 is anchored to regions of dsRNA, whereas the FKBP domain is free to interact with neighboring proteins. Apart from the identification of the BTHB as a new dsRNA-binding module, this domain adds to the growing list of auxiliary functions used by prolyl isomerases to define their primary cellular targets. PMID:29036638

  18. Tagatose: properties, applications, and biotechnological processes.

    Science.gov (United States)

    Oh, Deok-Kun

    2007-08-01

    D-Tagatose has attracted a great deal of attention in recent years due to its health benefits and similar properties to sucrose. D-Tagatose can be used as a low-calorie sweetener, as an intermediate for synthesis of other optically active compounds, and as an additive in detergent, cosmetic, and pharmaceutical formulation. Biotransformation of D-tagatose has been produced using several biocatalyst sources. Among the biocatalysts, L-arabinose isomerase has been mostly applied for D-tagatose production because of the industrial feasibility for the use of D-galactose as a substrate. In this article, the characterization of many L-arabinose isomerases and their D-tagatose production is compared. Protein engineering and immobilization of the enzyme for increasing the conversion rate of D-galactose to D-tagatose are also reviewed.

  19. The use of phosphomannose isomerase selection system for Agrobacterium-mediated transformation of tobacco and flax aimed for phytoremediation.

    Science.gov (United States)

    Hilgert, Jitka; Sura-De Jong, Martina; Fišer, Jiří; Tupá, Kateřina; Vrbová, Miroslava; Griga, Miroslav; Macek, Tomáš; Žiarovská, Jana

    2017-05-04

    A plant selection system based on the phosphomannose isomerase gene (pmi) as a selectable marker is often used to avoid selection using antibiotic resistance. Nevertheless, pmi gene is endogenous in several plant species and therefore difficult to use in such cases. Here we evaluated and compared Agrobacterium-mediated transformation of Linum usitatissimum breeding line AGT-952 (without endogenous pmi gene) and Nicotiana tabacum var. WSC-38 (with endogenous pmi gene). Transformation was evaluated for vectors bearing transgenes that have the potential to be involved in improved phytoremediation of contaminated environment. Tobacco regenerants selection resulted in 6.8% transformation efficiency when using a medium supplemented with 30 g/L mannose with stepwise decrease of the sucrose concentration. Similar transformation efficiency (5.3%) was achieved in transformation of flax. Relatively low selection efficiency was achieved (12.5% and 34.8%, respectively). The final detection of efficient pmi selection was conducted using PCR and the non-endogenous genes; pmi transgene for flax and todC2 transgene for tobacco plants.

  20. Role of protein disulfide isomerase and other thiol-reactive proteins in HIV-1 envelope protein-mediated fusion

    International Nuclear Information System (INIS)

    Ou Wu; Silver, Jonathan

    2006-01-01

    Cell-surface protein disulfide isomerase (PDI) has been proposed to promote disulfide bond rearrangements in HIV-1 envelope protein (Env) that accompany Env-mediated fusion. We evaluated the role of PDI in ways that have not been previously tested by downregulating PDI with siRNA and by overexpressing wild-type or variant forms of PDI in transiently and stably transfected cells. These manipulations, as well as treatment with anti-PDI antibodies, had only small effects on infection or cell fusion mediated by NL4-3 or AD8 strains of HIV-1. However, the cell-surface thiol-reactive reagent 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) had a much stronger inhibitory effect in our system, suggesting that cell-surface thiol-containing molecules other than PDI, acting alone or in concert, have a greater effect than PDI on HIV-1 Env-mediated fusion. We evaluated one such candidate, thioredoxin, a PDI family member reported to reduce a labile disulfide bond in CD4. We found that the ability of thioredoxin to reduce the disulfide bond in CD4 is enhanced in the presence of HIV-1 Env gp120 and that thioredoxin also reduces disulfide bonds in gp120 directly in the absence of CD4. We discuss the implications of these observations for identification of molecules involved in disulfide rearrangements in Env during fusion

  1. A novel potential biomarker for metabolic syndrome in Chinese adults: Circulating protein disulfide isomerase family A, member 4.

    Science.gov (United States)

    Chien, Chu-Yen; Hung, Yi-Jen; Shieh, Yi-Shing; Hsieh, Chang-Hsun; Lu, Chieh-Hua; Lin, Fu-Huang; Su, Sheng-Chiang; Lee, Chien-Hsing

    2017-01-01

    Protein disulfide isomerase (PDI) family members are specific endoplasmic reticulum proteins that are involved in the pathogenesis of numerous diseases including neurodegenerative diseases, cancer and obesity. However, the metabolic effects of PDIA4 remain unclear in humans. The aims of this study were to investigate the associations of serum PDIA4 with the metabolic syndrome (MetS) and its components in Chinese adults. A total of 669 adults (399 men and 270 women) were recruited. Serum PDIA4 concentrations and biochemical variables were recorded. Insulin sensitivity and β-cell function were examined by homeostasis model assessment. MetS was defined based on the modified National Cholesterol Education Program Adult Treatment Panel III criteria for Asia Pacific. The participants with MetS had significantly higher serum PDIA4 levels than those without MetS (Pmetabolic syndrome were 67 and 72%, respectively, in male patients and 60 and 78%, respectively, in female patients. Finally, the result showed that PDIA4 had a significantly higher area under the curve compared with blood pressure to detect MetS using receiver operating characteristic analysis. Serum PDIA4 concentrations are closely associated to MetS and its components in Chinese adults.

  2. Negative Regulation of the Stability and Tumor Suppressor Function of Fbw7 by the Pin1 Prolyl Isomerase

    Science.gov (United States)

    Min, Sang-Hyun; Lau, Alan W.; Lee, Tae Ho; Inuzuka, Hiroyuki; Wei, Shuo; Huang, Pengyu; Shaik, Shavali; Lee, Daniel Yenhong; Finn, Greg; Balastik, Martin; Chen, Chun-Hau; Luo, Manli; Tron, Adriana E.; DeCaprio, James A.; Zhou, Xiao Zhen; Wei, Wenyi; Lu, Kun Ping

    2012-01-01

    SUMMARY Fbw7 is the substrate recognition component of the SCF (Skp1-Cullin-F-box)-type E3 ligase complex and a well-characterized tumor suppressor that targets numerous oncoproteins for destruction. Genomic deletion or mutation of FBW7 has been frequently found in various types of human cancers, however, little is known about the upstream signaling pathway(s) governing Fbw7 stability and cellular functions. Here we report that Fbw7 protein destruction and tumor suppressor function are negatively regulated by the prolyl isomerase Pin1. Pin1 interacts with Fbw7 in a phoshorylation-dependent manner and promotes Fbw7 self-ubiquitination and protein degradation by disrupting Fbw7 dimerization. Consequently, over-expressing Pin1 reduces Fbw7 abundance and suppresses Fbw7’s ability to inhibit proliferation and transformation. By contrast, depletion of Pin1 in cancer cells leads to elevated Fbw7 expression, which subsequently reduces Mcl-1 abundance, sensitizing cancer cells to Taxol. Thus, Pin1-mediated inhibition of Fbw7 contributes to oncogenesis and Pin1 may be a promising drug target for anti-cancer therapy. PMID:22608923

  3. Characterizing the interactions between prolyl isomerase pin1 and phosphatase inhibitor-2 in living cells with FRET and FCS

    Science.gov (United States)

    Sun, Yuansheng; Wang, Lifu; Jyothikumar, Vinod; Brautigan, David L.; Periasamy, Ammasi

    2012-03-01

    Phosphatase inhibitor-2 (I2) was discovered as a regulator of protein Ser/Thr phosphatase-1 and is conserved from yeast to human. Binding between purified recombinant I2 from different species and the prolyl isomerase Pin1 has been demonstrated with pull-down assays, size exclusion chromatography and nuclear magnetic resonance spectroscopy. Despite this, questions persist as to whether these proteins associate together in living cells. In this study, we prepared fluorescent protein (FP) fusions of I2 and Pin1 and employed both Förster Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS) imaging techniques to characterize their interactions in living cells. In both intensity-based and time-resolved FRET studies, we observed FRET uniformly across whole cells co-expressing I2-Cerulean and Pin1-Venus that was significantly higher than in negative controls expressing Cerulean FP (without fusing to I2) as the FRET donor and Pin1-Venus, showing a specific interaction between I2-Cerulean and Pin1-Venus in living cells. We also observed the co-diffusion of I2-Cerulean and Pin1-mCherry in Fluorescence Cross Correlation Spectroscopy (FCCS) measurements. We further showed that I2 itself as well as I2-Pin1 formed complexes in living cells (predicted from in vitro studies) via a quantitative FRET assay, and demonstrated from FCS measurements that both I2 and Pin1 (fused to Cerulean) are highly mobile in living cells.

  4. Calculation of vibrational shifts of nitrile probes in the active site of ketosteroid isomerase upon ligand binding.

    Science.gov (United States)

    Layfield, Joshua P; Hammes-Schiffer, Sharon

    2013-01-16

    The vibrational Stark effect provides insight into the roles of hydrogen bonding, electrostatics, and conformational motions in enzyme catalysis. In a recent application of this approach to the enzyme ketosteroid isomerase (KSI), thiocyanate probes were introduced in site-specific positions throughout the active site. This paper implements a quantum mechanical/molecular mechanical (QM/MM) approach for calculating the vibrational shifts of nitrile (CN) probes in proteins. This methodology is shown to reproduce the experimentally measured vibrational shifts upon binding of the intermediate analogue equilinen to KSI for two different nitrile probe positions. Analysis of the molecular dynamics simulations provides atomistic insight into the roles that key residues play in determining the electrostatic environment and hydrogen-bonding interactions experienced by the nitrile probe. For the M116C-CN probe, equilinen binding reorients an active-site water molecule that is directly hydrogen-bonded to the nitrile probe, resulting in a more linear C≡N--H angle and increasing the CN frequency upon binding. For the F86C-CN probe, equilinen binding orients the Asp103 residue, decreasing the hydrogen-bonding distance between the Asp103 backbone and the nitrile probe and slightly increasing the CN frequency. This QM/MM methodology is applicable to a wide range of biological systems and has the potential to assist in the elucidation of the fundamental principles underlying enzyme catalysis.

  5. Structural and Genetic Studies Demonstrate Neurologic Dysfunction in Triosephosphate Isomerase Deficiency Is Associated with Impaired Synaptic Vesicle Dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Roland, Bartholomew P.; Zeccola, Alison M.; Larsen, Samantha B.; Amrich, Christopher G.; Talsma, Aaron D.; Stuchul, Kimberly A.; Heroux, Annie; Levitan, Edwin S.; VanDemark, Andrew P.; Palladino, Michael J.; Pallanck, Leo J.

    2016-03-31

    Triosephosphate isomerase (TPI) deficiency is a poorly understood disease characterized by hemolytic anemia, cardiomyopathy, neurologic dysfunction, and early death. TPI deficiency is one of a group of diseases known as glycolytic enzymopathies, but is unique for its severe patient neuropathology and early mortality. The disease is caused by missense mutations and dysfunction in the glycolytic enzyme, TPI. Previous studies have detailed structural and catalytic changes elicited by disease-associated TPI substitutions, and samples of patient erythrocytes have yielded insight into patient hemolytic anemia; however, the neuropathophysiology of this disease remains a mystery. This study combines structural, biochemical, and genetic approaches to demonstrate that perturbations of the TPI dimer interface are sufficient to elicit TPI deficiency neuropathogenesis. The present study demonstrates that neurologic dysfunction resulting from TPI deficiency is characterized by synaptic vesicle dysfunction, and can be attenuated with catalytically inactive TPI. Collectively, our findings are the first to identify, to our knowledge, a functional synaptic defect in TPI deficiency derived from molecular changes in the TPI dimer interface.

  6. The Role of S-Nitrosylation and S-Glutathionylation of Protein Disulphide Isomerase in Protein Misfolding and Neurodegeneration

    Directory of Open Access Journals (Sweden)

    M. Halloran

    2013-01-01

    Full Text Available Neurodegenerative diseases involve the progressive loss of neurons, and a pathological hallmark is the presence of abnormal inclusions containing misfolded proteins. Although the precise molecular mechanisms triggering neurodegeneration remain unclear, endoplasmic reticulum (ER stress, elevated oxidative and nitrosative stress, and protein misfolding are important features in pathogenesis. Protein disulphide isomerase (PDI is the prototype of a family of molecular chaperones and foldases upregulated during ER stress that are increasingly implicated in neurodegenerative diseases. PDI catalyzes the rearrangement and formation of disulphide bonds, thus facilitating protein folding, and in neurodegeneration may act to ameliorate the burden of protein misfolding. However, an aberrant posttranslational modification of PDI, S-nitrosylation, inhibits its protective function in these conditions. S-nitrosylation is a redox-mediated modification that regulates protein function by covalent addition of nitric oxide- (NO- containing groups to cysteine residues. Here, we discuss the evidence for abnormal S-nitrosylation of PDI (SNO-PDI in neurodegeneration and how this may be linked to another aberrant modification of PDI, S-glutathionylation. Understanding the role of aberrant S-nitrosylation/S-glutathionylation of PDI in the pathogenesis of neurodegenerative diseases may provide insights into novel therapeutic interventions in the future.

  7. Effect of pharmaceutical potential endocrine disruptor compounds on protein disulfide isomerase reductase activity using di-eosin-oxidized-glutathione.

    Directory of Open Access Journals (Sweden)

    Danièle Klett

    Full Text Available BACKGROUND: Protein Disulfide Isomerase (PDI in the endoplasmic reticulum of all cells catalyzes the rearrangement of disulfide bridges during folding of membrane and secreted proteins. As PDI is also known to bind various molecules including hormones such as estradiol and thyroxin, we considered the hypothesis that adverse effects of endocrine-disrupter compounds (EDC could be mediated through their interaction with PDI leading to defects in membrane or secreted proteins. METHODOLOGY/PRINCIPAL FINDINGS: Taking advantage of the recent description of the fluorescence self quenched substrate di-eosin-oxidized-glutathione (DiE-GSSG, we determined kinetically the effects of various potential pharmaceutical EDCs on the in-vitro reductase activity of bovine liver PDI by measuring the fluorescence of the reaction product (E-GSH. Our data show that estrogens (ethynylestradiol and bisphenol-A as well as indomethacin exert an inhibition whereas medroxyprogesteroneacetate and nortestosterone exert a potentiation of bovine PDI reductase activity. CONCLUSIONS: The present data indicate that the tested EDCs could not only affect endocrine target cells through nuclear receptors as previously shown, but could also affect these and all other cells by positively or negatively affecting PDI activity. The substrate DiE-GSSG has been demonstrated to be a convenient substrate to measure PDI reductase activity in the presence of various potential EDCs. It will certainly be usefull for the screening of potential effect of all kinds of chemicals on PDI reductase activity.

  8. The Expression of Millettia pinnata Chalcone Isomerase in Saccharomyces cerevisiae Salt-Sensitive Mutants Enhances Salt-Tolerance

    Directory of Open Access Journals (Sweden)

    Baiqu Huang

    2013-04-01

    Full Text Available The present study demonstrates a new Millettia pinnata chalcone isomerase (MpCHI whose transcription level in leaf was confirmed to be enhanced after being treated by seawater or NaCl (500 mM via transcriptome sequencing and Real-Time Quantitative Reverse Transcription PCR (QRT-PCR analyses. Its full length cDNA (666 bp was obtained by 3'-end and 5'-end Rapid Amplification of cDNA Ends (RACE. The analysis via NCBI BLAST indicates that both aminoacid sequence and nucleotide sequence of the MpCHI clone share high homology with other leguminous CHIs (73%–86%. Evolutionarily, the phylogenic analysis further revealed that the MpCHI is a close relative of leguminous CHIs. The MpCHI protein consists of 221 aminoacid (23.64 KDa, whose peptide length, amino acid residues of substrate-binding site and reactive site are very similar to other leguminous CHIs reported previously. Two pYES2-MpCHI transformed salt-sensitive Saccharomyces cerevisiae mutants (Δnha1 and Δnhx1 showed improved salt-tolerance significantly compared to pYES2-vector transformed yeast mutants, suggesting the MpCHI or the flavonoid biosynthesis pathway could regulate the resistance to salt stress in M. pinnata.

  9. The expression of Millettia pinnata chalcone isomerase in Saccharomyces cerevisiae salt-sensitive mutants enhances salt-tolerance.

    Science.gov (United States)

    Wang, Hui; Hu, Tangjin; Huang, Jianzi; Lu, Xiang; Huang, Baiqu; Zheng, Yizhi

    2013-04-24

    The present study demonstrates a new Millettia pinnata chalcone isomerase (MpCHI) whose transcription level in leaf was confirmed to be enhanced after being treated by seawater or NaCl (500 mM) via transcriptome sequencing and Real-Time Quantitative Reverse Transcription PCR (QRT-PCR) analyses. Its full length cDNA (666 bp) was obtained by 3'-end and 5'-end Rapid Amplification of cDNA Ends (RACE). The analysis via NCBI BLAST indicates that both aminoacid sequence and nucleotide sequence of the MpCHI clone share high homology with other leguminous CHIs (73%-86%). Evolutionarily, the phylogenic analysis further revealed that the MpCHI is a close relative of leguminous CHIs. The MpCHI protein consists of 221 aminoacid (23.64 KDa), whose peptide length, amino acid residues of substrate-binding site and reactive site are very similar to other leguminous CHIs reported previously. Two pYES2-MpCHI transformed salt-sensitive Saccharomyces cerevisiae mutants (Δnha1 and Δnhx1) showed improved salt-tolerance significantly compared to pYES2-vector transformed yeast mutants, suggesting the MpCHI or the flavonoid biosynthesis pathway could regulate the resistance to salt stress in M. pinnata.

  10. Site-Specific Measurement of Water Dynamics in the Substrate Pocket of Ketosteroid Isomerase Using Time-Resolved Vibrational Spectroscopy

    Science.gov (United States)

    Jha, Santosh Kumar; Ji, Minbiao; Gaffney, Kelly J.; Boxer, Steven G.

    2012-01-01

    Little is known about the reorganization capacity of water molecules at the active sites of enzymes and how this couples to the catalytic reaction. Here, we study the dynamics of water molecules at the active site of a highly proficient enzyme, Δ5-3-ketosteroid isomerase (KSI), during a light-activated mimic of its catalytic cycle. Photo-excitation of a nitrile containing photo-acid, coumarin183 (C183), mimics the change in charge density that occurs at the active site of KSI during the first step of the catalytic reaction. The nitrile of C183 is exposed to water when bound to the KSI active site, and we used time-resolved vibrational spectroscopy as a site-specific probe to study the solvation dynamics of water molecules in the vicinity of the nitrile. We observed that water molecules at the active site of KSI are highly rigid, during the light-activated catalytic cycle, compared to the solvation dynamics observed in bulk water. Based upon this result we hypothesize that rigid water dipoles at the active site might help in the maintenance of the pre-organized electrostatic environment required for efficient catalysis. The results also demonstrate the utility of nitrile probes in measuring the dynamics of local (H-bonded) water molecules in contrast to the commonly used fluorescence methods which measure the average behavior of primary and subsequent spheres of solvation. PMID:22931297

  11. Virtual screening and evaluation of Ketol-Acid Reducto-Isomerase (KARI as a putative drug target for Aspergillosis

    Directory of Open Access Journals (Sweden)

    Morya Vivek K

    2012-02-01

    Full Text Available Abstract Aspergillus is a leading causative agent for fungal morbidity and mortality in immuno-compromised patients. To identify a putative target to design or identify new antifungal drug, against Aspergillus is required. In our previous work, we have analyzed the various biochemical pathways, and we found Ketol Acid Reducto-Isomerase (KARI an enzyme involves in the amino acid biosynthesis, could be a better target. This enzyme was found to be unique by comparing to host proteome through BLASTp analysis. A homology based model of KARI was generated by Swiss model server. The generated model had been validated by PROCHECK and WHAT IF programs. The Zinc library was generated within the limitation of the Lipinski rule of five, for docking study. Based on the dock-score six molecules have been studied for ADME/TOX analysis and subjected for pharmacophore model generation. The Zinc ID of the potential inhibitors is ZINC00720614, ZINC01068126, ZINC0923, ZINC02090678, ZINC00663057 and ZINC02284065 and found to be pharmacologically active agonist and antagonist of KARI. This study is an attempt to Insilco evaluation of the KARI as a drug target and the screened inhibitors could help in the development of the better drug against Aspergillus.

  12. Crystallization and preliminary X-ray diffraction analysis of the peptidylprolyl isomerase Par27 of Bordetella pertussis

    International Nuclear Information System (INIS)

    Wohlkönig, Alexandre; Hodak, Hélène; Clantin, Bernard; Sénéchal, Magalie; Bompard, Coralie; Jacob-Dubuisson, Françoise; Villeret, Vincent

    2008-01-01

    Par27 from B. pertussis, the prototype of a new group of parvulins has been crystallized in two different crystal forms. Proteins with both peptidylprolyl isomerase (PPIase) and chaperone activities play a crucial role in protein folding in the periplasm of Gram-negative bacteria. Few such proteins have been structurally characterized and to date only the crystal structure of SurA from Escherichia coli has been reported. Par27, the prototype of a new group of parvulins, has recently been identified. Par27 exhibits both chaperone and PPIase activities in vitro and is the first identified parvulin protein that forms dimers in solution. Par27 has been expressed in E. coli. The protein was purified using affinity and gel-filtration chromatographic techniques and crystallized in two different crystal forms. Form A, which belongs to space group P2 (unit-cell parameters a = 42.2, b = 142.8, c = 56.0 Å, β = 95.1°), diffracts to 2.8 Å resolution, while form B, which belongs to space group C222 (unit-cell parameters a = 54.6, b = 214.1, c = 57.8 Å), diffracts to 2.2 Å resolution. Preliminary diffraction data analysis agreed with the presence of one monomer in the asymmetric unit of the orthorhombic crystal form and two in the monoclinic form

  13. TM0416, a Hyperthermophilic Promiscuous Nonphosphorylated Sugar Isomerase, Catalyzes Various C5 and C6 Epimerization Reactions.

    Science.gov (United States)

    Shin, Sun-Mi; Cao, Thinh-Phat; Choi, Jin Myung; Kim, Seong-Bo; Lee, Sang-Jae; Lee, Sung Haeng; Lee, Dong-Woo

    2017-05-15

    There is currently little information on nonphosphorylated sugar epimerases, which are of potential interest for producing rare sugars. We found a gene (the TM0416 gene) encoding a putative d-tagatose-3-epimerase-related protein from the hyperthermophilic bacterium Thermotoga maritima We overexpressed the TM0416 gene in Escherichia coli and purified the resulting recombinant protein for detailed characterization. Amino acid sequence alignment and a structural similarity search revealed that TM0416 is a putative nonphosphorylated sugar epimerase. The recombinant enzyme exhibited maximal C-3 epimerization of l-ribulose to l-xylulose at ∼80°C and pH 7 in the presence of 1 mM Mn 2+ In addition, this enzyme showed unusually high activity for the epimerization of d-tagatose to d-sorbose, with a conversion yield of 20% after 6 h at 80°C. Remarkably, the enzyme catalyzed the isomerization of d-erythrose or d-threose to d-erythrulose significantly, with conversion yields of 71% and 54.5%, respectively, after 6 h at 80°C at pH 7. To further investigate the substrate specificity of TM0416, we determined its crystal structures in complex with divalent metal ions and l-erythrulose at resolutions of 1.5 and 1.6 Å. Detailed inspection of the structural features and biochemical data clearly demonstrated that this metalloenzyme, with a freely accessible substrate-binding site and neighboring hydrophobic residues, exhibits different and promiscuous substrate preferences, compared with its mesophilic counterparts. Therefore, this study suggests that TM0416 can be functionally classified as a novel type of l-ribulose 3-epimerase (R3E) with d-erythrose isomerase activity. IMPORTANCE Rare sugars, which occur naturally in small amounts, have attracted considerable attention in the food and drug industries. However, there is little information on nonphosphorylated sugar epimerases, which might potentially be applied for the production of rare sugars. This study describes the

  14. An operon encoding three glycolytic enzymes in Lactobacillus delbrueckii subsp. bulgaricus: glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase.

    Science.gov (United States)

    Branny, P; de la Torre, F; Garel, J R

    1998-04-01

    The structural genes gap, pgk and tpi encoding three glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK) and triosephosphate isomerase (TPI), respectively, have been cloned and sequenced from Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). The genes were isolated after screening genomic sublibraries with specific gap and pgk probes obtained by PCR amplification of chromosomal DNA with degenerate primers corresponding to amino acid sequences highly conserved in GAPDHs and PGKs. Nucleotide sequencing revealed that the three genes were organized in the order gap-pgk-tpi. The translation start codons of the three genes were identified by alignment of the N-terminal sequences. These genes predicted polypeptide chains of 338, 403 and 252 amino acids for GAPDH, PGK and TPI, respectively, and they were separated by 96 bp between gap and pgk, and by only 18 bp between pgk and tpi. The codon usage in gap, pgk, tpi and three other glycolytic genes from L. bulgaricus differed, noticeably from that in other chromosomal genes. The site of transcriptional initiation was located by primer extension, and a probable promoter was identified for the gap-pgk-tpi operon. Northern hybridization of total RNA with specific probes showed two transcripts, an mRNA of 1.4 kb corresponding to the gap gene, and a less abundant mRNA of 3.4 kb corresponding to the gap-pgk-tpi cluster. The absence of a visible terminator in the 3'-end of the shorter transcript and the location of this 3'-end inside the pgk gene indicated that this shorter transcript was produced by degradation of the longer one, rather than by an early termination of transcription after the gap gene.

  15. Peptidyl-prolyl cis/trans-isomerase A1 (Pin1) is a target for modification by lipid electrophiles.

    Science.gov (United States)

    Aluise, Christopher D; Rose, Kristie; Boiani, Mariana; Reyzer, Michelle L; Manna, Joseph D; Tallman, Keri; Porter, Ned A; Marnett, Lawrence J

    2013-02-18

    Oxidation of membrane phospholipids is associated with inflammation, neurodegenerative disease, and cancer. Oxyradical damage to phospholipids results in the production of reactive aldehydes that adduct proteins and modulate their function. 4-Hydroxynonenal (HNE), a common product of oxidative damage to lipids, adducts proteins at exposed Cys, His, or Lys residues. Here, we demonstrate that peptidyl-prolyl cis/trans-isomerase A1 (Pin1), an enzyme that catalyzes the conversion of the peptide bond of pSer/pThr-Pro moieties in signaling proteins from cis to trans, is highly susceptible to HNE modification. Incubation of purified Pin1 with HNE followed by MALDI-TOF/TOF mass spectrometry resulted in detection of Michael adducts at the active site residues His-157 and Cys-113. Time and concentration dependencies indicate that Cys-113 is the primary site of HNE modification. Pin1 was adducted in MDA-MB-231 breast cancer cells treated with 8-alkynyl-HNE as judged by click chemistry conjugation with biotin followed by streptavidin-based pulldown and Western blotting with anti-Pin1 antibody. Furthermore, orbitrap MS data support the adduction of Cys-113 in the Pin1 active site upon HNE treatment of MDA-MB-231 cells. siRNA knockdown of Pin1 in MDA-MB-231 cells partially protected the cells from HNE-induced toxicity. Recent studies indicate that Pin1 is an important molecular target for the chemopreventive effects of green tea polyphenols. The present study establishes that it is also a target for electrophilic modification by products of lipid peroxidation.

  16. Comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Hahn-Hägerdal Bärbel

    2007-02-01

    Full Text Available Abstract Background Two heterologous pathways have been used to construct recombinant xylose-fermenting Saccharomyces cerevisiae strains: i the xylose reductase (XR and xylitol dehydrogenase (XDH pathway and ii the xylose isomerase (XI pathway. In the present study, the Pichia stipitis XR-XDH pathway and the Piromyces XI pathway were compared in an isogenic strain background, using a laboratory host strain with genetic modifications known to improve xylose fermentation (overexpressed xylulokinase, overexpressed non-oxidative pentose phosphate pathway and deletion of the aldose reductase gene GRE3. The two isogenic strains and the industrial xylose-fermenting strain TMB 3400 were studied regarding their xylose fermentation capacity in defined mineral medium and in undetoxified lignocellulosic hydrolysate. Results In defined mineral medium, the xylose consumption rate, the specific ethanol productivity, and the final ethanol concentration were significantly higher in the XR- and XDH-carrying strain, whereas the highest ethanol yield was achieved with the strain carrying XI. While the laboratory strains only fermented a minor fraction of glucose in the undetoxified lignocellulose hydrolysate, the industrial strain TMB 3400 fermented nearly all the sugar available. Xylitol was formed by the XR-XDH-carrying strains only in mineral medium, whereas in lignocellulose hydrolysate no xylitol formation was detected. Conclusion Despite by-product formation, the XR-XDH xylose utilization pathway resulted in faster ethanol production than using the best presently reported XI pathway in the strain background investigated. The need for robust industrial yeast strains for fermentation of undetoxified spruce hydrolysates was also confirmed.

  17. Triose phosphate isomerase deficiency is caused by altered dimerization--not catalytic inactivity--of the mutant enzymes.

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    Markus Ralser

    Full Text Available Triosephosphate isomerase (TPI deficiency is an autosomal recessive disorder caused by various mutations in the gene encoding the key glycolytic enzyme TPI. A drastic decrease in TPI activity and an increased level of its substrate, dihydroxyacetone phosphate, have been measured in unpurified cell extracts of affected individuals. These observations allowed concluding that the different mutations in the TPI alleles result in catalytically inactive enzymes. However, despite a high occurrence of TPI null alleles within several human populations, the frequency of this disorder is exceptionally rare. In order to address this apparent discrepancy, we generated a yeast model allowing us to perform comparative in vivo analyses of the enzymatic and functional properties of the different enzyme variants. We discovered that the majority of these variants exhibit no reduced catalytic activity per se. Instead, we observed, the dimerization behavior of TPI is influenced by the particular mutations investigated, and by the use of a potential alternative translation initiation site in the TPI gene. Additionally, we demonstrated that the overexpression of the most frequent TPI variant, Glu104Asp, which displays altered dimerization features, results in diminished endogenous TPI levels in mammalian cells. Thus, our results reveal that enzyme deregulation attributable to aberrant dimerization of TPI, rather than direct catalytic inactivation of the enzyme, underlies the pathogenesis of TPI deficiency. Finally, we discovered that yeast cells expressing a TPI variant exhibiting reduced catalytic activity are more resistant against oxidative stress caused by the thiol-oxidizing reagent diamide. This observed advantage might serve to explain the high allelic frequency of TPI null alleles detected among human populations.

  18. Phycourobilin in Trichromatic Phycocyanin from Oceanic Cyanobacteria Is Formed Post-translationally by a Phycoerythrobilin Lyase-Isomerase*S⃞

    Science.gov (United States)

    Blot, Nicolas; Wu, Xian-Jun; Thomas, Jean-Claude; Zhang, Juan; Garczarek, Laurence; Böhm, Stephan; Tu, Jun-Ming; Zhou, Ming; Plöscher, Matthias; Eichacker, Lutz; Partensky, Frédéric; Scheer, Hugo; Zhao, Kai-Hong

    2009-01-01

    Most cyanobacteria harvest light with large antenna complexes called phycobilisomes. The diversity of their constituting phycobiliproteins contributes to optimize the photosynthetic capacity of these microorganisms. Phycobiliprotein biosynthesis, which involves several post-translational modifications including covalent attachment of the linear tetrapyrrole chromophores (phycobilins) to apoproteins, begins to be well understood. However, the biosynthetic pathway to the blue-green-absorbing phycourobilin (λmax ∼ 495 nm) remained unknown, although it is the major phycobilin of cyanobacteria living in oceanic areas where blue light penetrates deeply into the water column. We describe a unique trichromatic phycocyanin, R-PC V, extracted from phycobilisomes of Synechococcus sp. strain WH8102. It is evolutionarily remarkable as the only chromoprotein known so far that absorbs the whole wavelength range between 450 and 650 nm. R-PC V carries a phycourobilin chromophore on its α-subunit, and this can be considered an extreme case of adaptation to blue-green light. We also discovered the enzyme, RpcG, responsible for its biosynthesis. This monomeric enzyme catalyzes binding of the green-absorbing phycoerythrobilin at cysteine 84 with concomitant isomerization to phycourobilin. This reaction is analogous to formation of the orange-absorbing phycoviolobilin from the red-absorbing phycocyanobilin that is catalyzed by the lyase-isomerase PecE/F in some freshwater cyanobacteria. The fusion protein, RpcG, and the heterodimeric PecE/F are mutually interchangeable in a heterologous expression system in Escherichia coli. The novel R-PC V likely optimizes rod-core energy transfer in phycobilisomes and thereby adaptation of a major phytoplankton group to the blue-green light prevailing in oceanic waters. PMID:19182270

  19. Variation in the Subcellular Localization and Protein Folding Activity among Arabidopsis thaliana Homologs of Protein Disulfide Isomerase

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    Christen Y. L. Yuen

    2013-10-01

    Full Text Available Protein disulfide isomerases (PDIs catalyze the formation, breakage, and rearrangement of disulfide bonds to properly fold nascent polypeptides within the endoplasmic reticulum (ER. Classical animal and yeast PDIs possess two catalytic thioredoxin-like domains (a, a′ and two non-catalytic domains (b, b′, in the order a-b-b′-a′. The model plant, Arabidopsis thaliana, encodes 12 PDI-like proteins, six of which possess the classical PDI domain arrangement (AtPDI1 through AtPDI6. Three additional AtPDIs (AtPDI9, AtPDI10, AtPDI11 possess two thioredoxin domains, but without intervening b-b′ domains. C-terminal green fluorescent protein (GFP fusions to each of the nine dual-thioredoxin PDI homologs localized predominantly to the ER lumen when transiently expressed in protoplasts. Additionally, expression of AtPDI9:GFP-KDEL and AtPDI10: GFP-KDDL was associated with the formation of ER bodies. AtPDI9, AtPDI10, and AtPDI11 mediated the oxidative folding of alkaline phosphatase when heterologously expressed in the Escherichia coli protein folding mutant, dsbA−. However, only three classical AtPDIs (AtPDI2, AtPDI5, AtPDI6 functionally complemented dsbA−. Interestingly, chemical inducers of the ER unfolded protein response were previously shown to upregulate most of the AtPDIs that complemented dsbA−. The results indicate that Arabidopsis PDIs differ in their localization and protein folding activities to fulfill distinct molecular functions in the ER.

  20. Post-duplication charge evolution of phosphoglucose isomerases in teleost fishes through weak selection on many amino acid sites

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    Sato Yukuto

    2007-10-01

    Full Text Available Abstract Background The partitioning of ancestral functions among duplicated genes by neutral evolution, or subfunctionalization, has been considered the primary process for the evolution of novel proteins (neofunctionalization. Nonetheless, how a subfunctionalized protein can evolve into a more adaptive protein is poorly understood, mainly due to the limitations of current analytical methods, which can detect only strong selection for amino acid substitutions involved in adaptive molecular evolution. In this study, we employed a comparative evolutionary approach to this question, focusing on differences in the structural properties of a protein, specifically the electric charge, encoded by fish-specific duplicated phosphoglucose isomerase (Pgi genes. Results Full-length cDNA cloning, RT-PCR based gene expression analyses, and comparative sequence analyses showed that after subfunctionalization with respect to the expression organ of duplicate Pgi genes, the net electric charge of the PGI-1 protein expressed mainly in internal tissues became more negative, and that of PGI-2 expressed mainly in muscular tissues became more positive. The difference in net protein charge was attributable not to specific amino acid sites but to the sum of various amino acid sites located on the surface of the PGI molecule. Conclusion This finding suggests that the surface charge evolution of PGI proteins was not driven by strong selection on individual amino acid sites leading to permanent fixation of a particular residue, but rather was driven by weak selection on a large number of amino acid sites and consequently by steady directional and/or purifying selection on the overall structural properties of the protein, which is derived from many modifiable sites. The mode of molecular evolution presented here may be relevant to various cases of adaptive modification in proteins, such as hydrophobic properties, molecular size, and electric charge.

  1. A zebrafish model of congenital disorders of glycosylation with phosphomannose isomerase deficiency reveals an early opportunity for corrective mannose supplementation

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    Jaime Chu

    2013-01-01

    Individuals with congenital disorders of glycosylation (CDG have recessive mutations in genes required for protein N-glycosylation, resulting in multi-systemic disease. Despite the well-characterized biochemical consequences in these individuals, the underlying cellular defects that contribute to CDG are not well understood. Synthesis of the lipid-linked oligosaccharide (LLO, which serves as the sugar donor for the N-glycosylation of secretory proteins, requires conversion of fructose-6-phosphate to mannose-6-phosphate via the phosphomannose isomerase (MPI enzyme. Individuals who are deficient in MPI present with bleeding, diarrhea, edema, gastrointestinal bleeding and liver fibrosis. MPI-CDG patients can be treated with oral mannose supplements, which is converted to mannose-6-phosphate through a minor complementary metabolic pathway, restoring protein glycosylation and ameliorating most symptoms, although liver disease continues to progress. Because Mpi deletion in mice causes early embryonic lethality and thus is difficult to study, we used zebrafish to establish a model of MPI-CDG. We used a morpholino to block mpi mRNA translation and established a concentration that consistently yielded 13% residual Mpi enzyme activity at 4 days post-fertilization (dpf, which is within the range of MPI activity detected in fibroblasts from MPI-CDG patients. Fluorophore-assisted carbohydrate electrophoresis detected decreased LLO and N-glycans in mpi morphants. These deficiencies resulted in 50% embryonic lethality by 4 dpf. Multi-systemic abnormalities, including small eyes, dysmorphic jaws, pericardial edema, a small liver and curled tails, occurred in 82% of the surviving larvae. Importantly, these phenotypes could be rescued with mannose supplementation. Thus, parallel processes in fish and humans contribute to the phenotypes caused by Mpi depletion. Interestingly, mannose was only effective if provided prior to 24 hpf. These data provide insight into treatment efficacy

  2. FabQ, a Dual-Function Dehydratase/Isomerase, Circumvents the Last Step of the Classical Fatty Acid Synthesis Cycle

    OpenAIRE

    Bi, Hongkai; Wang, Haihong; Cronan, John E.

    2013-01-01

    In the classical anaerobic pathway of unsaturated fatty acid biosynthesis, that of Escherichia coli, the double bond is introduced into the growing acyl chain by the FabA dehydratase/isomerase. Another dehydratase, FabZ, functions in the chain elongation cycle. In contrast, Aerococcus viridans has only a single FabA/FabZ homolog we designate FabQ. FabQ can not only replace the function of E. coli FabZ in vivo, but it also catalyzes the isomerization required for unsaturated fatty acid biosynt...

  3. Single zymomonas mobilis strain for xylose and arabinose fermentation

    Science.gov (United States)

    Zhang, Min; Chou, Yat-Chen; Picataggio, Stephen K.; Finkelstein, Mark

    1998-01-01

    This invention relates to single microorganisms which normally do not ferment pentose sugars which are genetically altered to ferment the pentose sugars, xylose and arabinose, to produce ethanol, and a fermentation process utilizing the same. Examples include Zymomonas mobilis which has been transformed with a combination of E. coli genes for xylose isomerase, xylulokinase, L-arabinose isomerase, L-ribulokinase, L-ribulose 5-phosphate 4-epimerase, transaldolase and transketolase. Expression of added genes are under the control of Z. mobilis promoters. These newly created microorganisms are useful for fermenting glucose, xylose and arabinose, produced by hydrolysis of hemicellulose and cellulose or starch, to produce ethanol.

  4. Functional and structural studies of the disulfide isomerase DsbC from the plant pathogen Xylella fastidiosa reveals a redox-dependent oligomeric modulation in vitro.

    Science.gov (United States)

    Santos, Clelton A; Toledo, Marcelo A S; Trivella, Daniela B B; Beloti, Lilian L; Schneider, Dilaine R S; Saraiva, Antonio M; Crucello, Aline; Azzoni, Adriano R; Souza, Alessandra A; Aparicio, Ricardo; Souza, Anete P

    2012-10-01

    Xylella fastidiosa is a Gram-negative bacterium that grows as a biofilm inside the xylem vessels of susceptible plants and causes several economically relevant crop diseases. In the present study, we report the functional and low-resolution structural characterization of the X. fastidiosa disulfide isomerase DsbC (XfDsbC). DsbC is part of the disulfide bond reduction/isomerization pathway in the bacterial periplasm and plays an important role in oxidative protein folding. In the present study, we demonstrate the presence of XfDsbC during different stages of X. fastidiosa biofilm development. XfDsbC was not detected during X. fastidiosa planktonic growth; however, after administering a sublethal copper shock, we observed an overexpression of XfDsbC that also occurred during planktonic growth. These results suggest that X. fastidiosa can use XfDsbC in vivo under oxidative stress conditions similar to those induced by copper. In addition, using dynamic light scattering and small-angle X-ray scattering, we observed that the oligomeric state of XfDsbC in vitro may be dependent on the redox environment. Under reducing conditions, XfDsbC is present as a dimer, whereas a putative tetrameric form was observed under nonreducing conditions. Taken together, our findings demonstrate the overexpression of XfDsbC during biofilm formation and provide the first structural model of a bacterial disulfide isomerase in solution. © 2012 The Authors Journal compilation © 2012 FEBS.

  5. PDILT, a divergent testis-specific protein disulfide isomerase with a non-classical SXXC motif that engages in disulfide-dependent interactions in the endoplasmic reticulum.

    Science.gov (United States)

    van Lith, Marcel; Hartigan, Nichola; Hatch, Jennifer; Benham, Adam M

    2005-01-14

    Protein disulfide isomerase (PDI) is the archetypal enzyme involved in the formation and reshuffling of disulfide bonds in the endoplasmic reticulum (ER). PDI achieves its redox function through two highly conserved thioredoxin domains, and PDI can also operate as an ER chaperone. The substrate specificities and the exact functions of most other PDI family proteins remain important unsolved questions in biology. Here, we characterize a new and striking member of the PDI family, which we have named protein disulfide isomerase-like protein of the testis (PDILT). PDILT is the first eukaryotic SXXC protein to be characterized in the ER. Our experiments have unveiled a novel, glycosylated PDI-like protein whose tissue-specific expression and unusual motifs have implications for the evolution, catalytic function, and substrate selection of thioredoxin family proteins. We show that PDILT is an ER resident glycoprotein that liaises with partner proteins in disulfide-dependent complexes within the testis. PDILT interacts with the oxidoreductase Ero1alpha, demonstrating that the N-terminal cysteine of the CXXC sequence is not required for binding of PDI family proteins to ER oxidoreductases. The expression of PDILT, in addition to PDI in the testis, suggests that PDILT performs a specialized chaperone function in testicular cells. PDILT is an unusual PDI relative that highlights the adaptability of chaperone and redox function in enzymes of the endoplasmic reticulum.

  6. On the structure and function of the phytoene desaturase CRTI from Pantoea ananatis, a membrane-peripheral and FAD-dependent oxidase/isomerase.

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    Patrick Schaub

    Full Text Available CRTI-type phytoene desaturases prevailing in bacteria and fungi can form lycopene directly from phytoene while plants employ two distinct desaturases and two cis-tans isomerases for the same purpose. This property renders CRTI a valuable gene to engineer provitamin A-formation to help combat vitamin A malnutrition, such as with Golden Rice. To understand the biochemical processes involved, recombinant CRTI was produced and obtained in homogeneous form that shows high enzymatic activity with the lipophilic substrate phytoene contained in phosphatidyl-choline (PC liposome membranes. The first crystal structure of apo-CRTI reveals that CRTI belongs to the flavoprotein superfamily comprising protoporphyrinogen IX oxidoreductase and monoamine oxidase. CRTI is a membrane-peripheral oxidoreductase which utilizes FAD as the sole redox-active cofactor. Oxygen, replaceable by quinones in its absence, is needed as the terminal electron acceptor. FAD, besides its catalytic role also displays a structural function by enabling the formation of enzymatically active CRTI membrane associates. Under anaerobic conditions the enzyme can act as a carotene cis-trans isomerase. In silico-docking experiments yielded information on substrate binding sites, potential catalytic residues and is in favor of single half-site recognition of the symmetrical C(40 hydrocarbon substrate.

  7. Ser46 phosphorylation and prolyl-isomerase Pin1-mediated isomerization of p53 are key events in p53-dependent apoptosis induced by mutant huntingtin.

    Science.gov (United States)

    Grison, Alice; Mantovani, Fiamma; Comel, Anna; Agostoni, Elena; Gustincich, Stefano; Persichetti, Francesca; Del Sal, Giannino

    2011-11-01

    Huntington disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the gene coding for huntingtin protein. Several mechanisms have been proposed by which mutant huntingtin (mHtt) may trigger striatal neurodegeneration, including mitochondrial dysfunction, oxidative stress, and apoptosis. Furthermore, mHtt induces DNA damage and activates a stress response. In this context, p53 plays a crucial role in mediating mHtt toxic effects. Here we have dissected the pathway of p53 activation by mHtt in human neuronal cells and in HD mice, with the aim of highlighting critical nodes that may be pharmacologically manipulated for therapeutic intervention. We demonstrate that expression of mHtt causes increased phosphorylation of p53 on Ser46, leading to its interaction with phosphorylation-dependent prolyl isomerase Pin1 and consequent dissociation from the apoptosis inhibitor iASPP, thereby inducing the expression of apoptotic target genes. Inhibition of Ser46 phosphorylation by targeting homeodomain-interacting protein kinase 2 (HIPK2), PKCδ, or ataxia telangiectasia mutated kinase, as well as inhibition of the prolyl isomerase Pin1, prevents mHtt-dependent apoptosis of neuronal cells. These results provide a rationale for the use of small-molecule inhibitors of stress-responsive protein kinases and Pin1 as a potential therapeutic strategy for HD treatment.

  8. Whole cell immobilization of refractory glucose isomerase using tris(hydroxymethyl)phosphine as crosslinker for preparation of high fructose corn syrup at elevated temperature.

    Science.gov (United States)

    Jia, Dong-Xu; Wang, Teng; Liu, Zi-Jian; Jin, Li-Qun; Li, Jia-Jia; Liao, Cheng-Jun; Chen, De-Shui; Zheng, Yu-Guo

    2018-04-04

    Glucose isomerase (GI) responsible for catalyzing the isomerization from d-glucose to d-fructose, was an important enzyme for producing high fructose corn syrup (HFCS). In a quest to prepare HFCS at elevated temperature and facilitate enzymatic recovery, an effective procedure for whole cell immobilization of refractory Thermus oshimai glucose isomerase (ToGI) onto Celite 545 using tris(hydroxymethyl)phosphine (THP) as crosslinker was established. The immobilized biocatalyst showed an activity of approximate 127.3 U/(g·immobilized product) via optimization in terms of cells loading, crosslinker concentration and crosslinking time. The pH optimum of the immobilized biocatalyst was displaced from pH 8.0 of native enzyme to neutral pH 7.0. Compared with conventional glutaraldehyde (GLU)-immobilized cells, it possessed the enhanced thermostability with 70.1% residual activity retaining after incubation at 90°C for 72 h. Moreover, the THP-immobilized biocatalyst exhibited superior operational stability, in which it retained 85.8% of initial activity after 15 batches of bioconversion at 85°C. This study paved a way for reducing catalysis cost for upscale preparation of HFCS with higher d-fructose concentration. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  9. Immobilized Trienzymatic System with Enhanced Stabilization for the Biotransformation of Lactose

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    Pedro Torres

    2017-02-01

    Full Text Available The use of ketohexose isomerases is a powerful tool in lactose whey processing, but these enzymes can be very sensitive and expensive. Development of immobilized/stabilized biocatalysts could be a further option to improve the process. In this work, β-galactosidase from Bacillus circulans, l-arabinose (d-galactose isomerase from Enterococcus faecium, and d-xylose (d-glucose isomerase from Streptomyces rubiginosus were immobilized individually onto Eupergit C and Eupergit C 250 L. Immobilized activity yields were over 90% in all cases. With the purpose of increasing thermostability of derivatives, two post-immobilization treatments were performed: alkaline incubation to favor the formation of additional covalent linkages, and blocking of excess oxirane groups by reacting with glycine. The greatest thermostability was achieved when alkaline incubation was carried out for 24 h, producing l-arabinose isomerase-Eupergit C derivatives with a half-life of 379 h and d-xylose isomerase-Eupergit C derivatives with a half-life of 554 h at 50 °C. Preliminary assays using immobilized and stabilized biocatalysts sequentially to biotransform lactose at pH 7.0 and 50 °C demonstrated improved performances as compared with soluble enzymes. Further improvements in ketohexose productivities were achieved when the three single-immobilizates were incubated simultaneously with lactose in a mono-reactor system.

  10. Bacterial xylose isomerases from the mammal gut Bacteroidetes cluster function in Saccharomyces cerevisiae for effective xylose fermentation.

    Science.gov (United States)

    Peng, Bingyin; Huang, Shuangcheng; Liu, Tingting; Geng, Anli

    2015-05-17

    Xylose isomerase (XI) catalyzes the conversion of xylose to xylulose, which is the key step for anaerobic ethanolic fermentation of xylose. Very few bacterial XIs can function actively in Saccharomyces cerevisiae. Here, we illustrate a group of XIs that would function for xylose fermentation in S. cerevisiae through phylogenetic analysis, recombinant yeast strain construction, and xylose fermentation. Phylogenetic analysis of deposited XI sequences showed that XI evolutionary relationship was highly consistent with the bacterial taxonomic orders and quite a few functional XIs in S. cerevisiae were clustered with XIs from mammal gut Bacteroidetes group. An XI from Bacteroides valgutus in this cluster was actively expressed in S. cerevisiae with an activity comparable to the fungal XI from Piromyces sp. Two XI genes were isolated from the environmental metagenome and they were clustered with XIs from environmental Bacteroidetes group. These two XIs could not be expressed in yeast with activity. With the XI from B. valgutus expressed in S. cerevisiae, background yeast strains were optimized by pentose metabolizing pathway enhancement and adaptive evolution in xylose medium. Afterwards, more XIs from the mammal gut Bacteroidetes group, including those from B. vulgatus, Tannerella sp. 6_1_58FAA_CT1, Paraprevotella xylaniphila and Alistipes sp. HGB5, were individually transformed into S. cerevisiae. The known functional XI from Orpinomyces sp. ukk1, a mammal gut fungus, was used as the control. All the resulting recombinant yeast strains were able to ferment xylose. The respiration-deficient strains harboring B. vulgatus and Alistipes sp. HGB5 XI genes respectively obtained specific xylose consumption rate of 0.662 and 0.704 g xylose gcdw(-1) h(-1), and ethanol specific productivity of 0.277 and 0.283 g ethanol gcdw(-1) h(-1), much comparable to those obtained by the control strain carrying Orpinomyces sp. ukk1 XI gene. This study demonstrated that XIs clustered in the

  11. Overexpression of an isopentenyl diphosphate isomerase gene to enhance trans-polyisoprene production in Eucommia ulmoides Oliver

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    Chen Ren

    2012-10-01

    Full Text Available Abstract Background Natural rubber produced by plants, known as polyisoprene, is the most widely used isoprenoid polymer. Plant polyisoprenes can be classified into two types; cis-polyisoprene and trans-polyisoprene, depending on the type of polymerization of the isoprene unit. More than 2000 species of higher plants produce latex consisting of cis-polyisoprene. Hevea brasiliensis (rubber tree produces cis-polyisoprene, and is the key source of commercial rubber. In contrast, relatively few plant species produce trans-polyisoprene. Currently, trans-polyisoprene is mainly produced synthetically, and no plant species is used for its commercial production. Results To develop a plant-based system suitable for large-scale production of trans-polyisoprene, we selected a trans-polyisoprene-producing plant, Eucommia ulmoides Oliver, as the target for genetic transformation. A full-length cDNA (designated as EuIPI, Accession No. AB041629 encoding isopentenyl diphosphate isomerase (IPI was isolated from E. ulmoides. EuIPI consisted of 1028 bp with a 675-bp open reading frame encoding a protein with 224 amino acid residues. EuIPI shared high identity with other plant IPIs, and the recombinant protein expressed in Escherichia coli showed IPI enzymatic activity in vitro. EuIPI was introduced into E. ulmoides via Agrobacterium-mediated transformation. Transgenic lines of E. ulmoides overexpressing EuIPI showed increased EuIPI expression (up to 19-fold that of the wild-type and a 3- to 4-fold increase in the total content of trans-polyisoprenes, compared with the wild-type (non-transgenic root line control. Conclusions Increasing the expression level of EuIPI by overexpression increased accumulation of trans-polyisoprenes in transgenic E. ulmoides. IPI catalyzes the conversion of isopentenyl diphosphate to its highly electrophilic isomer, dimethylallyl diphosphate, which is the first step in the biosynthesis of all isoprenoids, including polyisoprene. Our

  12. Molecular association of glucose-6-phosphate isomerase and pyruvate kinase M2 with glyceraldehyde-3-phosphate dehydrogenase in cancer cells

    International Nuclear Information System (INIS)

    Das, Mahua R.; Bag, Arup K.; Saha, Shekhar; Ghosh, Alok; Dey, Sumit K.; Das, Provas; Mandal, Chitra; Ray, Subhankar; Chakrabarti, Saikat; Ray, Manju; Jana, Siddhartha S.

    2016-01-01

    For a long time cancer cells are known for increased uptake of glucose and its metabolization through glycolysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key regulatory enzyme of this pathway and can produce ATP through oxidative level of phosphorylation. Previously, we reported that GAPDH purified from a variety of malignant tissues, but not from normal tissues, was strongly inactivated by a normal metabolite, methylglyoxal (MG). Molecular mechanism behind MG mediated GAPDH inhibition in cancer cells is not well understood. GAPDH was purified from Ehrlich ascites carcinoma (EAC) cells based on its enzymatic activity. GAPDH associated proteins in EAC cells and 3-methylcholanthrene (3MC) induced mouse tumor tissue were detected by mass spectrometry analysis and immunoprecipitation (IP) experiment, respectively. Interacting domains of GAPDH and its associated proteins were assessed by in silico molecular docking analysis. Mechanism of MG mediated GAPDH inactivation in cancer cells was evaluated by measuring enzyme activity, Circular dichroism (CD) spectroscopy, IP and mass spectrometry analyses. Here, we report that GAPDH is associated with glucose-6-phosphate isomerase (GPI) and pyruvate kinase M2 (PKM2) in Ehrlich ascites carcinoma (EAC) cells and also in 3-methylcholanthrene (3MC) induced mouse tumor tissue. Molecular docking analyses suggest C-terminal domain preference for the interaction between GAPDH and GPI. However, both C and N termini of PKM2 might be interacting with the C terminal domain of GAPDH. Expression of both PKM2 and GPI is increased in 3MC induced tumor compared with the normal tissue. In presence of 1 mM MG, association of GAPDH with PKM2 or GPI is not perturbed, but the enzymatic activity of GAPDH is reduced to 26.8 ± 5 % in 3MC induced tumor and 57.8 ± 2.3 % in EAC cells. Treatment of MG to purified GAPDH complex leads to glycation at R399 residue of PKM2 only, and changes the secondary structure of the protein complex. PKM2

  13. Synthesis and modifications of heterocyclic derivatives of D-arabinose: potential inhibitors of glucose-6-phosphate isomerase and glucosamine-6-phosphate synthase

    International Nuclear Information System (INIS)

    Viana, Renato Marcio Ribeiro; Prado, Maria Auxiliadora Fontes; Alves, Ricardo Jose

    2008-01-01

    The synthesis of -5-(D-arabino-1,2,3,4-tetrahydroxybutyl)tetrazole and -2-(d-arabino-1,2,3,4-tetra-acetoxybutyl)-5-methyl-1,3,4-oxadiazole from d-arabinose is described. Attempts at removing the protecting groups of the oxadiazole derivative were unsuccessful, leading to products resulting from the opening of the oxadiazole ring. The unprotected tetrazole derivative was selectively phosphorylated at the primary hydroxyl group with diethyl phosphoryl chloride. The resulting 5-[d-arabino-4-(diethylphosphoryloxy)-1,2,3-trihydroxybutyl]tetrazole is a protected form of a potential inhibitor of the enzymes glucose-6-phosphate isomerase and glucosamine synthase. (author)

  14. Distinguishing two types of gray mullet, Mugil cephalus L. (Mugiliformes: Mugilidae), by using glucose-6-phosphate isomerase (GPI) allozymes with special reference to enzyme activities.

    Science.gov (United States)

    Huang, C S; Weng, C F; Lee, S C

    2001-06-01

    The resident and migratory types of gray mullet, Mugil cephalus, on the coast of Taiwan can not be separated morphologically. Allozyme analysis was applied to estimate genetic variation between the two types of gray mullet and to test whether they belong to different populations. After starch gel electrophoresis, different allelic frequency spectra of glucose-6-phosphate isomerase-A (GPI-A) between stocks was observed. The resident stock contained Gpi-A(135) and Gpi-A(100), whereas the migratory type contained Gpi-A(100) only. In addition, GPI activities of locus A showed two distinct profiles between the two alleles. The results broadly revealed that Gpi-A allelic frequency was not regulated by temperature changes even after 6 months of thermal acclimation. This suggests that natural selection may play a role in shaping the allelic frequency change during the migratory journey. These findings suggest that the Gpi-A allelic difference can be used for population discrimination.

  15. Peptidyl prolyl isomerase Pin1-inhibitory activity of D-glutamic and D-aspartic acid derivatives bearing a cyclic aliphatic amine moiety.

    Science.gov (United States)

    Nakagawa, Hidehiko; Seike, Suguru; Sugimoto, Masatoshi; Ieda, Naoya; Kawaguchi, Mitsuyasu; Suzuki, Takayoshi; Miyata, Naoki

    2015-12-01

    Pin1 is a peptidyl prolyl isomerase that specifically catalyzes cis-trans isomerization of phosphorylated Thr/Ser-Pro peptide bonds in substrate proteins and peptides. Pin1 is involved in many important cellular processes, including cancer progression, so it is a potential target of cancer therapy. We designed and synthesized a novel series of Pin1 inhibitors based on a glutamic acid or aspartic acid scaffold bearing an aromatic moiety to provide a hydrophobic surface and a cyclic aliphatic amine moiety with affinity for the proline-binding site of Pin1. Glutamic acid derivatives bearing cycloalkylamino and phenylthiazole groups showed potent Pin1-inhibitory activity comparable with that of known inhibitor VER-1. The results indicate that steric interaction of the cyclic alkyl amine moiety with binding site residues plays a key role in enhancing Pin1-inhibitory activity. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  16. Active site mutations in yeast protein disulfide isomerase cause dithiothreitol sensitivity and a reduced rate of protein folding in the endoplasmic reticulum

    DEFF Research Database (Denmark)

    Holst, B; Tachibana, C; Winther, Jakob R.

    1997-01-01

    Aspects of protein disulfide isomerase (PDI) function have been studied in yeast in vivo. PDI contains two thioredoxin-like domains, a and a', each of which contains an active-site CXXC motif. The relative importance of the two domains was analyzed by rendering each one inactive by mutation to SGAS....... Such mutations had no significant effect on growth. The domains however, were not equivalent since the rate of folding of carboxypeptidase Y (CPY) in vivo was reduced by inactivation of the a domain but not the a' domain. To investigate the relevance of PDI redox potential, the G and H positions of each CGHC......-deleted strains overexpressing the yeast PDI homologue EUG1 are viable. Exchanging the wild-type Eug1p C(L/I)HS active site sequences for C(L/I)HC increased the growth rate significantly, however, further highlighting the importance of the oxidizing function for optimal growth....

  17. A high-throughput screen for inhibitors of the prolyl isomerase, Pin1, identifies a seaweed polyphenol that reduces adipose cell differentiation.

    Science.gov (United States)

    Mori, Tadashi; Hidaka, Masafumi; Ikuji, Hiroko; Yoshizawa, Ibuki; Toyohara, Haruhiko; Okuda, Toru; Uchida, Chiyoko; Asano, Tomoichiro; Yotsu-Yamashita, Mari; Uchida, Takafumi

    2014-01-01

    The peptidyl prolyl cis/trans isomerase Pin1 enhances the uptake of triglycerides and the differentiation of fibroblasts into adipose cells in response to insulin stimulation. Pin1 downregulation could be a potential approach to prevent and treat obesity-related disorders. In order to identify an inhibitor of Pin1 that exhibited minimal cytotoxicity, we established a high-throughput screen for Pin1 inhibitors and used this method to identify an inhibitor from 1,056 crude fractions of two natural product libraries. The candidate, a phlorotannin called 974-B, was isolated from the seaweed, Ecklonia kurome. 974-B inhibited the differentiation of mouse embryonic fibroblasts and 3T3-L1 cells into adipose cells without inducing cytotoxicity. We discovered the Pin1 inhibitor, 974-B, from the seaweed, E. kurome, and showed that it blocks the differentiation of fibroblasts into adipose cells, suggesting that 974-B could be a lead drug candidate for obesity-related disorders.

  18. 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.

  19. Discovery of ebselen as an inhibitor of Cryptosporidium parvum glucose-6-phosphate isomerase (CpGPI by high-throughput screening of existing drugs

    Directory of Open Access Journals (Sweden)

    Rana Eltahan

    2018-04-01

    Full Text Available Cryptosporidium parvum is a water-borne and food-borne apicomplexan pathogen. It is one of the top four diarrheal-causing pathogens in children under the age of five in developing countries, and an opportunistic pathogen in immunocompromised individuals. Unlike other apicomplexans, C. parvum lacks Kreb's cycle and cytochrome-based respiration, thus relying mainly on glycolysis to produce ATP. In this study, we characterized the primary biochemical features of the C. parvum glucose-6-phosphate isomerase (CpGPI and determined its Michaelis constant towards fructose-6-phosphate (Km = 0.309 mM, Vmax = 31.72 nmol/μg/min. We also discovered that ebselen, an organoselenium drug, was a selective inhibitor of CpGPI by high-throughput screening of 1200 known drugs. Ebselen acted on CpGPI as an allosteric noncompetitive inhibitor (IC50 = 8.33 μM; Ki = 36.33 μM, while complete inhibition of CpGPI activity was not achieved. Ebselen could also inhibit the growth of C. parvum in vitro (EC50 = 165 μM at concentrations nontoxic to host cells, albeit with a relatively small in vitro safety window of 4.2 (cytotoxicity TC50 on HCT-8 cells = 700 μM. Additionally, ebselen might also target other enzymes in the parasite, leading to the parasite growth reduction. Therefore, although ebselen is useful in studying the inhibition of CpGPI enzyme activity, further proof is needed to chemically and/or genetically validate CpGPI as a drug target. Keywords: Apicomplexan, Cryptosporidium parvum, Glucose-6-phosphate isomerase (GPI, Ebselen

  20. Prolyl isomerase Pin1 is highly expressed in Her2-positive breast cancer and regulates erbB2 protein stability

    Directory of Open Access Journals (Sweden)

    Lu Kun

    2008-12-01

    Full Text Available Abstract Overexpression of HER-2/Neu occurs in about 25–30% of breast cancer patients and is indicative of poor prognosis. While Her2/Neu overexpression is primarily a result of erbB2 amplification, it has recently been recognized that erbB2 levels are also regulated on the protein level. However, factors that regulate Her2/Neu protein stability are less well understood. The prolyl isomerase Pin1 catalyzes the isomerization of specific pSer/Thr-Pro motifs that have been phosphorylated in response to mitogenic signaling. We have previously reported that Pin1-catalyzed post-phosphorylational modification of signal transduction modulates the oncogenic pathways downstream from c-neu. The goal of this study was to examine the expression of prolyl isomerase Pin1 in human Her2+ breast cancer, and to study if Pin1 affects the expression of Her2/Neu itself. Methods Immunohistochemistry for Her2 and Pin1 were performed on two hundred twenty-three human breast cancers, with 59% of the specimen from primary cancers and 41% from metastatic sites. Pin1 inhibition was achieved using siRNA in Her2+ breast cancer cell lines, and its effects were studied using cell viability assays, immunoblotting and immunofluorescence. Results Sixty-four samples (28.7% stained positive for Her2 (IHC 3+, and 54% (122/223 of all breast cancers stained positive for Pin1. Of the Her2-positive cancers 40 (62.5% were also Pin1-positive, based on strong nuclear or nuclear and cytoplasmic staining. Inhibition of Pin1 via RNAi resulted in significant suppression of Her2-positive tumor cell growth in BT474, SKBR3 and AU565 cells. Pin1 inhibition greatly increased the sensitivity of Her2-positive breast cancer cells to the mTOR inhibitor Rapamycin, while it did not increase their sensitivity to Trastuzumab, suggesting that Pin1 might act on Her2 signaling. We found that Pin1 interacted with the protein complex that contains ubiquitinated erbB2 and that Pin1 inhibition accelerated erbB2

  1. Autoimmune gastro-pancreatitis with anti-protein disulfide isomerase-associated 2 autoantibody in Aire-deficient BALB/cAnN mice.

    Directory of Open Access Journals (Sweden)

    Hironori Kurisaki

    Full Text Available Although the autoimmune regulator (Aire knockout (KO mouse model has been reported to present various organ-specific autoimmune diseases depending on genetic background, autoimmune pancreatitis in mice of BALB/c background has not yet been reported. Here, we report that Aire KO mice with BALB/cAnN background showed significant lymphoid cell infiltration in the pancreas and stomach. To examine whether the phenotype in the pancreas and stomach is due to autoimmune reaction associated with autoantibody production, indirect immunofluorescence staining followed by Western blot analysis was performed. Consequently, the autoantibody against pancreas and stomach was detected in the sera of Aire KO mice, and the target antigen of the autoantibody was identified as protein disulfide isomerase-associated 2 (Pdia2, which was reported to be expressed preferentially in the pancreas and stomach. Thus, Aire KO mice of BALB/cAnN background can serve as a useful animal model for autoimmune gastro-pancreatitis with anti-Pdia2 autoantibody production.

  2. Autoimmune gastro-pancreatitis with anti-protein disulfide isomerase-associated 2 autoantibody in Aire-deficient BALB/cAnN mice.

    Science.gov (United States)

    Kurisaki, Hironori; Nagao, Yukihiro; Nagafuchi, Seiho; Mitsuyama, Masao

    2013-01-01

    Although the autoimmune regulator (Aire) knockout (KO) mouse model has been reported to present various organ-specific autoimmune diseases depending on genetic background, autoimmune pancreatitis in mice of BALB/c background has not yet been reported. Here, we report that Aire KO mice with BALB/cAnN background showed significant lymphoid cell infiltration in the pancreas and stomach. To examine whether the phenotype in the pancreas and stomach is due to autoimmune reaction associated with autoantibody production, indirect immunofluorescence staining followed by Western blot analysis was performed. Consequently, the autoantibody against pancreas and stomach was detected in the sera of Aire KO mice, and the target antigen of the autoantibody was identified as protein disulfide isomerase-associated 2 (Pdia2), which was reported to be expressed preferentially in the pancreas and stomach. Thus, Aire KO mice of BALB/cAnN background can serve as a useful animal model for autoimmune gastro-pancreatitis with anti-Pdia2 autoantibody production.

  3. Production of L-allose and D-talose from L-psicose and D-tagatose by L-ribose isomerase.

    Science.gov (United States)

    Terami, Yuji; Uechi, Keiko; Nomura, Saki; Okamoto, Naoki; Morimoto, Kenji; Takata, Goro

    2015-01-01

    L-ribose isomerase (L-RI) from Cellulomonas parahominis MB426 can convert L-psicose and D-tagatose to L-allose and D-talose, respectively. Partially purified recombinant L-RI from Escherichia coli JM109 was immobilized on DIAION HPA25L resin and then utilized to produce L-allose and D-talose. Conversion reaction was performed with the reaction mixture containing 10% L-psicose or D-tagatose and immobilized L-RI at 40 °C. At equilibrium state, the yield of L-allose and D-talose was 35.0% and 13.0%, respectively. Immobilized enzyme could convert L-psicose to L-allose without remarkable decrease in the enzyme activity over 7 times use and D-tagatose to D-talose over 37 times use. After separation and concentration, the mixture solution of L-allose and D-talose was concentrated up to 70% and crystallized by keeping at 4 °C. L-Allose and d-talose crystals were collected from the syrup by filtration. The final yield was 23.0% L-allose and 7.30% D-talose that were obtained from L-psicose and D-tagatose, respectively.

  4. The prolyl isomerase Pin1 acts synergistically with CDK2 to regulate the basal activity of estrogen receptor α in breast cancer.

    Directory of Open Access Journals (Sweden)

    Chiara Lucchetti

    Full Text Available In hormone receptor-positive breast cancers, most tumors in the early stages of development depend on the activity of the estrogen receptor and its ligand, estradiol. Anti-estrogens, such as tamoxifen, have been used as the first line of therapy for over three decades due to the fact that they elicit cell cycle arrest. Unfortunately, after an initial period, most cells become resistant to hormonal therapy. Peptidylprolyl isomerase 1 (Pin1, a protein overexpressed in many tumor types including breast, has been demonstrated to modulate ERalpha activity and is involved in resistance to hormonal therapy. Here we show a new mechanism through which CDK2 drives an ERalpha-Pin1 interaction under hormone- and growth factor-free conditions. The PI3K/AKT pathway is necessary to activate CDK2, which phosphorylates ERalphaSer294, and mediates the binding between Pin1 and ERalpha. Site-directed mutagenesis demonstrated that ERalphaSer294 is essential for Pin1-ERalpha interaction and modulates ERalpha phosphorylation on Ser118 and Ser167, dimerization and activity. These results open up new drug treatment opportunities for breast cancer patients who are resistant to anti-estrogen therapy.

  5. Ethylene Responses in Rice Roots and Coleoptiles Are Differentially Regulated by a Carotenoid Isomerase-Mediated Abscisic Acid Pathway[OPEN

    Science.gov (United States)

    Yin, Cui-Cui; Ma, Biao; Collinge, Derek Phillip; Pogson, Barry James; He, Si-Jie; Xiong, Qing; Duan, Kai-Xuan; Chen, Hui; Yang, Chao; Lu, Xiang; Wang, Yi-Qin; Zhang, Wan-Ke; Chu, Cheng-Cai; Sun, Xiao-Hong; Fang, Shuang; Chu, Jin-Fang; Lu, Tie-Gang; Chen, Shou-Yi; Zhang, Jin-Song

    2015-01-01

    Ethylene and abscisic acid (ABA) act synergistically or antagonistically to regulate plant growth and development. ABA is derived from the carotenoid biosynthesis pathway. Here, we analyzed the interplay among ethylene, carotenoid biogenesis, and ABA in rice (Oryza sativa) using the rice ethylene response mutant mhz5, which displays a reduced ethylene response in roots but an enhanced ethylene response in coleoptiles. We found that MHZ5 encodes a carotenoid isomerase and that the mutation in mhz5 blocks carotenoid biosynthesis, reduces ABA accumulation, and promotes ethylene production in etiolated seedlings. ABA can largely rescue the ethylene response of the mhz5 mutant. Ethylene induces MHZ5 expression, the production of neoxanthin, an ABA biosynthesis precursor, and ABA accumulation in roots. MHZ5 overexpression results in enhanced ethylene sensitivity in roots and reduced ethylene sensitivity in coleoptiles. Mutation or overexpression of MHZ5 also alters the expression of ethylene-responsive genes. Genetic studies revealed that the MHZ5-mediated ABA pathway acts downstream of ethylene signaling to inhibit root growth. The MHZ5-mediated ABA pathway likely acts upstream but negatively regulates ethylene signaling to control coleoptile growth. Our study reveals novel interactions among ethylene, carotenogenesis, and ABA and provides insight into improvements in agronomic traits and adaptive growth through the manipulation of these pathways in rice. PMID:25841037

  6. Protein disulfide isomerase-like protein 1-1 controls endosperm development through regulation of the amount and composition of seed proteins in rice.

    Directory of Open Access Journals (Sweden)

    Yeon Jeong Kim

    Full Text Available Protein disulfide isomerase (PDI is a chaperone protein involved in oxidative protein folding by acting as a catalyst and assisting folding in the endoplasmic reticulum (ER. A genome database search showed that rice contains 19 PDI-like genes. However, their functions are not clearly identified. This paper shows possible functions of rice PDI-like protein 1-1 (PDIL1-1 during seed development. Seeds of the T-DNA insertion PDIL1-1 mutant, PDIL1-1Δ, identified by genomic DNA PCR and western blot analysis, display a chalky phenotype and a thick aleurone layer. Protein content per seed was significantly lower and free sugar content higher in PDIL1-1Δ mutant seeds than in the wild type. Proteomic analysis of PDIL1-1Δ mutant seeds showed that PDIL1-1 is post-translationally regulated, and its loss causes accumulation of many types of seed proteins including glucose/starch metabolism- and ROS (reactive oxygen species scavenging-related proteins. In addition, PDIL1-1 strongly interacts with the cysteine protease OsCP1. Our data indicate that the opaque phenotype of PDIL1-1Δ mutant seeds results from production of irregular starch granules and protein body through loss of regulatory activity for various proteins involved in the synthesis of seed components.

  7. Novel Roles of the Non-catalytic Elements of Yeast Protein-disulfide Isomerase in Its Interplay with Endoplasmic Reticulum Oxidoreductin 1*

    Science.gov (United States)

    Niu, Yingbo; Zhang, Lihui; Yu, Jiaojiao; Wang, Chih-chen; Wang, Lei

    2016-01-01

    The formation of disulfide bonds in the endoplasmic reticulum (ER) of eukaryotic cells is catalyzed by the sulfhydryl oxidase, ER oxidoreductin 1 (Ero1), and protein-disulfide isomerase (PDI). PDI is oxidized by Ero1 to continuously introduce disulfides into substrates, and feedback regulates Ero1 activity by manipulating the regulatory disulfides of Ero1. In this study we find that yeast Ero1p is enzymatically active even with its regulatory disulfides intact, and further activation of Ero1p by reduction of the regulatory disulfides requires the reduction of non-catalytic Cys90-Cys97 disulfide in Pdi1p. The principal client-binding site in the Pdi1p b′ domain is necessary not only for the functional Ero1p-Pdi1p disulfide relay but also for the activation of Ero1p. We also demonstrate by complementary activation assays that the regulatory disulfides in Ero1p are much more stable than those in human Ero1α. These new findings on yeast Ero1p-Pdi1p interplay reveal significant differences from our previously identified mode of human Ero1α-PDI interplay and provide insights into the evolution of the eukaryotic oxidative protein folding pathway. PMID:26846856

  8. Conserved Residues Lys57 and Lys401 of Protein Disulfide Isomerase Maintain an Active Site Conformation for Optimal Activity: Implications for Post-Translational Regulation

    Directory of Open Access Journals (Sweden)

    Cody Caba

    2018-02-01

    Full Text Available Despite its study since the 1960's, very little is known about the post-translational regulation of the multiple catalytic activities performed by protein disulfide isomerase (PDI, the primary protein folding catalyst of the cell. This work identifies a functional role for the highly conserved CxxC-flanking residues Lys57 and Lys401 of human PDI in vitro. Mutagenesis studies have revealed these residues as modulating the oxidoreductase activity of PDI in a pH-dependent manner. Non-conservative amino acid substitutions resulted in enzyme variants upwards of 7-fold less efficient. This attenuated activity was found to translate into a 2-fold reduction of the rate of electron shuttling between PDI and the intraluminal endoplasmic reticulum oxidase, ERO1α, suggesting a functional significance to oxidative protein folding. In light of this, the possibility of lysine acetylation at residues Lys57 and Lys401 was assessed by in vitro treatment using acetylsalicylic acid (aspirin. A total of 28 acetyllysine residues were identified, including acLys57 and acLys401. The kinetic behavior of the acetylated protein form nearly mimicked that obtained with a K57/401Q double substitution variant providing an indication that acetylation of the active site-flanking lysine residues can act to reversibly modulate PDI activity.

  9. Conserved Residues Lys57 and Lys401 of Protein Disulfide Isomerase Maintain an Active Site Conformation for Optimal Activity: Implications for Post-Translational Regulation.

    Science.gov (United States)

    Caba, Cody; Ali Khan, Hyder; Auld, Janeen; Ushioda, Ryo; Araki, Kazutaka; Nagata, Kazuhiro; Mutus, Bulent

    2018-01-01

    Despite its study since the 1960's, very little is known about the post-translational regulation of the multiple catalytic activities performed by protein disulfide isomerase (PDI), the primary protein folding catalyst of the cell. This work identifies a functional role for the highly conserved CxxC-flanking residues Lys 57 and Lys 401 of human PDI in vitro . Mutagenesis studies have revealed these residues as modulating the oxidoreductase activity of PDI in a pH-dependent manner. Non-conservative amino acid substitutions resulted in enzyme variants upwards of 7-fold less efficient. This attenuated activity was found to translate into a 2-fold reduction of the rate of electron shuttling between PDI and the intraluminal endoplasmic reticulum oxidase, ERO1α, suggesting a functional significance to oxidative protein folding. In light of this, the possibility of lysine acetylation at residues Lys 57 and Lys 401 was assessed by in vitro treatment using acetylsalicylic acid (aspirin). A total of 28 acetyllysine residues were identified, including acLys 57 and acLys 401 . The kinetic behavior of the acetylated protein form nearly mimicked that obtained with a K57/401Q double substitution variant providing an indication that acetylation of the active site-flanking lysine residues can act to reversibly modulate PDI activity.

  10. Hepatitis C virus NS5A protein is a substrate for the peptidyl-prolyl cis/trans isomerase activity of cyclophilins A and B.

    Science.gov (United States)

    Hanoulle, Xavier; Badillo, Aurélie; Wieruszeski, Jean-Michel; Verdegem, Dries; Landrieu, Isabelle; Bartenschlager, Ralf; Penin, François; Lippens, Guy

    2009-05-15

    We report here a biochemical and structural characterization of domain 2 of the nonstructural 5A protein (NS5A) from the JFH1 Hepatitis C virus strain and its interactions with cyclophilins A and B (CypA and CypB). Gel filtration chromatography, circular dichroism spectroscopy, and finally NMR spectroscopy all indicate the natively unfolded nature of this NS5A-D2 domain. Because mutations in this domain have been linked to cyclosporin A resistance, we used NMR spectroscopy to investigate potential interactions between NS5A-D2 and cellular CypA and CypB. We observed a direct molecular interaction between NS5A-D2 and both cyclophilins. The interaction surface on the cyclophilins corresponds to their active site, whereas on NS5A-D2, it proved to be distributed over the many proline residues of the domain. NMR heteronuclear exchange spectroscopy yielded direct evidence that many proline residues in NS5A-D2 form a valid substrate for the enzymatic peptidyl-prolyl cis/trans isomerase (PPIase) activity of CypA and CypB.

  11. Characterization of the guinea pig 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase expressed in the adrenal gland and gonads.

    Science.gov (United States)

    Durocher, Francine; Sanchez, Rocio; Ricketts, Marie-Louise; Labrie, Yvan; Laudet, Vincent; Simard, Jacques

    2005-11-01

    The guinea pig adrenal gland, analogous to the human, possesses the capacity to synthesize C(19) steroids. In order to further understand the control of guinea pig adrenal steroidogenesis we undertook the characterization of the guinea pig 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3beta-HSD) expressed in the adrenal gland. A cDNA clone encoding guinea pig 3beta-HSD isolated from a guinea pig adrenal library is predicted to encode a protein of 373 amino acid residues and 41,475Da. Ribonuclease protection assay suggests that this cDNA corresponds to the predominant, if not the sole, mRNA species detectable in total RNA from the guinea pig adrenal gland, ovary and testis. The guinea pig 3beta-HSD shows a similar affinity for both pregnenolone and dehydroepiandrosterone, and in addition, a 17beta-HSD type II-like activity was also observed. A phylogenetical analysis of the 3beta-HSD gene family demonstrates that the guinea pig is in a parallel branch to the myomorpha group supporting the hypothesis that the guinea pig lineage has branched off after the divergence among primates, artiodactyls and rodents, suggesting the paraphyly of the order rodentia.

  12. Analysis of the relationship between Chalcone Isomerase gene expression level and rutin production in Ficus deltoidea var. deltoidea and F. deltoidea var. angustifolia

    Science.gov (United States)

    Najid, Najihah Mohd; Zain, Che Radziah Che Mohd; Zainal, Zamri

    2016-11-01

    Ficus deltoidea (moraceae) is a herbal plant with medicinal values. Previous studies reported that the F. deltoidea contains a high level of bioactive compounds such as flavonoids. A cDNA encodes for chalcone isomerase was identified from F. deltoidea, designated as FdCHI, which involved in the isomerization of naringenin chalcone to naringenin. Naringenin is a key branch point for the synthesis of rutin, which is believed involved in defense mechanism in the plant. Therefore, we hypothesized that there might be a direct relationship between FdCHI expression level and rutin production in leaves of F. deltoidea var. deltoidea (FDD) and F. deltoidea var. angustifolia (FDA). Our result showed that expression level of FdCHI in leaves FDD was greater than FDA. Analysis of High Performance Liquid Chromatography (HPLC) revealed that rutin was only detected in FDA leaves. Based on the results between FdCHI expression and rutin production, this study concluded that there is no relationship between FdCHI expression and rutin production in leaves of FDA and FDD.

  13. Co-expression of D-glucose isomerase and D-psicose 3-epimerase: development of an efficient one-step production of D-psicose.

    Science.gov (United States)

    Men, Yan; Zhu, Yueming; Zeng, Yan; Izumori, Ken; Sun, Yuanxia; Ma, Yanhe

    2014-10-01

    D-Psicose has been attracting attention in recent years because of its alimentary activities and is used as an ingredient in a range of foods and dietary supplements. To develop a one-step enzymatic process of D-psicose production, thermoactive D-glucose isomerase and the D-psicose 3-epimerase obtained from Bacillus sp. and Ruminococcus sp., respectively, were successfully co-expressed in Escherichia coli BL21 strain. The substrate of one-step enzymatic process was D-glucose. The co-expression system exhibited maximum activity at 65 °C and pH 7.0. Mg(2+) could enhance the output of D-psicose by 2.32 fold to 1.6 g/L from 10 g/L of D-glucose. When using high-fructose corn syrup (HFCS) as substrate, 135 g/L D-psicose was produced under optimum conditions. The mass ratio of D-glucose, D-fructose, and D-psicose was almost 3.0:2.7:1.0, when the reaction reached equilibrium after an 8h incubation time. This co-expression system approaching to produce D-psicose has potential application in food and beverage products, especially softdrinks. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Improvement and characterization of a hyperthermophilic glucose isomerase from Thermoanaerobacter ethanolicus and its application in production of high fructose corn syrup.

    Science.gov (United States)

    Liu, Zhi-Qiang; Zheng, Wei; Huang, Jian-Feng; Jin, Li-Qun; Jia, Dong-Xu; Zhou, Hai-Yan; Xu, Jian-Miao; Liao, Cheng-Jun; Cheng, Xin-Ping; Mao, Bao-Xing; Zheng, Yu-Guo

    2015-08-01

    High fructose corn syrup (HFCS) is an alternative of liquid sweetener to sucrose that is isomerized by commercial glucose isomerase (GI). One-step production of 55 % HFCS by thermostable GI has been drawn more and more attentions. In this study, a new hyperthermophilic GI from Thermoanaerobacter ethanolicus CCSD1 (TEGI) was identified by genome mining, and then a 1317 bp fragment encoding the TEGI was synthesized and expressed in Escherichia coli BL21(DE3). To improve the activity of TEGI, two amino acid residues, Trp139 and Val186, around the active site and substrate-binding pocket based on the structural analysis and molecular docking were selected for site-directed mutagenesis. The specific activity of mutant TEGI-W139F/V186T was 2.3-fold and the value of k cat/K m was 1.86-fold as compared to the wild type TEGI, respectively. Thermostability of mutant TEGI-W139F/V186T at 90 °C for 24 h showed 1.21-fold extension than that of wild type TEGI. During the isomerization of glucose to fructose, the yield of fructose could maintain above 55.4 % by mutant TEGI-W139F/V186T as compared to 53.8 % by wild type TEGI at 90 °C. This study paved foundation for the production of 55 % HFCS using the thermostable TEGI.

  15. 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.

  16. Performance of Glutamate Dehydrogenase and Triose Phosphate Isomerase Genes in the Analysis of Genotypic Variability of Isolates of Giardia duodenalis from Livestocks

    Science.gov (United States)

    Fava, Natália M. N.; Soares, Rodrigo M.; Scalia, Luana A. M.; Kalapothakis, Evanguedes; Pena, Isabella F.; Vieira, Carlos U.; Faria, Elaine S. M.; Cunha, Maria J.; Couto, Talles R.; Cury, Márcia Cristina

    2013-01-01

    Giardia duodenalis is a small intestinal protozoan parasite of several terrestrial vertebrates. This work aims to assess the genotypic variability of Giardia duodenalis isolates from cattle, sheep and pigs in the Southeast of Brazil, by comparing the standard characterization between glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) primers. Fecal samples from the three groups of animals were analyzed using the zinc sulphate centrifugal flotation technique. Out of 59 positive samples, 30 were from cattle, 26 from sheep and 3 from pigs. Cyst pellets were stored and submitted to PCR and nested-PCR reactions with gdh and tpi primers. Fragment amplification of gdh and tpi genes was observed in 25 (42.4%) and 36 (61.0%) samples, respectively. Regarding the sequencing, 24 sequences were obtained with gdh and 20 with tpi. For both genes, there was a prevalence of E specific species assemblage, although some isolates have been identified as A and B, by the tpi sequencing. This has also shown a larger number of heterogeneous sequences, which have been attribute to mixed infections between assemblages B and E. The largest variability of inter-assemblage associated to the frequency of heterogeneity provided by tpi sequencing reinforces the polymorphic nature of this gene and makes it an excellent target for studies on molecular epidemiology. PMID:24308010

  17. Differential Selectivity of the Escherichia coli Cell Membrane Shifts the Equilibrium for the Enzyme-Catalyzed Isomerization of Galactose to Tagatose▿

    Science.gov (United States)

    Kim, Jin-Ha; Lim, Byung-Chul; Yeom, Soo-Jin; Kim, Yeong-Su; Kim, Hye-Jung; Lee, Jung-Kul; Lee, Sook-Hee; Kim, Seon-Won; Oh, Deok-Kun

    2008-01-01

    An Escherichia coli galactose kinase gene knockout (ΔgalK) strain, which contains the l-arabinose isomerase gene (araA) to isomerize d-galactose to d-tagatose, showed a high conversion yield of tagatose compared with the original galK strain because galactose was not metabolized by endogenous galactose kinase. In whole cells of the ΔgalK strain, the isomerase-catalyzed reaction exhibited an equilibrium shift toward tagatose, producing a tagatose fraction of 68% at 37°C, whereas the purified l-arabinose isomerase gave a tagatose equilibrium fraction of 36%. These equilibrium fractions are close to those predicted from the measured equilibrium constants of the isomerization reaction catalyzed in whole cells and by the purified enzyme. The equilibrium shift in these cells resulted from the higher uptake and lower release rates for galactose, which is a common sugar substrate, than for tagatose, which is a rare sugar product. A ΔmglB mutant had decreased uptake rates for galactose and tagatose, indicating that a methylgalactoside transport system, MglABC, is the primary contributing transporter for the sugars. In the present study, whole-cell conversion using differential selectivity of the cell membrane was proposed as a method for shifting the equilibrium in sugar isomerization reactions. PMID:18263746

  18. Differential selectivity of the Escherichia coli cell membrane shifts the equilibrium for the enzyme-catalyzed isomerization of galactose to tagatose.

    Science.gov (United States)

    Kim, Jin-Ha; Lim, Byung-Chul; Yeom, Soo-Jin; Kim, Yeong-Su; Kim, Hye-Jung; Lee, Jung-Kul; Lee, Sook-Hee; Kim, Seon-Won; Oh, Deok-Kun

    2008-04-01

    An Escherichia coli galactose kinase gene knockout (DeltagalK) strain, which contains the l-arabinose isomerase gene (araA) to isomerize d-galactose to d-tagatose, showed a high conversion yield of tagatose compared with the original galK strain because galactose was not metabolized by endogenous galactose kinase. In whole cells of the DeltagalK strain, the isomerase-catalyzed reaction exhibited an equilibrium shift toward tagatose, producing a tagatose fraction of 68% at 37 degrees C, whereas the purified l-arabinose isomerase gave a tagatose equilibrium fraction of 36%. These equilibrium fractions are close to those predicted from the measured equilibrium constants of the isomerization reaction catalyzed in whole cells and by the purified enzyme. The equilibrium shift in these cells resulted from the higher uptake and lower release rates for galactose, which is a common sugar substrate, than for tagatose, which is a rare sugar product. A DeltamglB mutant had decreased uptake rates for galactose and tagatose, indicating that a methylgalactoside transport system, MglABC, is the primary contributing transporter for the sugars. In the present study, whole-cell conversion using differential selectivity of the cell membrane was proposed as a method for shifting the equilibrium in sugar isomerization reactions.

  19. Prokaryotic soluble overexpression and purification of bioactive human growth hormone by fusion to thioredoxin, maltose binding protein, and protein disulfide isomerase.

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    Minh Tan Nguyen

    Full Text Available Human growth hormone (hGH is synthesized by somatotroph cells of the anterior pituitary gland and induces cell proliferation and growth. This protein has been approved for the treatment of various conditions, including hGH deficiency, chronic renal failure, and Turner syndrome. Efficient production of hGH in Escherichia coli (E. coli has proven difficult because the E. coli-expressed hormone tends to aggregate and form inclusion bodies, resulting in poor solubility. In this study, seven N-terminal fusion partners, hexahistidine (His6, thioredoxin (Trx, glutathione S-transferase (GST, maltose-binding protein (MBP, N-utilization substance protein A (NusA, protein disulfide bond isomerase (PDI, and the b'a' domain of PDI (PDIb'a', were tested for soluble overexpression of codon-optimized hGH in E. coli. We found that MBP and hPDI tags significantly increased the solubility of the hormone. In addition, lowering the expression temperature to 18°C also dramatically increased the solubility of all the fusion proteins. We purified hGH from MBP-, PDIb'a'-, or Trx-tagged hGH expressed at 18°C in E. coli using simple chromatographic techniques and compared the final purity, yield, and activity of hGH to assess the impact of each partner protein. Purified hGH was highly pure on silver-stained gel and contained very low levels of endotoxin. On average, ∼37 mg, ∼12 mg, and ∼7 mg of hGH were obtained from 500 mL-cell cultures of Trx-hGH, MBP-hGH, and PDIb'a'-hGH, respectively. Subsequently, hGH was analyzed using mass spectroscopy to confirm the presence of two intra-molecular disulfide bonds. The bioactivity of purified hGHs was demonstrated using Nb2-11 cell.

  20. Spectroscopic investigation of new water soluble Mn(II)(2) and Mg(II)(2) complexes for the substrate binding models of xylose/glucose isomerases.

    Science.gov (United States)

    Patra, Ayan; Bera, Manindranath

    2014-01-30

    In methanol, the reaction of stoichiometric amounts of Mn(OAc)(2)·4H(2)O and the ligand H(3)hpnbpda [H(3)hpnbpda=N,N'-bis(2-pyridylmethyl)-2-hydroxy-1,3-propanediamine-N,N'-diacetic acid] in the presence of NaOH, afforded a new water soluble dinuclear manganese(II) complex, [Mn2(hpnbpda)(μ-OAc)] (1). Similarly, the reaction of Mg(OAc)(2)·4H(2)O and the ligand H3hpnbpda in the presence of NaOH, in methanol, yielded a new water soluble dinuclear magnesium(II) complex, [Mg2(hpnbpda)(μ-OAc)(H2O)2] (2). DFT calculations have been performed for the structural optimization of complexes 1 and 2. The DFT optimized structure of complex 1 shows that two manganese(II) centers are in a distorted square pyramidal geometry, whereas the DFT optimized structure of complex 2 reveals that two magnesium(II) centers adopt a six-coordinate distorted octahedral geometry. To understand the mode of substrate binding and the mechanistic details of the active site metals in xylose/glucose isomerases (XGI), we have investigated the binding interactions of biologically important monosaccharides d-glucose and d-xylose with complexes 1 and 2, in aqueous alkaline solution by a combined approach of FTIR, UV-vis, fluorescence, and (13)C NMR spectroscopic techniques. Fluorescence spectra show the binding-induced gradual decrease in emission of complexes 1 and 2 accompanied by a significant blue shift upon increasing the concentration of sugar substrates. The binding modes of d-glucose and d-xylose with complex 2 are indicated by their characteristic coordination induced shift (CIS) values in (13)C NMR spectra for C1 and C2 carbon atoms. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Enolase 1 (ENO1 and protein disulfide-isomerase associated 3 (PDIA3 regulate Wnt/β-catenin-driven trans-differentiation of murine alveolar epithelial cells

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    Kathrin Mutze

    2015-08-01

    Full Text Available The alveolar epithelium represents a major site of tissue destruction during lung injury. It consists of alveolar epithelial type I (ATI and type II (ATII cells. ATII cells are capable of self-renewal and exert progenitor function for ATI cells upon alveolar epithelial injury. Cell differentiation pathways enabling this plasticity and allowing for proper repair, however, are poorly understood. Here, we applied proteomics, expression analysis and functional studies in primary murine ATII cells to identify proteins and molecular mechanisms involved in alveolar epithelial plasticity. Mass spectrometry of cultured ATII cells revealed a reduction of carbonyl reductase 2 (CBR2 and an increase in enolase 1 (ENO1 and protein disulfide-isomerase associated 3 (PDIA3 protein expression during ATII-to-ATI cell trans-differentiation. This was accompanied by increased Wnt/β-catenin signaling, as analyzed by qRT-PCR and immunoblotting. Notably, ENO1 and PDIA3, along with T1α (podoplanin; an ATI cell marker, exhibited decreased protein expression upon pharmacological and molecular Wnt/β-catenin inhibition in cultured ATII cells, whereas CBR2 levels were stabilized. Moreover, we analyzed primary ATII cells from mice with bleomycin-induced lung injury, a model exhibiting activated Wnt/β-catenin signaling in vivo. We observed reduced CBR2 significantly correlating with surfactant protein C (SFTPC, whereas ENO1 and PDIA3 along with T1α were increased in injured ATII cells. Finally, siRNA-mediated knockdown of ENO1, as well as PDIA3, in primary ATII cells led to reduced T1α expression, indicating diminished cell trans-differentiation. Our data thus identified proteins involved in ATII-to-ATI cell trans-differentiation and suggest a Wnt/β-catenin-driven functional role of ENO1 and PDIA3 in alveolar epithelial cell plasticity in lung injury and repair.

  2. TAL effectors target the C-terminal domain of RNA polymerase II (CTD by inhibiting the prolyl-isomerase activity of a CTD-associated cyclophilin.

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    Mariane Noronha Domingues

    Full Text Available Transcriptional activator-like (TAL effectors of plant pathogenic bacteria function as transcription factors in plant cells. However, how TAL effectors control transcription in the host is presently unknown. Previously, we showed that TAL effectors of the citrus canker pathogen Xanthomonas citri, named PthAs, targeted the citrus protein complex comprising the thioredoxin CsTdx, ubiquitin-conjugating enzymes CsUev/Ubc13 and cyclophilin CsCyp. Here we show that CsCyp complements the function of Cpr1 and Ess1, two yeast cyclophilins that regulate transcription by the isomerization of proline residues of the regulatory C-terminal domain (CTD of RNA polymerase II. We also demonstrate that CsCyp, CsTdx, CsUev and four PthA variants interact with the citrus CTD and that CsCyp co-immunoprecipitate with the CTD in citrus cell extracts and with PthA2 transiently expressed in sweet orange epicotyls. The interactions of CsCyp with the CTD and PthA2 were inhibited by cyclosporin A (CsA, a cyclophilin inhibitor. Moreover, we present evidence that PthA2 inhibits the peptidyl-prolyl cis-trans isomerase (PPIase activity of CsCyp in a similar fashion as CsA, and that silencing of CsCyp, as well as treatments with CsA, enhance canker lesions in X. citri-infected leaves. Given that CsCyp appears to function as a negative regulator of cell growth and that Ess1 negatively regulates transcription elongation in yeast, we propose that PthAs activate host transcription by inhibiting the PPIase activity of CsCyp on the CTD.

  3. Structural analysis of substrate recognition by glucose isomerase in Mn2+ binding mode at M2 site in S. rubiginosus.

    Science.gov (United States)

    Bae, Ji-Eun; Hwang, Kwang Yeon; Nam, Ki Hyun

    2018-06-16

    Glucose isomerase (GI) catalyzes the reversible enzymatic isomerization of d-glucose and d-xylose to d-fructose and d-xylulose, respectively. This is one of the most important enzymes in the production of high-fructose corn syrup (HFCS) and biofuel. We recently determined the crystal structure of GI from S. rubiginosus (SruGI) complexed with a xylitol inhibitor in one metal binding mode. Although we assessed inhibitor binding at the M1 site, the metal binding at the M2 site and the substrate recognition mechanism for SruGI remains the unclear. Here, we report the crystal structure of the two metal binding modes of SruGI and its complex with glucose. This study provides a snapshot of metal binding at the SruGI M2 site in the presence of Mn 2+ , but not in the presence of Mg 2+ . Metal binding at the M2 site elicits a configuration change at the M1 site. Glucose molecule can only bind to the M1 site in presence of Mn 2+ at the M2 site. Glucose and Mn 2+ at the M2 site were bridged by water molecules using a hydrogen bonding network. The metal binding geometry of the M2 site indicates a distorted octahedral coordination with an angle of 55-110°, whereas the M1 site has a relatively stable octahedral coordination with an angle of 85-95°. We suggest a two-step sequential process for SruGI substrate recognition, in Mn 2+ binding mode, at the M2 site. Our results provide a better understanding of the molecular role of the M2 site in GI substrate recognition. Copyright © 2018. Published by Elsevier Inc.

  4. Proline substitutions in a Mip-like peptidyl-prolyl cis-trans isomerase severely affect its structure, stability, shape and activity

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    Soumitra Polley

    2015-01-01

    Full Text Available FKBP22, an Escherichia coli-specific peptidyl-prolyl cis-trans isomerase, shows substantial homology with the Mip-like virulence factors. Mip-like proteins are homodimeric and possess a V-shaped conformation. Their N-terminal domains form dimers, whereas their C-terminal domains bind protein/peptide substrates and distinct inhibitors such as rapamycin and FK506. Interestingly, the two domains of the Mip-like proteins are separated by a lengthy, protease-susceptible α-helix. To delineate the structural requirement of this domain-connecting region in Mip-like proteins, we have investigated a recombinant FKBP22 (rFKBP22 and its three point mutants I65P, V72P and A82P using different probes. Each mutant harbors a Pro substitution mutation at a distinct location in the hinge region. We report that the three mutants are not only different from each other but also different from rFKBP22 in structure and activity. Unlike rFKBP22, the three mutants were unfolded by a non-two state mechanism in the presence of urea. In addition, the stabilities of the mutants, particularly I65P and V72P, differed considerably from that of rFKBP22. Conversely, the rapamycin binding affinity of no mutant was different from that of rFKBP22. Of the mutants, I65P showed the highest levels of structural/functional loss and dissociated partly in solution. Our computational study indicated a severe collapse of the V-shape in I65P due to the anomalous movement of its C-terminal domains. The α-helical nature of the domain-connecting region is, therefore, critical for the Mip-like proteins.

  5. Inhibition of the functional interplay between endoplasmic reticulum (ER) oxidoreduclin-1α (Ero1α) and protein-disulfide isomerase (PDI) by the endocrine disruptor bisphenol A.

    Science.gov (United States)

    Okumura, Masaki; Kadokura, Hiroshi; Hashimoto, Shoko; Yutani, Katsuhide; Kanemura, Shingo; Hikima, Takaaki; Hidaka, Yuji; Ito, Len; Shiba, Kohei; Masui, Shoji; Imai, Daiki; Imaoka, Susumu; Yamaguchi, Hiroshi; Inaba, Kenji

    2014-09-26

    Bisphenol A (BPA) is an endocrine disruptor that may have adverse effects on human health. We recently isolated protein-disulfide isomerase (PDI) as a BPA-binding protein from rat brain homogenates and found that BPA markedly inhibited PDI activity. To elucidate mechanisms of this inhibition, detailed structural, biophysical, and functional analyses of PDI were performed in the presence of BPA. BPA binding to PDI induced significant rearrangement of the N-terminal thioredoxin domain of PDI, resulting in more compact overall structure. This conformational change led to closure of the substrate-binding pocket in b' domain, preventing PDI from binding to unfolded proteins. The b' domain also plays an essential role in the interplay between PDI and ER oxidoreduclin 1α (Ero1α), a flavoenzyme responsible for reoxidation of PDI. We show that BPA inhibited Ero1α-catalyzed PDI oxidation presumably by inhibiting the interaction between the b' domain of PDI and Ero1α; the phenol groups of BPA probably compete with a highly conserved tryptophan residue, located in the protruding β-hairpin of Ero1α, for binding to PDI. Consistently, BPA slowed down the reoxidation of PDI and caused the reduction of PDI in HeLa cells, indicating that BPA has a great impact on the redox homeostasis of PDI within cells. However, BPA had no effect on the interaction between PDI and peroxiredoxin-4 (Prx4), another PDI family oxidase, suggesting that the interaction between Prx4 and PDI is different from that of Ero1α and PDI. These results indicate that BPA, a widely distributed and potentially harmful chemical, inhibits Ero1-PDI-mediated disulfide bond formation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Inhibition of the Functional Interplay between Endoplasmic Reticulum (ER) Oxidoreduclin-1α (Ero1α) and Protein-disulfide Isomerase (PDI) by the Endocrine Disruptor Bisphenol A*

    Science.gov (United States)

    Okumura, Masaki; Kadokura, Hiroshi; Hashimoto, Shoko; Yutani, Katsuhide; Kanemura, Shingo; Hikima, Takaaki; Hidaka, Yuji; Ito, Len; Shiba, Kohei; Masui, Shoji; Imai, Daiki; Imaoka, Susumu; Yamaguchi, Hiroshi; Inaba, Kenji

    2014-01-01

    Bisphenol A (BPA) is an endocrine disruptor that may have adverse effects on human health. We recently isolated protein-disulfide isomerase (PDI) as a BPA-binding protein from rat brain homogenates and found that BPA markedly inhibited PDI activity. To elucidate mechanisms of this inhibition, detailed structural, biophysical, and functional analyses of PDI were performed in the presence of BPA. BPA binding to PDI induced significant rearrangement of the N-terminal thioredoxin domain of PDI, resulting in more compact overall structure. This conformational change led to closure of the substrate-binding pocket in b′ domain, preventing PDI from binding to unfolded proteins. The b′ domain also plays an essential role in the interplay between PDI and ER oxidoreduclin 1α (Ero1α), a flavoenzyme responsible for reoxidation of PDI. We show that BPA inhibited Ero1α-catalyzed PDI oxidation presumably by inhibiting the interaction between the b′ domain of PDI and Ero1α; the phenol groups of BPA probably compete with a highly conserved tryptophan residue, located in the protruding β-hairpin of Ero1α, for binding to PDI. Consistently, BPA slowed down the reoxidation of PDI and caused the reduction of PDI in HeLa cells, indicating that BPA has a great impact on the redox homeostasis of PDI within cells. However, BPA had no effect on the interaction between PDI and peroxiredoxin-4 (Prx4), another PDI family oxidase, suggesting that the interaction between Prx4 and PDI is different from that of Ero1α and PDI. These results indicate that BPA, a widely distributed and potentially harmful chemical, inhibits Ero1-PDI-mediated disulfide bond formation. PMID:25122773

  7. Modulation of neutrophil superoxide generation by inhibitors of protein kinase C, calmodulin, diacylglycerol and myosin light chain kinases, and peptidyl prolyl cis-trans isomerase.

    Science.gov (United States)

    Bergstrand, H; Eriksson, T; Hallberg, A; Johansson, B; Karabelas, K; Michelsen, P; Nybom, A

    1992-12-01

    To assess the role of protein kinase C (PKC) in the respiratory burst of adherent human polymorphonuclear leukocytes (PMNL), reduction of ferricytochrome C by cells triggered with a phorbol ester (PMA), ionophore A23187, serum-treated zymosan (STZ) or three lipid derivatives, 3-decanoyl-sn-glycerol (G-3-OCOC9), (R,R)-1,4-diethyl-2-O-decyl-L-tartrate (Tt-2-OC10) and 3-decyloxy-5-hydroxymethylphenol (DHP) was examined in a microtiter plate procedure in the presence of inhibitors of PKC and, for comparison, inhibitors of calmodulin, diacylglycerol and myosin light chain kinases and the peptidyl-prolyl cis-trans isomerase activity of fujiphilin. 1) Of the protein kinase inhibitors examined, Ro 31-7549 and staurosporine reduced responses to all stimuli except possibly STZ; in contrast, K252a and the myosin light chain kinase inhibitors ML-7 and ML-9 blocked responses to A23187 and STZ better than those triggered by PMA. H-7 reduced responses to A23187, DHP and G-3-OCOC9, and calphostin, palmitoyl carnitine, sphingosine and the multifunctional drugs TMB-8 and W-7 reduced A23187; they also, when examined, reduced decane derivative-induced O2- production more effectively than PMA- and STZ-triggered responses. Polymyxin B, 4 alpha-PMA and retinal displayed no inhibitory capacity. 2) Of the selective calmodulin antagonists, CGS 9343B, Ro 22-4839 and calmidazolium did not inhibit the oxidative response irrespective of the stimulus used, whereas metofenazate reduced those evoked by A23187, DHP, G-3-OCOC9 and STZ.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. The Glycolytic Enzyme Triosephosphate Isomerase of Trichomonas vaginalis Is a Surface-Associated Protein Induced by Glucose That Functions as a Laminin- and Fibronectin-Binding Protein.

    Science.gov (United States)

    Miranda-Ozuna, Jesús F T; Hernández-García, Mar S; Brieba, Luis G; Benítez-Cardoza, Claudia G; Ortega-López, Jaime; González-Robles, Arturo; Arroyo, Rossana

    2016-10-01

    Triosephosphate isomerase of Trichomonas vaginalis (TvTIM) is a 27-kDa cytoplasmic protein encoded by two genes, tvtim1 and tvtim2, that participates in glucose metabolism. TvTIM is also localized to the parasite surface. Thus, the goal of this study was to identify the novel functions of the surface-associated TvTIM in T. vaginalis and to assess the effect of glucose as an environmental factor that regulates its expression and localization. Reverse transcription-PCR (RT-PCR) showed that the tvtim genes were differentially expressed in response to glucose concentration. tvtim1 was overexpressed under glucose-restricted (GR) conditions, whereas tvtim2 was overexpressed under glucose-rich, or high-glucose (HG), conditions. Western blot and indirect immunofluorescence assays also showed that glucose positively affected the amount and surface localization of TvTIM in T. vaginalis Affinity ligand assays demonstrated that the recombinant TvTIM1 and TvTIM2 proteins bound to laminin (Lm) and fibronectin (Fn) but not to plasminogen. Moreover, higher levels of adherence to Lm and Fn were detected in parasites grown under HG conditions than in those grown under GR conditions. Furthermore, pretreatment of trichomonads with an anti-TvTIMr polyclonal antibody or pretreatment of Lm- or Fn-coated wells with both recombinant proteins (TvTIM1r and TvTIM2r) specifically reduced the binding of live parasites to Lm and Fn in a concentration-dependent manner. Moreover, T. vaginalis was exposed to different glucose concentrations during vaginal infection of women with trichomoniasis. Our data indicate that TvTIM is a surface-associated protein under HG conditions that mediates specific binding to Lm and Fn as a novel virulence factor of T. vaginalis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  9. Structure, expression profile and phylogenetic inference of chalcone isomerase-like genes from the narrow-leafed lupin (Lupinus angustifolius L. genome

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    Łucja ePrzysiecka

    2015-04-01

    Full Text Available Lupins, like other legumes, have a unique biosynthesis scheme of 5-deoxy-type flavonoids and isoflavonoids. A key enzyme in this pathway is chalcone isomerase (CHI, a member of CHI-fold protein family, encompassing subfamilies of CHI1, CHI2, CHI-like (CHIL, and fatty acid-binding (FAP proteins. Here, two Lupinus angustifolius (narrow-leafed lupin CHILs, LangCHIL1 and LangCHIL2, were identified and characterized using DNA fingerprinting, cytogenetic and linkage mapping, sequencing and expression profiling. Clones carrying CHIL sequences were assembled into two contigs. Full gene sequences were obtained from these contigs, and mapped in two L. angustifolius linkage groups by gene-specific markers. Bacterial artificial chromosome fluorescence in situ hybridization approach confirmed the localization of two LangCHIL genes in distinct chromosomes. The expression profiles of both LangCHIL isoforms were very similar. The highest level of transcription was in the roots of the third week of plant growth; thereafter, expression declined. The expression of both LangCHIL genes in leaves and stems was similar and low. Comparative mapping to reference legume genome sequences revealed strong syntenic links; however, LangCHIL2 contig had a much more conserved structure than LangCHIL1. LangCHIL2 is assumed to be an ancestor gene, whereas LangCHIL1 probably appeared as a result of duplication. As both copies are transcriptionally active, questions arise concerning their hypothetical functional divergence. Screening of the narrow-leafed lupin genome and transcriptome with CHI-fold protein sequences, followed by Bayesian inference of phylogeny and cross-genera synteny survey, identified representatives of all but one (CHI1 main subfamilies. They are as follows: two copies of CHI2, FAPa2 and CHIL, and single copies of FAPb and FAPa1. Duplicated genes are remnants of whole genome duplication which is assumed to have occurred after the divergence of Lupinus, Arachis

  10. Discovery of ebselen as an inhibitor of Cryptosporidium parvum glucose-6-phosphate isomerase (CpGPI) by high-throughput screening of existing drugs.

    Science.gov (United States)

    Eltahan, Rana; Guo, Fengguang; Zhang, Haili; Xiang, Lixin; Zhu, Guan

    2018-04-01

    Cryptosporidium parvum is a water-borne and food-borne apicomplexan pathogen. It is one of the top four diarrheal-causing pathogens in children under the age of five in developing countries, and an opportunistic pathogen in immunocompromised individuals. Unlike other apicomplexans, C. parvum lacks Kreb's cycle and cytochrome-based respiration, thus relying mainly on glycolysis to produce ATP. In this study, we characterized the primary biochemical features of the C. parvum glucose-6-phosphate isomerase (CpGPI) and determined its Michaelis constant towards fructose-6-phosphate (K m  = 0.309 mM, V max  = 31.72 nmol/μg/min). We also discovered that ebselen, an organoselenium drug, was a selective inhibitor of CpGPI by high-throughput screening of 1200 known drugs. Ebselen acted on CpGPI as an allosteric noncompetitive inhibitor (IC 50  = 8.33 μM; K i  = 36.33 μM), while complete inhibition of CpGPI activity was not achieved. Ebselen could also inhibit the growth of C. parvum in vitro (EC 50  = 165 μM) at concentrations nontoxic to host cells, albeit with a relatively small in vitro safety window of 4.2 (cytotoxicity TC 50 on HCT-8 cells = 700 μM). Additionally, ebselen might also target other enzymes in the parasite, leading to the parasite growth reduction. Therefore, although ebselen is useful in studying the inhibition of CpGPI enzyme activity, further proof is needed to chemically and/or genetically validate CpGPI as a drug target. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. Enzymatic characterization and gene identification of aconitate isomerase, an enzyme involved in assimilation of trans-aconitic acid, from Pseudomonas sp. WU-0701.

    Science.gov (United States)

    Yuhara, Kahori; Yonehara, Hiromi; Hattori, Takasumi; Kobayashi, Keiichi; Kirimura, Kohtaro

    2015-11-01

    trans-Aconitic acid is an unsaturated organic acid that is present in some plants such as soybean and wheat; however, it remains unclear how trans-aconitic acid is degraded and/or assimilated by living cells in nature. From soil, we isolated Pseudomonas sp. WU-0701 assimilating trans-aconitic acid as a sole carbon source. In the cell-free extract of Pseudomonas sp. WU-0701, aconitate isomerase (AI; EC 5.3.3.7) activity was detected. Therefore, it seems likely that strain Pseudomonas sp. WU-0701 converts trans-aconitic acid to cis-aconitic acid with AI, and assimilates this via the tricarboxylic acid cycle. For the characterization of AI from Pseudomonas sp. WU-0701, we performed purification, determination of enzymatic properties and gene identification of AI. The molecular mass of AI purified from cell-free extract was estimated to be ~ 25 kDa by both SDS/PAGE and gel filtration analyses, indicating that AI is a monomeric enzyme. The optimal pH and temperature of purified AI for the reaction were 6.0 °C and 37 °C, respectively. The gene ais encoding AI was cloned on the basis of the N-terminal amino acid sequence of the protein, and Southern blot analysis revealed that only one copy of ais is located on the bacterial genome. The gene ais contains an ORF of 786 bp, encoding a polypeptide of 262 amino acids, including the N-terminal 22 amino acids as a putative periplasm-targeting signal peptide. It is noteworthy that the amino acid sequence of AI shows 90% and 74% identity with molybdenum ABC transporter substrate-binding proteins of Pseudomonas psychrotolerans and Xanthomonas albilineans, respectively. This is the first report on purification to homogeneity, characterization and gene identification of AI. The nucleotide sequence of ais described in this article is available in the DDBJ/EMBL/GenBank nucleotide sequence databases under the Accession No. LC010980. © 2015 FEBS.

  12. Structural modeling and docking studies of ribose 5-phosphate isomerase from Leishmania major and Homo sapiens: a comparative analysis for Leishmaniasis treatment.

    Science.gov (United States)

    Capriles, Priscila V S Z; Baptista, Luiz Phillippe R; Guedes, Isabella A; Guimarães, Ana Carolina R; Custódio, Fabio L; Alves-Ferreira, Marcelo; Dardenne, Laurent E

    2015-02-01

    Leishmaniases are caused by protozoa of the genus Leishmania and are considered the second-highest cause of death worldwide by parasitic infection. The drugs available for treatment in humans are becoming ineffective mainly due to parasite resistance; therefore, it is extremely important to develop a new chemotherapy against these parasites. A crucial aspect of drug design development is the identification and characterization of novel molecular targets. In this work, through an in silico comparative analysis between the genomes of Leishmania major and Homo sapiens, the enzyme ribose 5-phosphate isomerase (R5PI) was indicated as a promising molecular target. R5PI is an important enzyme that acts in the pentose phosphate pathway and catalyzes the interconversion of d-ribose-5-phosphate (R5P) and d-ribulose-5-phosphate (5RP). R5PI activity is found in two analogous groups of enzymes called RpiA (found in H. sapiens) and RpiB (found in L. major). Here, we present the first report of the three-dimensional (3D) structures and active sites of RpiB from L. major (LmRpiB) and RpiA from H. sapiens (HsRpiA). Three-dimensional models were constructed by applying a hybrid methodology that combines comparative and ab initio modeling techniques, and the active site was characterized based on docking studies of the substrates R5P (furanose and ring-opened forms) and 5RP. Our comparative analyses show that these proteins are structural analogs and that distinct residues participate in the interconversion of R5P and 5RP. We propose two distinct reaction mechanisms for the reversible isomerization of R5P to 5RP, which is catalyzed by LmRpiB and HsRpiA. We expect that the present results will be important in guiding future molecular modeling studies to develop new drugs that are specially designed to inhibit the parasitic form of the enzyme without significant effects on the human analog. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production in Trichoderma reesei Rut-C30

    Science.gov (United States)

    2011-01-01

    Background Cellulase and hemicellulase genes in the fungus Trichoderma reesei are repressed by glucose and induced by lactose. Regulation of the cellulase genes is mediated by the repressor CRE1 and the activator XYR1. T. reesei strain Rut-C30 is a hypercellulolytic mutant, obtained from the natural strain QM6a, that has a truncated version of the catabolite repressor gene, cre1. It has been previously shown that bacterial mutants lacking phosphoglucose isomerase (PGI) produce more nucleotide precursors and amino acids. PGI catalyzes the second step of glycolysis, the formation of fructose-6-P from glucose-6-P. Results We deleted the gene pgi1, encoding PGI, in the T. reesei strain Rut-C30 and we introduced the cre1 gene in a Δpgi1 mutant. Both Δpgi1 and cre1+Δpgi1 mutants showed a pellet-like and growth as well as morphological alterations compared with Rut-C30. None of the mutants grew in media with fructose, galactose, xylose, glycerol or lactose but they grew in media with glucose, with fructose and glucose, with galactose and fructose or with lactose and fructose. No growth was observed in media with xylose and glucose. On glucose, Δpgi1 and cre1+Δpgi1 mutants showed higher cellulase activity than Rut-C30 and QM6a, respectively. But in media with lactose, none of the mutants improved the production of the reference strains. The increase in the activity did not correlate with the expression of mRNA of the xylanase regulator gene, xyr1. Δpgi1 mutants were also affected in the extracellular β-galactosidase activity. Levels of mRNA of the glucose 6-phosphate dehydrogenase did not increase in Δpgi1 during growth on glucose. Conclusions The ability to grow in media with glucose as the sole carbon source indicated that Trichoderma Δpgi1 mutants were able to use the pentose phosphate pathway. But, they did not increase the expression of gpdh. Morphological characteristics were the result of the pgi1 deletion. Deletion of pgi1 in Rut-C30 increased cellulase

  14. An Intracellular Peptidyl-Prolyl cis/trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease.

    Science.gov (United States)

    Wiemels, Richard E; Cech, Stephanie M; Meyer, Nikki M; Burke, Caleb A; Weiss, Andy; Parks, Anastacia R; Shaw, Lindsey N; Carroll, Ronan K

    2017-01-01

    Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus IMPORTANCE: Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently

  15. Atypical protein disulfide isomerases (PDI: Comparison of the molecular and catalytic properties of poplar PDI-A and PDI-M with PDI-L1A.

    Directory of Open Access Journals (Sweden)

    Benjamin Selles

    Full Text Available Protein disulfide isomerases are overwhelmingly multi-modular redox catalysts able to perform the formation, reduction or isomerisation of disulfide bonds. We present here the biochemical characterization of three different poplar PDI isoforms. PDI-A is characterized by a single catalytic Trx module, the so-called a domain, whereas PDI-L1a and PDI-M display an a-b-b'-a' and a°-a-b organisation respectively. Their activities have been tested in vitro using purified recombinant proteins and a series of model substrates as insulin, NADPH thioredoxin reductase, NADP malate dehydrogenase (NADP-MDH, peroxiredoxins or RNase A. We demonstrated that PDI-A exhibited none of the usually reported activities, although the cysteines of the WCKHC active site signature are able to form a disulfide with a redox midpoint potential of -170 mV at pH 7.0. The fact that it is able to bind a [Fe2S2] cluster upon Escherichia coli expression and anaerobic purification might indicate that it does not have a function in dithiol-disulfide exchange reactions. The two other proteins were able to catalyze oxidation or reduction reactions, PDI-L1a being more efficient in most cases, except that it was unable to activate the non-physiological substrate NADP-MDH, in contrast to PDI-M. To further evaluate the contribution of the catalytic domains of PDI-M, the dicysteinic motifs have been independently mutated in each a domain. The results indicated that the two a domains seem interconnected and that the a° module preferentially catalyzed oxidation reactions whereas the a module catalyzed reduction reactions, in line with the respective redox potentials of -170 mV and -190 mV at pH 7.0. Overall, these in vitro results illustrate that the number and position of a and b domains influence the redox properties and substrate recognition (both electron donors and acceptors of PDI which contributes to understand why this protein family expanded along evolution.

  16. Insights into the evolution of enzyme substrate promiscuity after the discovery of (βα)₈ isomerase evolutionary intermediates from a diverse metagenome.

    Science.gov (United States)

    Noda-García, Lianet; Juárez-Vázquez, Ana L; Ávila-Arcos, María C; Verduzco-Castro, Ernesto A; Montero-Morán, Gabriela; Gaytán, Paul; Carrillo-Tripp, Mauricio; Barona-Gómez, Francisco

    2015-06-10

    Current sequence-based approaches to identify enzyme functional shifts, such as enzyme promiscuity, have proven to be highly dependent on a priori functional knowledge, hampering our ability to reconstruct evolutionary history behind these mechanisms. Hidden Markov Model (HMM) profiles, broadly used to classify enzyme families, can be useful to distinguish between closely related enzyme families with different specificities. The (βα)8-isomerase HisA/PriA enzyme family, involved in L-histidine (HisA, mono-substrate) biosynthesis in most bacteria and plants, but also in L-tryptophan (HisA/TrpF or PriA, dual-substrate) biosynthesis in most Actinobacteria, has been used as model system to explore evolutionary hypotheses and therefore has a considerable amount of evolutionary, functional and structural knowledge available. We searched for functional evolutionary intermediates between the HisA and PriA enzyme families in order to understand the functional divergence between these families. We constructed a HMM profile that correctly classifies sequences of unknown function into the HisA and PriA enzyme sub-families. Using this HMM profile, we mined a large metagenome to identify plausible evolutionary intermediate sequences between HisA and PriA. These sequences were used to perform phylogenetic reconstructions and to identify functionally conserved amino acids. Biochemical characterization of one selected enzyme (CAM1) with a mutation within the functionally essential N-terminus phosphate-binding site, namely, an alanine instead of a glycine in HisA or a serine in PriA, showed that this evolutionary intermediate has dual-substrate specificity. Moreover, site-directed mutagenesis of this alanine residue, either backwards into a glycine or forward into a serine, revealed the robustness of this enzyme. None of these mutations, presumably upon functionally essential amino acids, significantly abolished its enzyme activities. A truncated version of this enzyme (CAM2

  17. Autophagy regulated by prolyl isomerase Pin1 and phospho-Ser-GSK3αβ involved in protection of oral squamous cell carcinoma against cadmium toxicity

    Energy Technology Data Exchange (ETDEWEB)

    So, Keum-Young [Department of Anesthesiology and Pain Medicine College of Dentistry, Chosun University, 309 Pilmundaero, Dong-gu, Gwangju 501-759 (Korea, Republic of); Ahn, Sang-Gun [Department of Pathology, College of Dentistry, Chosun University, 309 Pilmundaero, Dong-gu, Gwangju 501-759 (Korea, Republic of); Oh, Seon-Hee, E-mail: seonh@chosun.ac.kr [Department of Premedicine, School of Medicine, College of Dentistry, Chosun University, 309 Pilmundaero, Dong-gu, Gwangju 501-759 (Korea, Republic of)

    2015-10-23

    Prolyl isomerase Pin1 plays an important role in cell proliferation and is overexpressed in many human tumors. However, its role in autophagy induction remains undefined. Here we show that Pin1 regulates cell survival via autophagy in cadmium (Cd)-exposed oral squamous cell carcinoma (OSCC). OSCC exposure to Cd induced autophagy, as demonstrated by the formation of green fluorescent punctae in transfected cells expressing GFP-conjugated microtubule-associated protein light chain 3 (LC3) and by LC3 flux in the presence of autophagy inhibitors. Suppression of Atg5 enhanced Cd-induced apoptosis, indicating that autophagy is involved in cell protection. In dose–response experiments, cleavage of procaspase-3, PARP-1, and LC3-II was induced by Cd with an IC{sub 50} of 45 μM. Expression of Pin1 was decreased at or above the Cd IC{sub 50} value and was inversely correlated with the level of phospho(p)-Ser-GSK3αβ. Genetic or pharmacologic inhibition of Pin1 suppressed Cd-induced autophagy, but increased p-Akt-mediated p-Ser-GSK3αβ; this was reversed by overexpression of Pin1. However, suppression of GSK3αβ inhibited Cd-induced autophagy and induced apoptosis, which could be reversed by overexpression of GSK3β. The PI3K inhibitor Ly294002 blocked p-Akt-mediated increases in p-Ser-GSK3αβ and autophagy and induced apoptosis. Therefore, p-Ser-GSK3αβ can directly regulate Cd-induced autophagy, although its function is suppressed by Pin1. Collectively, the present results indicate that targeting Pin1 and GSK3αβ at the same time could be an effective therapeutic tool for Cd-induced carcinogenesis. - Highlights: • Pin1 regulated autophagy to protect cells from cadmium toxicity. • Pin1 suppression inhibited cadmium-induced autophagy and induced apoptosis. • Pin1 inhibited the function of p-Ser-GSK3αβ in autophagy regulation. • p-Ser-GSK3αβ regulated autophagy independently of Pin1.

  18. Autophagy regulated by prolyl isomerase Pin1 and phospho-Ser-GSK3αβ involved in protection of oral squamous cell carcinoma against cadmium toxicity

    International Nuclear Information System (INIS)

    So, Keum-Young; Ahn, Sang-Gun; Oh, Seon-Hee

    2015-01-01

    Prolyl isomerase Pin1 plays an important role in cell proliferation and is overexpressed in many human tumors. However, its role in autophagy induction remains undefined. Here we show that Pin1 regulates cell survival via autophagy in cadmium (Cd)-exposed oral squamous cell carcinoma (OSCC). OSCC exposure to Cd induced autophagy, as demonstrated by the formation of green fluorescent punctae in transfected cells expressing GFP-conjugated microtubule-associated protein light chain 3 (LC3) and by LC3 flux in the presence of autophagy inhibitors. Suppression of Atg5 enhanced Cd-induced apoptosis, indicating that autophagy is involved in cell protection. In dose–response experiments, cleavage of procaspase-3, PARP-1, and LC3-II was induced by Cd with an IC_5_0 of 45 μM. Expression of Pin1 was decreased at or above the Cd IC_5_0 value and was inversely correlated with the level of phospho(p)-Ser-GSK3αβ. Genetic or pharmacologic inhibition of Pin1 suppressed Cd-induced autophagy, but increased p-Akt-mediated p-Ser-GSK3αβ; this was reversed by overexpression of Pin1. However, suppression of GSK3αβ inhibited Cd-induced autophagy and induced apoptosis, which could be reversed by overexpression of GSK3β. The PI3K inhibitor Ly294002 blocked p-Akt-mediated increases in p-Ser-GSK3αβ and autophagy and induced apoptosis. Therefore, p-Ser-GSK3αβ can directly regulate Cd-induced autophagy, although its function is suppressed by Pin1. Collectively, the present results indicate that targeting Pin1 and GSK3αβ at the same time could be an effective therapeutic tool for Cd-induced carcinogenesis. - Highlights: • Pin1 regulated autophagy to protect cells from cadmium toxicity. • Pin1 suppression inhibited cadmium-induced autophagy and induced apoptosis. • Pin1 inhibited the function of p-Ser-GSK3αβ in autophagy regulation. • p-Ser-GSK3αβ regulated autophagy independently of Pin1.

  19. Ribose catabolism of Escherichia coli: characterization of the rpiB gene encoding ribose phosphate isomerase B and of the rpiR gene, which is involved in regulation of rpiB expression

    DEFF Research Database (Denmark)

    Sørensen, Kim I.; Hove-Jensen, Bjarne

    1996-01-01

    . The rpiB gene resided on a 4.6-kbp HindIII-EcoRV DNA fragment from phage lambda 10H5 (642) of the Kohara gene library and mapped at 92.85 min. Consistent with this map position, the cloned DNA fragment contained two divergent open reading frames of 149 and 296 codons, encoding ribose phosphate isomerase B...

  20. Novel 9-cis/all-trans β-carotene isomerases from plastidic oil bodies in Dunaliella bardawil catalyze the conversion of all-trans to 9-cis β-carotene.

    Science.gov (United States)

    Davidi, Lital; Pick, Uri

    2017-06-01

    We identified and demonstrated the function of 9-cis/all-trans β-carotene isomerases in plastidic globules of Dunaliella bardawil, the species accumulating the highest levels of 9-cis β-carotene that is essential for humans. The halotolerant alga Dunaliella bardawil is unique in that it accumulates under light stress high levels of β-carotene in plastidic lipid globules. The pigment is composed of two major isomers: all-trans β-carotene, the common natural form of this pigment, and 9-cis β-carotene. The biosynthetic pathway of β-carotene is known, but it is not clear how the 9-cis isomer is formed. We identified in plastidic lipid globules that were isolated from D. bardawil two proteins with high sequence homology to the D27 protein-a 9-cis/all-trans β-carotene isomerase from rice (Alder et al. Science 335:1348-1351, 2012). The proteins are enriched in the oil globules by 6- to 17-fold compared to chloroplast proteins. The expression of the corresponding genes, 9-cis-βC-iso1 and 9-cis-βC-iso2, is enhanced under light stress. The synthetic proteins catalyze in vitro conversion of all-trans to 9-cis β-carotene. Expression of the 9-cis-βC-iso1 or of 9-cis-βC-iso2 genes in an E. coli mutant line that harbors β-carotene biosynthesis genes enhanced the conversion of all-trans into 9-cis β-carotene. These results suggest that 9-cis-βC-ISO1 and 9-cis-βC-ISO2 proteins are responsible for the formation of 9-cis β-carotene in D. bardawil under stress conditions.

  1. Receptor type I and type II binding regions and the peptidyl-prolyl isomerase site of cyclophilin B are required for enhancement of T-lymphocyte adhesion to fibronectin.

    Science.gov (United States)

    Carpentier, Mathieu; Allain, Fabrice; Slomianny, Marie-Christine; Durieux, Sandrine; Vanpouille, Christophe; Haendler, Bernard; Spik, Geneviève

    2002-04-23

    Cyclophilin B (CyPB), a cyclosporin A (CsA) binding protein, interacts with two types of binding sites at the surface of T-lymphocytes. The type I sites correspond to functional receptors involved in endocytosis and the type II sites to sulfated glycosaminoglycans (GAGs). Mutational analysis of CyPB has revealed that W128, which is part of the CsA-binding pocket, is implicated in the binding to the functional type I receptors and that two amino acid clusters located in the N-terminus ensure the binding to GAGs. The peptidyl-prolyl isomerase activity of CyPB is not required for receptor binding. We have recently demonstrated that CyPB enhances adhesion of peripheral blood T-lymphocytes to fibronectin, a component of the extracellular matrix. We intended to identify additional amino acids involved in the binding of CyPB to its functional type I receptor and to determine regions responsible for the stimulation of peripheral blood T-lymphocyte adhesion. We determined that residues R76, G77, K132, D155, and D158 of the calcineurin (CN) interacting region were implicated in the recognition of type I receptor but not of GAGs. We also found that two different changes in the N-terminal extension that abated binding to GAGs prevented adhesion of peripheral blood T-lymphocytes to coated CyPB, whereas abbrogation of the PPIase activity had no effect. On the other hand, the adhesion of peripheral blood T-lymphocytes to coated fibronectin was not stimulated by CyPB mutants devoid of either type I receptor or GAGs binding activity or by mutants of the PPIase site. Altogether, the results demonstrate that different regions of CyPB are involved in peripheral blood T-lymphocyte activation and imply a novel important physiological function for peptidyl-prolyl isomerase activity.

  2. Spectroscopic and computational studies of cobalamin species with variable lower axial ligation: implications for the mechanism of Co-C bond activation by class I cobalamin-dependent isomerases.

    Science.gov (United States)

    Conrad, Karen S; Jordan, Christopher D; Brown, Kenneth L; Brunold, Thomas C

    2015-04-20

    5'-deoxyadenosylcobalamin (coenzyme B12, AdoCbl) serves as the cofactor for several enzymes that play important roles in fermentation and catabolism. All of these enzymes initiate catalysis by promoting homolytic cleavage of the cofactor's Co-C bond in response to substrate binding to their active sites. Despite considerable research efforts, the role of the lower axial ligand in facilitating Co-C bond homolysis remains incompletely understood. In the present study, we characterized several derivatives of AdoCbl and its one-electron reduced form, Co(II)Cbl, by using electronic absorption and magnetic circular dichroism spectroscopies. To complement our experimental data, we performed computations on these species, as well as additional Co(II)Cbl analogues. The geometries of all species investigated were optimized using a quantum mechanics/molecular mechanics method, and the optimized geometries were used to compute absorption spectra with time-dependent density functional theory. Collectively, our results indicate that a reduction in the basicity of the lower axial ligand causes changes to the cofactor's electronic structure in the Co(II) state that replicate the effects seen upon binding of Co(II)Cbl to Class I isomerases, which replace the lower axial dimethylbenzimidazole ligand of AdoCbl with a protein-derived histidine (His) residue. Such a reduction of the basicity of the His ligand in the enzyme active site may be achieved through proton uptake by the catalytic triad of conserved residues, DXHXGXK, during Co-C bond homolysis.

  3. Increased Production of Food-Grade d-Tagatose from d-Galactose by Permeabilized and Immobilized Cells of Corynebacterium glutamicum, a GRAS Host, Expressing d-Galactose Isomerase from Geobacillus thermodenitrificans.

    Science.gov (United States)

    Shin, Kyung-Chul; Sim, Dong-Hyun; Seo, Min-Ju; Oh, Deok-Kun

    2016-11-02

    The generally recognized as safe microorganism Corynebacterium glutamicum expressing Geobacillus thermodenitrificans d-galactose isomerase (d-GaI) was an efficient host for the production of d-tagatose, a functional sweetener. The d-tagatose production at 500 g/L d-galactose by the host was 1.4-fold higher than that by Escherichia coli expressing d-GaI. The d-tagatose-producing activity of permeabilized C. glutamicum (PCG) cells treated with 1% (w/v) Triton X-100 was 2.1-fold higher than that of untreated cells. Permeabilized and immobilized C. glutamicum (PICG) cells in 3% (w/v) alginate showed a 3.1-fold longer half-life at 50 °C and 3.1-fold higher total d-tagatose concentration in repeated batch reactions than PCG cells. PICG cells, which produced 165 g/L d-tagatose after 3 h, with a conversion of 55% (w/w) and a productivity of 55 g/L/h, showed significantly higher d-tagatose productivity than that reported for other cells. Thus, d-tagatose production by PICG cells may be an economical process to produce food-grade d-tagatose.

  4. Down-regulation of triose phosphate isomerase in Vineristine-resistant gastric cancer SGC7901 cell line identified by immobilized pH gradient two-dimensional gel electrophoresis and mierosequencing

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective:To exkplore new multidrug-resistance-related proteins in gastric SC7901 cells and clarify their mechanisms.Methods:Two-dimensional(2-D) polyacrylamide gel electrophoresis with immobilized pH gradients(IPG) was applied to compare the differential expression of multidrug-resistance-related proteins in gastric cancer SGC7901 cells and Vineristine-resistant SGC7901 cells (SGC7901/VCR) induced by vincristine sulfate.The 2-D gels were silver-stained.Then,preparative 2-D PAGE was performed.The differential proteins of PVDF membranes were cxcised and identified by N-terminal microsequencing.The mRNA expressions of differential proteins were detected in SGC 7901 cells and SGC7901/VCR cells by RT-PCR.Results:Approximatedly 680 protein sports were resolved on each 2-D gel by silver staining.Most protein spots showed no difference in composition,shape or density.25 proteins differed in abundance (6 higher in SGC7901/VCR cells;19 higher in 7901 cells);5 proteins were unique to one kind of cell or the othe(3 in SGC7901/VRC cells,2 in 7901 cells).One drug-resistance-related protein,which was down-regulated in SGC7901/VCR cells,was identified as trisephosphate isomerase(TPI),a glycolytic pathway enzyme.Conclusions:the results suggest that these differential proteins including TPI may be related to the Vincristine-resistant mechanism in human gastric cancer SGC7901/VCR cell line.

  5. Mutation in cyclophilin B that causes hyperelastosis cutis in American Quarter Horse does not affect peptidylprolyl cis-trans isomerase activity but shows altered cyclophilin B-protein interactions and affects collagen folding.

    Science.gov (United States)

    Ishikawa, Yoshihiro; Vranka, Janice A; Boudko, Sergei P; Pokidysheva, Elena; Mizuno, Kazunori; Zientek, Keith; Keene, Douglas R; Rashmir-Raven, Ann M; Nagata, Kazuhiro; Winand, Nena J; Bächinger, Hans Peter

    2012-06-22

    The rate-limiting step of folding of the collagen triple helix is catalyzed by cyclophilin B (CypB). The G6R mutation in cyclophilin B found in the American Quarter Horse leads to autosomal recessive hyperelastosis cutis, also known as hereditary equine regional dermal asthenia. The mutant protein shows small structural changes in the region of the mutation at the side opposite the catalytic domain of CypB. The peptidylprolyl cis-trans isomerase activity of the mutant CypB is normal when analyzed in vitro. However, the biosynthesis of type I collagen in affected horse fibroblasts shows a delay in folding and secretion and a decrease in hydroxylysine and glucosyl-galactosyl hydroxylysine. This leads to changes in the structure of collagen fibrils in tendon, similar to those observed in P3H1 null mice. In contrast to cyclophilin B null mice, where little 3-hydroxylation was found in type I collagen, 3-hydroxylation of type I collagen in affected horses is normal. The mutation disrupts the interaction of cyclophilin B with the P-domain of calreticulin, with lysyl hydroxylase 1, and probably other proteins, such as the formation of the P3H1·CypB·cartilage-associated protein complex, resulting in less effective catalysis of the rate-limiting step in collagen folding in the rough endoplasmic reticulum.

  6. Mutation in Cyclophilin B That Causes Hyperelastosis Cutis in American Quarter Horse Does Not Affect Peptidylprolyl cis-trans Isomerase Activity but Shows Altered Cyclophilin B-Protein Interactions and Affects Collagen Folding*

    Science.gov (United States)

    Ishikawa, Yoshihiro; Vranka, Janice A.; Boudko, Sergei P.; Pokidysheva, Elena; Mizuno, Kazunori; Zientek, Keith; Keene, Douglas R.; Rashmir-Raven, Ann M.; Nagata, Kazuhiro; Winand, Nena J.; Bächinger, Hans Peter

    2012-01-01

    The rate-limiting step of folding of the collagen triple helix is catalyzed by cyclophilin B (CypB). The G6R mutation in cyclophilin B found in the American Quarter Horse leads to autosomal recessive hyperelastosis cutis, also known as hereditary equine regional dermal asthenia. The mutant protein shows small structural changes in the region of the mutation at the side opposite the catalytic domain of CypB. The peptidylprolyl cis-trans isomerase activity of the mutant CypB is normal when analyzed in vitro. However, the biosynthesis of type I collagen in affected horse fibroblasts shows a delay in folding and secretion and a decrease in hydroxylysine and glucosyl-galactosyl hydroxylysine. This leads to changes in the structure of collagen fibrils in tendon, similar to those observed in P3H1 null mice. In contrast to cyclophilin B null mice, where little 3-hydroxylation was found in type I collagen, 3-hydroxylation of type I collagen in affected horses is normal. The mutation disrupts the interaction of cyclophilin B with the P-domain of calreticulin, with lysyl hydroxylase 1, and probably other proteins, such as the formation of the P3H1·CypB·cartilage-associated protein complex, resulting in less effective catalysis of the rate-limiting step in collagen folding in the rough endoplasmic reticulum. PMID:22556420

  7. The effect of a moderate zinc deficiency and dietary fat source on the activity and expression of the Δ(3)Δ (2)-enoyl-CoA isomerase in the liver of growing rats.

    Science.gov (United States)

    Justus, Jennifer; Weigand, Edgar

    2014-06-01

    Auxiliary enzymes participate in β-oxidation of unsaturated fatty acids. The objective of the study was to investigate the impact of a moderate zinc deficiency and a high intake of polyunsaturated fat on Δ(3)Δ(2)-enoyl-CoA isomerase (ECI) in the liver and other tissues. Five groups of eight weanling rats each were fed moderately zinc-deficient (ZD) or zinc-adequate (ZA) semisynthetic diets (7 or 50 mg Zn/kg) enriched with 22 % cocoa butter (CB) or 22 % safflower oil (SO) for 4 weeks: (1) ZD-CB, fed free choice; (2) ZA-CBR, ZA-CB diet fed in equivalent amounts consumed by the ZD-CB group; (3) ZD-SO, fed free choice; (4) ZA-SOR, ZA-SO diet fed in equivalent amounts consumed by the ZD-SO group; and (5) ZA-SO, fed free choice. Growth and Zn status markers were markedly reduced in the ZD groups. ECI activity in the liver of the animals fed the ZD- and ZA-SO diets were significantly higher (approximately 2- and 3-fold, respectively) as compared with the CB-fed animals, whereas activities in extrahepatic tissues (kidneys, heart, skeletal muscle, testes, adipose tissue) were not altered by dietary treatments. Transcript levels of the mitochondrial Eci gene in the liver did not significantly differ between ZD and ZA rats, but were 1.6-fold higher in the ZA-SO- than in the ZD-CB-fed animals (P safflower oil as a source high in linoleic acid induce markedly increased hepatic ECI activities and that a moderate Zn deficiency does not affect transcription of the mitochondrial Eci gene in the liver.

  8. DLX5, FGF8 and the Pin1 isomerase control ΔNp63α protein stability during limb development: a regulatory loop at the basis of the SHFM and EEC congenital malformations

    Science.gov (United States)

    Restelli, Michela; Lopardo, Teresa; Lo Iacono, Nadia; Garaffo, Giulia; Conte, Daniele; Rustighi, Alessandra; Napoli, Marco; Del Sal, Giannino; Perez-Morga, David; Costanzo, Antonio; Merlo, Giorgio Roberto; Guerrini, Luisa

    2014-01-01

    Ectrodactyly, or Split-Hand/Foot Malformation (SHFM), is a congenital condition characterized by the loss of central rays of hands and feet. The p63 and the DLX5;DLX6 transcription factors, expressed in the embryonic limb buds and ectoderm, are disease genes for these conditions. Mutations of p63 also cause the ectodermal dysplasia–ectrodactyly–cleft lip/palate (EEC) syndrome, comprising SHFM. Ectrodactyly is linked to defects of the apical ectodermal ridge (AER) of the developing limb buds. FGF8 is the key signaling molecule in this process, able to direct proximo-distal growth and patterning of the skeletal primordial of the limbs. In the limb buds of both p63 and Dlx5;Dlx6 murine models of SHFM, the AER is poorly stratified and FGF8 expression is severely reduced. We show here that the FGF8 locus is a downstream target of DLX5 and that FGF8 counteracts Pin1–ΔNp63α interaction. In vivo, lack of Pin1 leads to accumulation of the p63 protein in the embryonic limbs and ectoderm. We show also that ΔNp63α protein stability is negatively regulated by the interaction with the prolyl-isomerase Pin1, via proteasome-mediated degradation; p63 mutant proteins associated with SHFM or EEC syndromes are resistant to Pin1 action. Thus, DLX5, p63, Pin1 and FGF8 participate to the same time- and location-restricted regulatory loop essential for AER stratification, hence for normal patterning and skeletal morphogenesis of the limb buds. These results shed new light on the molecular mechanisms at the basis of the SHFM and EEC limb malformations. PMID:24569166

  9. Binding of 5-phospho-D-arabinonohydroxamate and 5-phospho-D-arabinonate inhibitors to zinc phosphomannose isomerase from Candida albicans studied by polarizable molecular mechanics and quantum mechanics.

    Science.gov (United States)

    Roux, Celine; Gresh, Nohad; Perera, Lalith E; Piquemal, Jean-Philip; Salmon, Laurent

    2007-04-15

    Type I phosphomannose isomerase (PMI) is a Zn-dependent metalloenzyme involved in the isomerization of D-fructose 6-phosphate to D-mannose 6-phosphate. One of our laboratories has recently designed and synthesized 5-phospho-D-arabinonohydroxamate (5PAH), an inhibitor endowed with a nanomolar affinity for PMI (Roux et al., Biochemistry 2004, 43, 2926). By contrast, the 5-phospho-D-arabinonate (5PAA), in which the hydroxamate moiety is replaced by a carboxylate one, is devoid of inhibitory potency. Subsequent biochemical studies showed that in its PMI complex, 5PAH binds Zn(II) through its hydroxamate moiety rather than through its phosphate. These results have stimulated the present theoretical investigation in which we resort to the SIBFA polarizable molecular mechanics procedure to unravel the structural and energetical aspects of 5PAH and 5PAA binding to a 164-residue model of PMI. Consistent with the experimental results, our theoretical studies indicate that the complexation of PMI by 5PAH is much more favorable than by 5PAA, and that in the 5PAH complex, Zn(II) ligation by hydroxamate is much more favorable than by phosphate. Validations by parallel quantum-chemical computations on model of the recognition site extracted from the PMI-inhibitor complexes, and totaling up to 140 atoms, showed the values of the SIBFA intermolecular interaction energies in such models to be able to reproduce the quantum-chemistry ones with relative errors < 3%. On the basis of the PMI-5PAH SIBFA energy-minimized structure, we report the first hypothesis of a detailed view of the active site of the zinc PMI complexed to the high-energy intermediate analogue inhibitor, which allows us to identify active site residues likely involved in the proton transfer between the two adjacent carbons of the substrates. (c) 2007 Wiley Periodicals, Inc.

  10. DLX5, FGF8 and the Pin1 isomerase control ΔNp63α protein stability during limb development: a regulatory loop at the basis of the SHFM and EEC congenital malformations.

    Science.gov (United States)

    Restelli, Michela; Lopardo, Teresa; Lo Iacono, Nadia; Garaffo, Giulia; Conte, Daniele; Rustighi, Alessandra; Napoli, Marco; Del Sal, Giannino; Perez-Morga, David; Costanzo, Antonio; Merlo, Giorgio Roberto; Guerrini, Luisa

    2014-07-15

    Ectrodactyly, or Split-Hand/Foot Malformation (SHFM), is a congenital condition characterized by the loss of central rays of hands and feet. The p63 and the DLX5;DLX6 transcription factors, expressed in the embryonic limb buds and ectoderm, are disease genes for these conditions. Mutations of p63 also cause the ectodermal dysplasia-ectrodactyly-cleft lip/palate (EEC) syndrome, comprising SHFM. Ectrodactyly is linked to defects of the apical ectodermal ridge (AER) of the developing limb buds. FGF8 is the key signaling molecule in this process, able to direct proximo-distal growth and patterning of the skeletal primordial of the limbs. In the limb buds of both p63 and Dlx5;Dlx6 murine models of SHFM, the AER is poorly stratified and FGF8 expression is severely reduced. We show here that the FGF8 locus is a downstream target of DLX5 and that FGF8 counteracts Pin1-ΔNp63α interaction. In vivo, lack of Pin1 leads to accumulation of the p63 protein in the embryonic limbs and ectoderm. We show also that ΔNp63α protein stability is negatively regulated by the interaction with the prolyl-isomerase Pin1, via proteasome-mediated degradation; p63 mutant proteins associated with SHFM or EEC syndromes are resistant to Pin1 action. Thus, DLX5, p63, Pin1 and FGF8 participate to the same time- and location-restricted regulatory loop essential for AER stratification, hence for normal patterning and skeletal morphogenesis of the limb buds. These results shed new light on the molecular mechanisms at the basis of the SHFM and EEC limb malformations. © The Author 2014. Published by Oxford University Press.

  11. The nairovirus nairobi sheep disease virus/ganjam virus induces the translocation of protein disulphide isomerase-like oxidoreductases from the endoplasmic reticulum to the cell surface and the extracellular space.

    Science.gov (United States)

    Lasecka, Lidia; Baron, Michael D

    2014-01-01

    Nairobi sheep disease virus (NSDV) of the genus Nairovirus causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%; the virus is found in East and Central Africa, and in India, where the virus is called Ganjam virus. NSDV is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus, which also causes a haemorrhagic disease. As with other nairoviruses, replication of NSDV takes place in the cytoplasm and the new virus particles bud into the Golgi apparatus; however, the effect of viral replication on cellular compartments has not been studied extensively. We have found that the overall structure of the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment and the Golgi were unaffected by infection with NSDV. However, we observed that NSDV infection led to the loss of protein disulphide isomerase (PDI), an oxidoreductase present in the lumen of the endoplasmic reticulum (ER) and which assists during protein folding, from the ER. Further investigation showed that NSDV-infected cells have high levels of PDI at their surface, and PDI is also secreted into the culture medium of infected cells. Another chaperone from the PDI family, ERp57, was found to be similarly affected. Analysis of infected cells and expression of individual viral glycoproteins indicated that the NSDV PreGn glycoprotein is involved in redistribution of these soluble ER oxidoreductases. It has been suggested that extracellular PDI can activate integrins and tissue factor, which are involved respectively in pro-inflammatory responses and disseminated intravascular coagulation, both of which manifest in many viral haemorrhagic fevers. The discovery of enhanced PDI secretion from NSDV-infected cells may be an important finding for understanding the mechanisms underlying the pathogenicity of haemorrhagic nairoviruses.

  12. The nairovirus nairobi sheep disease virus/ganjam virus induces the translocation of protein disulphide isomerase-like oxidoreductases from the endoplasmic reticulum to the cell surface and the extracellular space.

    Directory of Open Access Journals (Sweden)

    Lidia Lasecka

    Full Text Available Nairobi sheep disease virus (NSDV of the genus Nairovirus causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%; the virus is found in East and Central Africa, and in India, where the virus is called Ganjam virus. NSDV is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus, which also causes a haemorrhagic disease. As with other nairoviruses, replication of NSDV takes place in the cytoplasm and the new virus particles bud into the Golgi apparatus; however, the effect of viral replication on cellular compartments has not been studied extensively. We have found that the overall structure of the endoplasmic reticulum (ER, the ER-Golgi intermediate compartment and the Golgi were unaffected by infection with NSDV. However, we observed that NSDV infection led to the loss of protein disulphide isomerase (PDI, an oxidoreductase present in the lumen of the endoplasmic reticulum (ER and which assists during protein folding, from the ER. Further investigation showed that NSDV-infected cells have high levels of PDI at their surface, and PDI is also secreted into the culture medium of infected cells. Another chaperone from the PDI family, ERp57, was found to be similarly affected. Analysis of infected cells and expression of individual viral glycoproteins indicated that the NSDV PreGn glycoprotein is involved in redistribution of these soluble ER oxidoreductases. It has been suggested that extracellular PDI can activate integrins and tissue factor, which are involved respectively in pro-inflammatory responses and disseminated intravascular coagulation, both of which manifest in many viral haemorrhagic fevers. The discovery of enhanced PDI secretion from NSDV-infected cells may be an important finding for understanding the mechanisms underlying the pathogenicity of haemorrhagic nairoviruses.

  13. Determination of trace impurities in uranium-transition metal alloy fuels by ICP-MS using extended common analyte internal standardization (ECAIS) technique

    International Nuclear Information System (INIS)

    Saha, Abhijit; Deb, S.B.; Nagar, B.K.; Saxena, M.K.

    2015-01-01

    An analytical methodology was developed for the determination of eight trace impurities viz, Al, B, Cd, Co, Cu, Mg, Mn and Ni in three different uranium-transition metal alloy fuels (U-Me; Me = Ti, Zr and Mo) employing inductively coupled plasma mass spectrometry (ICP-MS). The well known common analyte internal standardization (CAIS) chemometric technique was modified and then employed to minimize and account for the matrix effect on analyte intensity. Standard addition of analytes to the pure synthetic U-Me sample solutions and subsequently their ≥ 94% recovery by the ICP-MS measurement validates the proposed methodology. One real sample of each of these alloys was analyzed by the developed analytical methodology and the %RSD observed was in the range of 5-8%. The method detection limits were found to be within 4-10 μg L -1 . (author)

  14. The dehydrogenase region of the NADPH oxidase component Nox2 acts as a protein disulfide isomerase (PDI) resembling PDIA3 with a role in the binding of the activator protein p67phox

    Science.gov (United States)

    Bechor, Edna; Dahan, Iris; Fradin, Tanya; Berdichevsky, Yevgeny; Zahavi, Anat; Rafalowski, Meirav; Federman-Gross, Aya; Pick, Edgar

    2015-02-01

    The superoxide (O2.-)-generating NADPH oxidase of phagocytes consists of a membrane component, cytochrome b558 (a heterodimer of Nox2 and p22phox), and four cytosolic components, p47phox, p67phox, p40phox, and Rac. The catalytic component, responsible for O2.- generation, is Nox2. It is activated by the interaction of the dehydrogenase region (DHR) of Nox2 with the cytosolic components, principally with p67phox. Using a peptide-protein binding assay, we found that Nox2 peptides containing a 369CysGlyCys371 triad (CGC) bound p67phox with high affinity, dependent upon the establishment of a disulfide bond between the two cysteines. Serially truncated recombinant Nox2 DHR proteins bound p67phox only when they comprised the CGC triad. CGC resembles the catalytic motif (CGHC) of protein disulfide isomerases (PDIs). This led to the hypothesis that Nox2 establishes disulfide bonds with p67phox via a thiol-dilsulfide exchange reaction and, thus, functions as a PDI. Evidence for this was provided by the following: 1. Recombinant Nox2 protein, which contained the CGC triad, exhibited PDI-like disulfide reductase activity; 2. Truncation of Nox2 C-terminal to the CGC triad or mutating C369 and C371 to R, resulted in loss of PDI activity; 3. Comparison of the sequence of the DHR of Nox2 with PDI family members revealed three small regions of homology with PDIA3; 4. Two monoclonal anti-Nox2 antibodies, with epitopes corresponding to regions of Nox2/PDIA3 homology, reacted with PDIA3 but not with PDIA1; 5. A polyclonal anti-PDIA3 (but not an anti-PDIA1) antibody reacted with Nox2; 6. p67phox, in which all cysteines were mutated to serines, lost its ability to bind to a Nox2 peptide containing the CGC triad and had an impaired capacity to support oxidase activity in vitro. We propose a model of oxidase assembly in which binding of p67phox to Nox2 via disulfide bonds, by virtue of the intrinsic PDI activity of Nox2, stabilizes the primary interaction between the two components.

  15. Relationships between the H and A-O blood types, phosphohexose isomerase and 6-phosphogluconate dehydrogenase red cell enzyme systems and halothane sensitivity, and economic traits in a superior and an inferior selection line of swiss landrace pigs.

    Science.gov (United States)

    Vögeli, P; Stranzinger, G; Schneebeli, H; Hagger, C; Künzi, N; Gerwig, C

    1984-12-01

    Associations between production traits and the genes for halothane sensitivity (HAL), S, A and H blood group systems and phosphohexose isomerase (PHI) and 6-phosphogluconate dehydrogenase (6-PGD) enzyme systems were investigated in two lines of pigs selected for an index. The phenotypic variance-covariance matrix of the index included backfat thickness and daily gain, whereas the genetic variance-covariance matrix included daily gain, feed conversion and percentage of lean meat. The experiment was conducted at the experimental station of the Institute of Animal Production and has been underway since 1973. The same index was applied but in two opposite directions to give a superior and inferior line in relation to the production traits. One hundred twenty-nine animals of the superior line in the seventh generation and 88 animals of the inferior line in the sixth generation were studied. Forty-two percent (54/129) of the animals of the superior line were halothane-positive. No animals in the inferior line were halothane reactors. Of the halothane-positive pigs, 70.4% (38/54) in the superior line had the HaHa and 94.4% (51/54) had the SsSs genotype, whereas only 4% (3/75) of the HaHa and 12% (9/75) of the SsSs pigs were halothane-negative. By practicing selection at the H and S loci, it seems possible to efficiently reduce halothane sensitivity in Swiss Landrace pigs. In pigs of the superior line, there were significant differences in percentage of lean meat, carcass length, pH1 (pH value at 45 min to 1 h postmortem, M. longissimus) and reflectance values among genotypes of the HAL, S and H systems and among some genotypes of the 6-PGD system. Poorest meat quality, highest percentage of lean meat and shortest carcass length were observed in pigs homozygous for the alleles HALn, Ss, Ha, PHIB and 6-PGDA. In the inferior line, these associations were absent. As the HAL locus is associated with the above mentioned production traits, linkage disequilibria may explain the

  16. In vivo adenylate cyclase activity in ultraviolet- and gamma-irradiated Escherichia coli

    International Nuclear Information System (INIS)

    Chatterjee, A.; Bhattacharya, A.K.

    1988-01-01

    The incorporation of [ 14 C]adenine into the cyclic AMP fraction by whole cells of Escherichia coli B/r was taken as a measure of the in vivo adenylate cyclase activity. This activity was significantly inhibited by irradiation of the cells either with 60 Co γ-rays or with UV light from a germicidal lamp, suggesting inhibition of cyclic AMP synthesis. The incubation of cells after irradiation with lower doses (50-100 Gy) of γ-rays produced a significant increase of in vivo adenylate cyclase activity, whereas there was no significant change after high doses (150 Gy and above). Dark incubation of cells after irradiation with UV light (54 J m -2 ) led to recovery of enzyme activity to the level measured in unirradiated cells. Thus it appears that the catabolite repression of L-arabinose isomerase induced by UV light, as well as γ-irradiation, is due to reduced cyclic AMP synthesis in irradiated cells. (author)

  17. In vivo adenylate cyclase activity in ultraviolet- and gamma-irradiated Escherichia coli.

    Science.gov (United States)

    Chatterjee, A; Bhattacharya, A K

    1988-06-01

    The incorporation of [14C]adenine into the cyclic AMP fraction by whole cells of Escherichia coli B/r was taken as a measure of the in vivo adenylate cyclase activity. This activity was significantly inhibited by irradiation of the cells either with 60Co gamma-rays or with UV light from a germicidal lamp, suggesting inhibition of cyclic AMP synthesis. The incubation of cells after irradiation with lower doses (50-100 Gy) of gamma-rays produced a significant increase of in vivo adenylate cyclase activity, whereas there was no significant change after higher doses (150 Gy and above). Dark incubation of cells after irradiation with UV light (54 J m-2) led to recovery of enzyme activity to the level measured in unirradiated cells. Thus it appears that the catabolite repression of L-arabinose isomerase induced by UV light, as well as gamma-irradiation, is due to reduced cyclic AMP synthesis in irradiated cells.

  18. Genetics Home Reference: glucose phosphate isomerase deficiency

    Science.gov (United States)

    ... glycolytic pathway; in this step, a molecule called glucose-6-phosphate is converted to another molecule called fructose-6-phosphate. When GPI remains a single molecule (a monomer) it is involved in the development and maintenance of nerve cells ( neurons ). In this context, it is often known as ...

  19. Genetics Home Reference: triosephosphate isomerase deficiency

    Science.gov (United States)

    ... movement problems are caused by impairment of motor neurons, which are specialized nerve cells in the brain ... known as glycolysis. During glycolysis, the simple sugar glucose is broken down to produce energy for cells. ...

  20. Understanding and Measuring Evaluation Capacity: A Model and Instrument Validation Study

    Science.gov (United States)

    Taylor-Ritzler, Tina; Suarez-Balcazar, Yolanda; Garcia-Iriarte, Edurne; Henry, David B.; Balcazar, Fabricio E.

    2013-01-01

    This study describes the development and validation of the Evaluation Capacity Assessment Instrument (ECAI), a measure designed to assess evaluation capacity among staff of nonprofit organizations that is based on a synthesis model of evaluation capacity. One hundred and sixty-nine staff of nonprofit organizations completed the ECAI. The 68-item…

  1. Characteristics of chalcone isomerase promoter in crabapple leaves ...

    African Journals Online (AJOL)

    Administrator

    2011-09-05

    Sep 5, 2011 ... in flavonoid biosynthetic pathway, can convert chalcone to (2S)-naringenin in the ... Construction of expression vectors with McCHI promoter fragment ... Each sample was bombarded three times with tungsten particles coated ...

  2. Cloning and characterization of peptidylprolyl isomerase B in the ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-12-15

    Dec 15, 2009 ... 1Institute of Life Sciences, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, P. R. China. 2Beijing ... 6 Tianshuiyuan Street, Chaoyang District, Beijing 100026, P. R. China. ... Kang et al., 2008). Cyclophilins were ..... Xu YX, Manley JL (2007). ... Yao Q, Li M, Yang H, Chai H, Fisher W, Chen C (2005).

  3. 21 CFR 184.1372 - Insoluble glucose isomerase enzyme preparations.

    Science.gov (United States)

    2010-04-01

    ... high fructose corn syrup described in § 184.1866. They are derived from recognized species of precisely... ingredient is used as an enzyme, as defined in § 170.3(o)(9) of this chapter, to convert glucose to fructose. (2) The ingredient is used in high fructose corn syrup, at levels not to exceed current good...

  4. In vivo adenylate cyclase activity in ultraviolet- and gamma-irradiated Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Chatterjee, A; Bhattacharya, A K

    1988-06-01

    The incorporation of (/sup 14/C)adenine into the cyclic AMP fraction by whole cells of Escherichia coli B/r was taken as a measure of the in vivo adenylate cyclase activity. This activity was significantly inhibited by irradiation of the cells either with /sup 60/Co ..gamma..-rays or with UV light from a germicidal lamp, suggesting inhibition of cyclic AMP synthesis. The incubation of cells after irradiation with lower doses (50-100 Gy) of ..gamma..-rays produced a significant increase of in vivo adenylate cyclase activity, whereas there was no significant change after high doses (150 Gy and above). Dark incubation of cells after irradiation with UV light (54 J m/sup -2/) led to recovery of enzyme activity to the level measured in unirradiated cells. Thus it appears that the catabolite repression of L-arabinose isomerase induced by UV light, as well as ..gamma..-irradiation, is due to reduced cyclic AMP synthesis in irradiated cells.

  5. High production of D-tagatose by the addition of boric acid.

    Science.gov (United States)

    Lim, Byung-Chul; Kim, Hye-Jung; Oh, Deok-Kun

    2007-01-01

    An L-arabinose isomerase mutant enzyme from Geobacillus thermodenitrificans was used to catalyze the isomerization of D-galactose to D-tagatose with boric acid. Maximum production of D-tagatose occurred at pH 8.5-9.0, 60 degrees C, and 0.4 molar ratio of boric acid to D-galactose, and the production increased with increasing enzyme concentration. Under the optimum conditions, the enzyme (10.8 units/mL) converted 300 g/L D-galactose to 230 g/L D-tagatose for 20 h with a yield of 77% (w/w); the production and conversion yield with boric acid were 1.5-fold and 24% higher than without boric acid, respectively. In 24 h, the enzyme produced 370 g/L D-tagatose from 500 g/L D-galactose with boric acid, corresponding to a conversion yield of 74% (w/w) and a production rate of 15.4 g/L.h. The production and yield of D-tagatose obtained in this study are unprecedented.

  6. Efficient production of D-tagatose using a food-grade surface display system.

    Science.gov (United States)

    Liu, Yi; Li, Sha; Xu, Hong; Wu, Lingtian; Xu, Zheng; Liu, Jing; Feng, Xiaohai

    2014-07-16

    D-tagatose, a functional sweetener, is commonly transformed from D-galactose by L-arabinose isomerase (L-AI). In this study, a novel type of biocatalyst, L-AI from Lactobacillus fermentum CGMCC2921 displayed on the spore surface of Bacillus subtilis 168, was developed for producing D-tagatose. The anchored L-AI, exhibiting the relatively high bioactivity, suggested that the surface display system using CotX as the anchoring protein was successfully constructed. The stability of the anchored L-AI was significantly improved. Specifically, the consolidation of thermal stability representing 87% of relative activity was retained even at 80 °C for 30 min, which remarkably favored the production of D-tagatose. Under the optimal conditions, the robust spores can convert 75% D-galactose (100 g/L) into D-tagatose after 24 h, and the conversion rate remained at 56% at the third cycle. Therefore, this biocatalysis system, which could express the target enzyme on the food-grade vector, was an alternative method for the value-added production of D-tagatose.

  7. d-Tagatose production by permeabilized and immobilized Lactobacillus plantarum using whey permeate.

    Science.gov (United States)

    Jayamuthunagai, J; Srisowmeya, G; Chakravarthy, M; Gautam, P

    2017-07-01

    The aim of the work is to produce d-Tagatose by direct addition of alginate immobilized Lactobacillus plantarum cells to lactose hydrolysed whey permeate. The cells were untreated and immobilized (UIC), permeabilized and immobilized (PIC) and the relative activities were compared with purified l-arabinose isomerase (l-AI) for d-galactose isomerization. Successive lactose hydrolysis by β-galactosidase from Escherichia coli and d-galactose isomerization using l-AI from Lactobacillus plantarum was performed to investigate the in vivo production of d-tagatose in whey permeate. In whey permeate, maximum conversion of 38% and 33% (w/w) d-galactose isomerization by PIC and UIC has been obtained. 162mg/g and 141mg/g of d-tagatose production was recorded in a 48h reaction time at 50°C, pH 7.0 with 5mM Mn 2+ ion concentration in the initial substrate mixture. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Production of D-tagatose and bioethanol from onion waste by an intergrating bioprocess.

    Science.gov (United States)

    Kim, Ho Myeong; Song, Younho; Wi, Seung Gon; Bae, Hyeun-Jong

    2017-10-20

    The rapid increase of agricultural waste is becoming a burgeoning problem and considerable efforts are being made by numerous researchers to convert it into a high-value resource material. Onion waste is one of the biggest issues in a world of dwindling resource. In this study, the potential of onion juice residue (OJR) for producing valuable rare sugar or bioethanol was evaluated. Purified Paenibacillus polymyxaL-arabinose isomerase (PPAI) has a molecular weight of approximately 53kDa, and exhibits maximal activity at 30°C and pH 7.5 in the presence of 0.8mM Mn 2+ . PPAI can produce 0.99g D-tagatose from 10g OJR. In order to present another application for OJR, we produced 1.56g bioethanol from 10g OJR through a bioconversion and fermentation process. These results indicate that PPAI can be used for producing rare sugars in an industrial setting, and OJR can be converted to D-tagatose and bioethanol. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Las poblaciones de Phytophthora infestans presentes en papa en el altiplano Cundiboyacense en 1996 son monomórficas para la enzima glucosa-6-fosfato Isomerasa Populations of Phytophthora infestans present on potato in the Cundinamarca and Boyacá plateau in 1996 are monomorphic for glucose-6-phosphate isomerase

    Directory of Open Access Journals (Sweden)

    Gualtero Cúellar Elsa Janeth

    1998-06-01

    ólo genotipo. Esta homogeneidad, en lo que se refiere a GPI en la población, permite concluir que en esta zona predomina la reproducción asexual, a través de la cual la variación genética es mínima o no se presenta. Resultados alternativos como la aparición de genotipos nuevos apoyarían la existencia de migraciones de otras poblaciones o la recombinación sexual explicada por la presencia de los tipos de apareamiento A1 y A2.
    Potato late blight, a disease caused by the Oomycete Phytophthora infestans, is responsible in great proportion for severe decrements in potato production in the Cundinamarca and Boyacá plateaus. Until now, late blight control has been done mainly with fungicides. The widened genetic variability in populations of this organism for a number of traits, including sensitivity to commercially available fungicides, observed in a world-wide perspective, has shown the need to research the genetic structure of local populations. This study was launched to characterize the populations of P. infestans in Cundinamarca and Boyacá through the polymorphism of glucose-6-phosfate isomerase (GPI. The results pointed at a clonal nature of these populations. All the local isolates were homozygous monomorphic for GPI, with genotype 100/100. Isolate Ro showed genotype 86/100 that corresponds to lineage US-1. Isolate MT2 showed genotype 84/100. These iso lates correspond to heterozygous populations that may have resulted from sexual reproduction. Isolate HIN had genotype 100/100, coinciding with local isolates. This isolate belongs to mating type A1 and corresponds to lineage US-6. This lineage represents one of the earliest migrations from Mexico to the United States, Europe and the rest of the world. Prior to the migrations of mating type A2. Results indicate that local populations are not too diverse, and suggest a clonal orrqtn. These results agree with the evaluation of this same population as regards sensitivity to metalaxil and mating type (Gonzalez, 1997

  10. Xylitol production from xylose mother liquor: a novel strategy that combines the use of recombinant Bacillus subtilis and Candida maltosa

    Science.gov (United States)

    2011-01-01

    Background Xylose mother liquor has high concentrations of xylose (35%-40%) as well as other sugars such as L-arabinose (10%-15%), galactose (8%-10%), glucose (8%-10%), and other minor sugars. Due to the complexity of this mother liquor, further isolation of xylose by simple method is not possible. In China, more than 50,000 metric tons of xylose mother liquor was produced in 2009, and the management of sugars like xylose that present in the low-cost liquor is a problem. Results We designed a novel strategy in which Bacillus subtilis and Candida maltosa were combined and used to convert xylose in this mother liquor to xylitol, a product of higher value. First, the xylose mother liquor was detoxified with the yeast C. maltosa to remove furfural and 5-hydromethylfurfural (HMF), which are inhibitors of B. subtilis growth. The glucose present in the mother liquor was also depleted by this yeast, which was an added advantage because glucose causes carbon catabolite repression in B. subtilis. This detoxification treatment resulted in an inhibitor-free mother liquor, and the C. maltosa cells could be reused as biocatalysts at a later stage to reduce xylose to xylitol. In the second step, a recombinant B. subtilis strain with a disrupted xylose isomerase gene was constructed. The detoxified xylose mother liquor was used as the medium for recombinant B. subtilis cultivation, and this led to L-arabinose depletion and xylose enrichment of the medium. In the third step, the xylose was further reduced to xylitol by C. maltosa cells, and crystallized xylitol was obtained from this yeast transformation medium. C. maltosa transformation of the xylose-enriched medium resulted in xylitol with 4.25 g L-1·h-1 volumetric productivity and 0.85 g xylitol/g xylose specific productivity. Conclusion In this study, we developed a biological method for the purification of xylose from xylose mother liquor and subsequent preparation of xylitol by C. maltosa-mediated biohydrogenation of xylose

  11. Xylitol production from xylose mother liquor: a novel strategy that combines the use of recombinant Bacillus subtilis and Candida maltosa

    Directory of Open Access Journals (Sweden)

    Jiang Mingguo

    2011-02-01

    Full Text Available Abstract Background Xylose mother liquor has high concentrations of xylose (35%-40% as well as other sugars such as L-arabinose (10%-15%, galactose (8%-10%, glucose (8%-10%, and other minor sugars. Due to the complexity of this mother liquor, further isolation of xylose by simple method is not possible. In China, more than 50,000 metric tons of xylose mother liquor was produced in 2009, and the management of sugars like xylose that present in the low-cost liquor is a problem. Results We designed a novel strategy in which Bacillus subtilis and Candida maltosa were combined and used to convert xylose in this mother liquor to xylitol, a product of higher value. First, the xylose mother liquor was detoxified with the yeast C. maltosa to remove furfural and 5-hydromethylfurfural (HMF, which are inhibitors of B. subtilis growth. The glucose present in the mother liquor was also depleted by this yeast, which was an added advantage because glucose causes carbon catabolite repression in B. subtilis. This detoxification treatment resulted in an inhibitor-free mother liquor, and the C. maltosa cells could be reused as biocatalysts at a later stage to reduce xylose to xylitol. In the second step, a recombinant B. subtilis strain with a disrupted xylose isomerase gene was constructed. The detoxified xylose mother liquor was used as the medium for recombinant B. subtilis cultivation, and this led to L-arabinose depletion and xylose enrichment of the medium. In the third step, the xylose was further reduced to xylitol by C. maltosa cells, and crystallized xylitol was obtained from this yeast transformation medium. C. maltosa transformation of the xylose-enriched medium resulted in xylitol with 4.25 g L-1·h-1 volumetric productivity and 0.85 g xylitol/g xylose specific productivity. Conclusion In this study, we developed a biological method for the purification of xylose from xylose mother liquor and subsequent preparation of xylitol by C. maltosa

  12. A mutant crp allele that differentially activates the operons of the fuc regulon in Escherichia coli.

    Science.gov (United States)

    Zhu, Y; Lin, E C

    1988-05-01

    L-Fucose is used by Escherichia coli through an inducible pathway mediated by a fucP-encoded permease, a fucI-encoded isomerase, a fucK-encoded kinase, and a fucA-encoded aldolase. The adolase catalyzes the formation of dihydroxyacetone phosphate and L-lactaldehyde. Anaerobically, lactaldehyde is converted by a fucO-encoded oxidoreductase to L-1,2-propanediol, which is excreted. The fuc genes belong to a regulon comprising four linked operons: fucO, fucA, fucPIK, and fucR. The positive regulator encoded by fucR responds to fuculose 1-phosphate as the effector. Mutants serially selected for aerobic growth on propanediol became constitutive in fucO and fucA [fucO(Con) fucA(Con)], but noninducible in fucPIK [fucPIK(Non)]. An external suppressor mutation that restored growth on fucose caused constitutive expression of fucPIK. Results from this study indicate that this suppressor mutation occurred in crp, which encodes the cyclic AMP-binding (or receptor) protein. When the suppressor allele (crp-201) was transduced into wild-type strains, the recipient became fucose negative and fucose sensitive (with glycerol as the carbon and energy source) because of impaired expression of fucA. The fucPIK operon became hyperinducible. The growth rate on maltose was significantly reduced, but growth on L-rhamnose, D-galactose, L-arabinose, glycerol, or glycerol 3-phosphate was close to normal. Lysogenization of fuc+ crp-201 cells by a lambda bacteriophage bearing crp+ restored normal growth ability on fucose. In contrast, lysogenization of [fucO(Con)fucA(Con)fucPIK(Non)crp-201] cells by the same phage retarded their growth on fucose.

  13. Phosphomannose isomerase gene for selection in lettuce (Lactuca sativa L.) transformation

    Czech Academy of Sciences Publication Activity Database

    Bříza, Jindřich; Růžičková, N.; Niedermeierová, Hana; Dusbábková, Jana; Vlasák, Josef

    2010-01-01

    Roč. 57, č. 1 (2010), s. 63-68 ISSN 0001-527X Institutional research plan: CEZ:AV0Z50510513 Keywords : pmi * npt II * Lactuca sativa Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.234, year: 2010

  14. Overexpression of petunia chalcone isomerase in tomato results in fruit containing increased levels of flavonols

    NARCIS (Netherlands)

    Muir, S.R.; Collins, G.J.; Robinson, S.; Hughes, S.G.; Bovy, A.G.; Vos, de C.H.R.; Tunen, van A.J.; Verhoeven, M.E.

    2001-01-01

    Tomatoes are an excellent source of the carotenoid lycopene, a compound that is thought to be protective against prostate cancer. They also contain small amounts of flavonoids in their peel (|[sim]|5–10 mg/kg fresh weight), mainly naringenin chalcone and the flavonol rutin, a quercetin glycoside.

  15. Alfalfa contains substantial 9-hydroperoxide lyase activity and a 3Z:2E-enal isomerase

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Noordermeer, M.A.; Veldink, G.A.

    1999-01-01

    Fatty acid hydroperoxides formed by lipoxygenase can be cleaved by hydroperoxide lyase resulting in the formation of short-chain aldehydes and omega-oxo acids. Plant hydroperoxide lyases use 13- or 9-hydroperoxy linoleic and linolenic acid as substrates. Alfalfa (Medicago sativa L.) has been

  16. Development of Efficient Xylose Fermentation in Saccharomyces cerevisiae : Xylose Isomerase as a Key Component

    NARCIS (Netherlands)

    Van Maris, A.J.A.; Winkler, A.A.; Kuyper, M.; De Laat, W.T.; Van Dijken, J.P.; Pronk, J.T.

    2007-01-01

    Metabolic engineering of Saccharomyces cerevisiae for ethanol production from d-xylose, an abundant sugar in plant biomass hydrolysates, has been pursued vigorously for the past 15 years. Whereas wild-type S. cerevisiae cannot ferment d-xylose, the ketoisomer d-xylulose can be metabolised slowly.

  17. Pearl millet transformation system using the positive selectable marker gene phosphomannose isomerase

    CSIR Research Space (South Africa)

    O'Kennedy, MM

    2004-04-01

    Full Text Available of the transgene. Similar to results obtained from previous studies with maize and wheat, the manA gene was shown to be a superior selectable marker gene for improving transformation efficiencies when compared to antibiotic or herbicide selectable marker genes....

  18. Arabidopsis Responds to Alternaria alternata Volatiles by Triggering Plastid Phosphoglucose Isomerase-Independent Mechanisms

    Czech Academy of Sciences Publication Activity Database

    Sanchez-Lopez, A.M.; Bahaji, A.; De Diego, N.; Baslam, M.; Li, J.; Munoz, F.J.; Almagro, G.; Garcia-Gomez, P.; Ameztoy, K.; Ricarte-Bermejo, A.; Novák, Ondřej; Humplík, J.F.; Spíchal, L.; Doležal, Karel; Ciordia, S.; Mena, M. C.; Navajas, R.; Baroja-Fernandez, E.; Pozueta-Romero, J.

    2016-01-01

    Roč. 172, č. 3 (2016), s. 1989-2001 ISSN 0032-0889 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : tandem mass-spectrometry * exceptionally high-levels * starch biosynthesis * functional-characterization * glucose translocator * thaliana * mutants * cytokinin * tissues * leaves Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.456, year: 2016

  19. Modified prokaryotic glucose isomerase enzymes with altered pH activity profiles

    NARCIS (Netherlands)

    Lambeir, Anne-Marie; Lasters, Ignace; Mrabet, Nadir; Quax, Wim; Van Der Laan, Jan M.; Misset, Onno

    1994-01-01

    A method for selecting amino acid residues is disclosed which upon replacement will give rise to an enzyme with an altered pH optimum. The method is specific for metalloenzymes which are inactivated at low pH due to the dissociation of the metal ions. The method is based on altering the pKa of the

  20. The First Workshop on Artificial Intelligence Techniques for Ambient Intelligence (AITAmI '06)

    OpenAIRE

    Augusto, Juan Carlos; Shapiro, Daniel

    2007-01-01

    The first annual workshop on the role of AI in ambient intelligence was held in Riva de Garda, Italy, on August 29, 2006. The workshop was colocated with the European Conference on Artificial Intelligence (ECAI 2006). It provided an opportunity for researchers in a variety of AI subfields together with representatives of commercial interests to explore ambient intelligence technology and applications.

  1. Use of phosphomannose isomerase-based selection system for Agrobacterium-mediated transformation of tomato and potato

    Czech Academy of Sciences Publication Activity Database

    Bříza, Jindřich; Pavingerová, Daniela; Přikrylová, P.; Gazdová, J.; Vlasák, Josef; Niedermeierová, Hana

    2008-01-01

    Roč. 52, č. 3 (2008), s. 453-461 ISSN 0006-3134 R&D Projects: GA ČR GA521/05/2092 Institutional research plan: CEZ:AV0Z50510513 Keywords : transgenic plants * trnasgenic potato * transgenic tomato Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.426, year: 2008

  2. Functional characterisation of parvulin-type peptidyl prolyl cis-trans isomerase, PinA in Dictyostelium discoideum

    International Nuclear Information System (INIS)

    Haokip, Nemneineng; Naorem, Aruna

    2017-01-01

    Pin1-type parvulins are unique among PPIases that can catalyse an otherwise slow cis-trans isomerisation of phosphorylated peptide bond preceding proline in target proteins. This prolyl isomerisation process can regulate activity, stability and localisation of target proteins and thus control cellular processes like eukaryotic cell proliferation, cell cycle progression and gene regulation. Towards understanding the function of Pin1-type prolyl isomerisation in Dictyostelium discoideum, a slime mould with distinct growth and developmental phases, we identified PinA as a novel Pin1-type parvulin by its ability to complement the temperature sensitivity phenotype associated with a mutation in ESS1 in S. cerevisiae. In D. discoideum, pinA is temporally and spatially regulated during growth and development. PinA is both nuclear as well as cytoplasmic in the growing cells. We further show that loss of pinA (pinA − ) leads to decreased growth rate, reduced spore formation and abnormal prespore-prestalk patterning. We conclude that PinA is required for normal growth as well as development in D. discoideum. - Highlights: • PinA is a bona fide homologue of S. cerevisiae Ess1. • PinA is required for normal cell proliferation of D. discoideum. • PinA is spatially localised in developmental structures. • PinA is important for cell differentiation and patterning.

  3. Autocrine motility factor (neuroleukin, phosphohexose isomerase) induces cell movement through 12-lipoxygenase-dependent tyrosine phosphorylation and serine dephosphorylation events.

    Science.gov (United States)

    Timár, J; Tóth, S; Tóvári, J; Paku, S; Raz, A

    1999-01-01

    Autocrine motility factor (AMF) is one of the motility cytokines regulating tumor cell migration, therefore identification of the signaling pathway coupled with it has critical importance. Previous studies revealed several elements of this pathway predominated by lipoxygenase-PKC activations but the role for tyrosine kinases remained questionable. Motility cytokines frequently have mitogenic effect as well, producing activation of overlapping signaling pathways therefore we have used B16a melanoma cells as models where AMF has exclusive motility effect. Our studies revealed that in B16a cells AMF initiated rapid (1-5 min) activation of the protein tyrosine kinase (PTK) cascade inducing phosphorylation of 179, 125, 95 and 40/37 kD proteins which was mediated by upstream cyclo- and lipoxygenases. The phosphorylated proteins were localized to the cortical actin-stress fiber attachment zones in situ by confocal microscopy. On the other hand, AMF receptor activation induced significant decrease in overall serine-phosphorylation level of cellular proteins accompanied by serine phosphorylation of 200, 90, 78 and 65 kd proteins. The decrease in serine phosphorylation was independent of PTKs, PKC as well as cyclo- and lipoxygenases. However, AMF induced robust translocation of PKCalpha to the stress fibers and cortical actin suggesting a critical role for this kinase in the generation of the motility signal. Based on the significant decrease in serine phosphorylation after AMF stimulus in B16a cells we postulated the involvement of putative serine/threonine phosphatase(s) upstream lipoxygenase and activation of the protein tyrosine kinase cascade downstream cyclo- and lipoxygenase(s) in the previously identified autocrine motility signal.

  4. Progranulin, a Glycoprotein Deficient in Frontotemporal Dementia, Is a Novel Substrate of Several Protein Disulfide Isomerase Family Proteins

    OpenAIRE

    Almeida, Sandra; Zhou, Lijuan; Gao, Fen-Biao

    2011-01-01

    The reduced production or activity of the cysteine-rich glycoprotein progranulin is responsible for about 20% of cases of familial frontotemporal dementia. However, little is known about the molecular mechanisms that govern the level and secretion of progranulin. Here we show that progranulin is expressed in mouse cortical neurons and more prominently in mouse microglia in culture and is abundant in the endoplasmic reticulum (ER) and Golgi. Using chemical crosslinking, immunoprecipitation, an...

  5. Combined enzyme mediated fermentation of cellulose and xylose to ethanol by Schizosaccharomyces pombe, cellulase, [beta]-glucosidase, and xylose isomerase

    Science.gov (United States)

    Lastick, S.M.; Mohagheghi, A.; Tucker, M.P.; Grohmann, K.

    1994-12-13

    A process for producing ethanol from mixed sugar streams from pretreated biomass comprising xylose and cellulose using enzymes to convert these substrates to fermentable sugars; selecting and isolating a yeast Schizosaccharomyces pombe ATCC No. 2476, having the ability to ferment these sugars as they are being formed to produce ethanol; loading the substrates with the fermentation mix composed of yeast, enzymes and substrates; fermenting the loaded substrates and enzymes under anaerobic conditions at a pH range of between about 5.0 to about 6.0 and at a temperature range of between about 35 C to about 40 C until the fermentation is completed, the xylose being isomerized to xylulose, the cellulose being converted to glucose, and these sugars being concurrently converted to ethanol by yeast through means of the anaerobic fermentation; and recovering the ethanol. 2 figures.

  6. A disulphide isomerase gene (PDI-V) from Haynaldia villosa contributes to powdery mildew resistance in common wheat.

    Science.gov (United States)

    Faheem, Muhammad; Li, Yingbo; Arshad, Muhammad; Jiangyue, Cheng; Jia, Zhao; Wang, Zongkuan; Xiao, Jin; Wang, Haiyan; Cao, Aizhong; Xing, Liping; Yu, Feifei; Zhang, Ruiqi; Xie, Qi; Wang, Xiue

    2016-04-13

    In this study, we report the contribution of a PDI-like gene from wheat wild relative Haynaldia villosa in combating powdery mildew. PDI-V protein contains two conserved thioredoxin (TRX) active domains (a and a') and an inactive domain (b). PDI-V interacted with E3 ligase CMPG1-V protein, which is a positive regulator of powdery mildew response. PDI-V was mono-ubiquitinated by CMPG1-V without degradation being detected. PDI-V was located on H. villosa chromosome 5V and encoded for a protein located in the endoplasmic reticulum. Bgt infection in leaves of H. villosa induced PDI-V expression. Virus induced gene silencing of PDIs in a T. durum-H. villosa amphiploid compromised the resistance. Single cell transient over-expression of PDI-V or a truncated version containing the active TXR domain a decreased the haustorial index in moderately susceptible wheat cultivar Yangmai 158. Stable transgenic lines over-expressing PDI-V in Yangmai 158 displayed improved powdery mildew resistance at both the seedling and adult stages. By contrast over-expression of point-mutated PDI-V(C57A) did not increase the level of resistance in Yangmai 158. The above results indicate a pivotal role of PDI-V in powdery mildew resistance and showed that conserved TRX domain a is critical for its function.

  7. Co-expression of sulphydryl oxidase and protein disulphide isomerase in Escherichia coli allows for production of soluble CRM197

    CSIR Research Space (South Africa)

    Roth, Robyn L

    2017-04-01

    Full Text Available The aim of this article is to investigate the production of soluble cross-reacting material 197 (CRM(sub197)) in Escherichia coli, a safe and effective T-cell-dependent protein carrier for polysaccharides used in the manufacture and application...

  8. Functional properties of the two redox-active sites in yeast protein disulphide isomerase in vitro and in vivo

    DEFF Research Database (Denmark)

    Westphal, V; Darby, N J; Winther, Jakob R.

    1999-01-01

    to that of human PDI, both in rearrangement and oxidation reactions. However, while the a domain active site of the human enzyme is more active than the a'-site, the reverse is the case for yPDI. This prompted us to set up an assay to investigate whether the situation would be different with a native yeast......-site to be most important. We furthermore show that the apparent difference between in vivo and in vitro activities is not due to catalytic contributions from the other PDI homologues found in yeast....

  9. Rodentibacter gen. nov including Rodentibacter pneumotropicus comb. nov., Rodentibacter heylii sp nov., Rodentibacter myodis sp nov., Rodentibacter ratti sp nov., Rodentibacter heidelbergensis sp nov., Rodentibacter trehalosifermentans sp nov., Rodentibacter rarus sp nov., Rodentibacter mrazii and two genomospecies

    DEFF Research Database (Denmark)

    Adhikary, Sadhana; Nicklas, Werner; Bisgaard, Magne

    2017-01-01

    -galactosidase and in acid formation from (+)-l-arabinose, (−)-d-ribose, (+)-d-xylose, myo-inositol, (−)-d-mannitol, lactose, melibiose and trehalose. Forty-six strains including taxon 48 of Bisgaard formed a monophyletic group by rpoB and 16S rRNA gene sequence analysis, but could not be separated phenotypically from R...

  10. Structure of the effector-binding domain of the arabinose repressor AraR from Bacillus subtilis

    Energy Technology Data Exchange (ETDEWEB)

    Procházková, Kateřina; Čermáková, Kateřina [Academy of Sciences of the Czech Republic, Flemingovo nam. 2, Prague 6 (Czech Republic); Pachl, Petr; Sieglová, Irena [Academy of Sciences of the Czech Republic, Flemingovo nam. 2, Prague 6 (Czech Republic); Academy of Sciences of the Czech Republic, Videnska 1083, Prague 4 (Czech Republic); Fábry, Milan [Academy of Sciences of the Czech Republic, Videnska 1083, Prague 4 (Czech Republic); Otwinowski, Zbyszek [UT Southwestern Medical Center, Dallas, Texas (United States); Řezáčová, Pavlína, E-mail: rezacova@uochb.cas.cz [Academy of Sciences of the Czech Republic, Flemingovo nam. 2, Prague 6 (Czech Republic); Academy of Sciences of the Czech Republic, Videnska 1083, Prague 4 (Czech Republic)

    2012-02-01

    The crystal structure of the effector-binding domain of the transcriptional repressor AraR from B. subtilis in complex with the effector molecule (l-arabinose) was determined at 2.2 Å resolution. A detailed analysis of the crystal identified a dimer organization that is distinctive from that of other members of the GalR/LacI family. In Bacillus subtilis, the arabinose repressor AraR negatively controls the expression of genes in the metabolic pathway of arabinose-containing polysaccharides. The protein is composed of two domains of different phylogenetic origin and function: an N-terminal DNA-binding domain belonging to the GntR family and a C-terminal effector-binding domain that shows similarity to members of the GalR/LacI family. The crystal structure of the C-terminal effector-binding domain of AraR in complex with the effector l-arabinose has been determined at 2.2 Å resolution. The l-arabinose binding affinity was characterized by isothermal titration calorimetry and differential scanning fluorimetry; the K{sub d} value was 8.4 ± 0.4 µM. The effect of l-arabinose on the protein oligomeric state was investigated in solution and detailed analysis of the crystal identified a dimer organization which is distinctive from that of other members of the GalR/LacI family.

  11. Arabinase induction and carbon catabolite repression in Aspergillus niger and Aspergillus nidulans

    NARCIS (Netherlands)

    Veen, van der P.

    1995-01-01

    The first aim of this thesis was to get a better understanding of the properties and the induction features of arabinan degrading enzymes and enzymes involved in the intracellular L-arabinose catabolic pathway in Aspergillus niger. The second aim was to understand the

  12. Synthesis and characterization of arabinose-palmitic acid esters by enzymatic esterification

    NARCIS (Netherlands)

    Pappalardo, Valeria M.; Boeriu, Carmen G.; Zaccheria, Federica; Ravasio, Nicoletta

    2017-01-01

    The direct esterification of palmitic acid with L-(+)-arabinose has been carried out. The use of Candida antartica lipase B as the catalyst and the choice of suitable solvent and experimental conditions allowed carrying out the reaction successfully. In particular 10% dimethyl-sulfoxide in

  13. The ß-1,4-endogalactanase A gene from Aspergillus niger is specifically induced on arabinose and galacturonic acid and plays an important role in the degradation of pectic hairy regions

    NARCIS (Netherlands)

    Vries, de R.P.; Parenicova, L.; Hinz, S.W.A.; Kester, H.C.M.; Beldman, G.; Benen, J.A.E.; Visser, J.

    2002-01-01

    The Aspergillus nigerß-1,4-endogalactanase encoding gene (galA) was cloned and characterized. The expression of galA in A. niger was only detected in the presence of sugar beet pectin, d-galacturonic acid and l-arabinose, suggesting that galA is coregulated with both the pectinolytic genes as well

  14. Induction, purification, and characterization of two extracellular alpha-L-arabinofuranosidases from Fusarium oxysporum

    DEFF Research Database (Denmark)

    Panagiotou, Gianni; Topakas, E.; Economou, L.

    2003-01-01

    In the presence of L-arabinose as sole carbon source, Fusarium oxysporum produces two alpha-L-arabinofuranosidases (ABFs) named ABF1 and ABF2, with molecular masses of 200 and 180 kDa, respectively. The two F. oxysporum proteins have been purified to homogeneity. The purified enzymes are composed...

  15. Synthesis of arabinoxylan fragments

    DEFF Research Database (Denmark)

    Underlin, Emilie Nørmølle; Böhm, Maximilian F.; Madsen, Robert

    , or production of commercial chemicals which are mainly obtained from fossil fuels today.The arbinoxylan fragments have a backbone of β-1,4-linked xylans with α-L-arabinose units attached at specific positions. The synthesis ultilises an efficient synthetic route, where all the xylan units can be derived from D...

  16. Immediate perception of a reward is distinct from the reward’s long-term salience

    Science.gov (United States)

    McGinnis, John P; Jiang, Huoqing; Agha, Moutaz Ali; Sanchez, Consuelo Perez; Lange, Jeff; Yu, Zulin; Marion-Poll, Frederic; Si, Kausik

    2016-01-01

    Reward perception guides all aspects of animal behavior. However, the relationship between the perceived value of a reward, the latent value of a reward, and the behavioral response remains unclear. Here we report that, given a choice between two sweet and chemically similar sugars—L- and D-arabinose—Drosophila melanogaster prefers D- over L- arabinose, but forms long-term memories of L-arabinose more reliably. Behavioral assays indicate that L-arabinose-generated memories require sugar receptor Gr43a, and calcium imaging and electrophysiological recordings indicate that L- and D-arabinose differentially activate Gr43a-expressing neurons. We posit that the immediate valence of a reward is not always predictive of the long-term reinforcement value of that reward, and that a subset of sugar-sensing neurons may generate distinct representations of similar sugars, allowing for rapid assessment of the salient features of various sugar rewards and generation of reward-specific behaviors. However, how sensory neurons communicate information about L-arabinose quality and concentration—features relevant for long-term memory—remains unknown. DOI: http://dx.doi.org/10.7554/eLife.22283.001 PMID:28005005

  17. Effect of temperature and hydraulic retention time on hydrogen producing granules: Homoacetogenesis and morphological characteristics

    International Nuclear Information System (INIS)

    Abreu, A. A.; Danko, A. S.; Alves, M. M.

    2009-01-01

    The effect of temperature and hydraulic retention time (HRT) on the homoacetogenesisi and on the morphological characteristics of hydrogen producing granules was investigated. Hydrogen was produced using an expanded granular sludge blanket (EGSB) reactor, fed with glucose and L-arabinose, under mesophilic (37 degree centigrade), thermophilic (55 degree centigrade), and hyper thermophilic (70 degree centigrade) conditions. (Author)

  18. Otariodibacter oris gen. nov., sp. nov., a member of the family Pasteurellaceae isolated from the oral cavity of pinnipeds

    DEFF Research Database (Denmark)

    Hansen, Mie Johanne; Bertelsen, Mads Frost; Christensen, Henrik

    2012-01-01

    from existing genera of the Pasteurellaceae by the following tests: positive reactions for catalase, oxidase, Voges-Proskauer and indole; no X- or V-factor dependency; and acid production from L-arabinose (slow), L-fucose, maltose and trehalose, but not from dulcitol, D-mannitol, D-mannose or sucrose...

  19. Genome-scale consequences of cofactor balancing in engineered pentose utilization pathways in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Amit Ghosh

    Full Text Available Biofuels derived from lignocellulosic biomass offer promising alternative renewable energy sources for transportation fuels. Significant effort has been made to engineer Saccharomyces cerevisiae to efficiently ferment pentose sugars such as D-xylose and L-arabinose into biofuels such as ethanol through heterologous expression of the fungal D-xylose and L-arabinose pathways. However, one of the major bottlenecks in these fungal pathways is that the cofactors are not balanced, which contributes to inefficient utilization of pentose sugars. We utilized a genome-scale model of S. cerevisiae to predict the maximal achievable growth rate for cofactor balanced and imbalanced D-xylose and L-arabinose utilization pathways. Dynamic flux balance analysis (DFBA was used to simulate batch fermentation of glucose, D-xylose, and L-arabinose. The dynamic models and experimental results are in good agreement for the wild type and for the engineered D-xylose utilization pathway. Cofactor balancing the engineered D-xylose and L-arabinose utilization pathways simulated an increase in ethanol batch production of 24.7% while simultaneously reducing the predicted substrate utilization time by 70%. Furthermore, the effects of cofactor balancing the engineered pentose utilization pathways were evaluated throughout the genome-scale metabolic network. This work not only provides new insights to the global network effects of cofactor balancing but also provides useful guidelines for engineering a recombinant yeast strain with cofactor balanced engineered pathways that efficiently co-utilizes pentose and hexose sugars for biofuels production. Experimental switching of cofactor usage in enzymes has been demonstrated, but is a time-consuming effort. Therefore, systems biology models that can predict the likely outcome of such strain engineering efforts are highly useful for motivating which efforts are likely to be worth the significant time investment.

  20. The 2002 Starting Artificial Intelligence Researchers Symposium

    OpenAIRE

    Vidal, Thierry

    2003-01-01

    During the 2002 European Conference on Artificial Intelligence (ECAI-02) was introduced the Starting Artificial Intelligence Researchers Symposium STAIRS), the first-ever international symposium specifically aimed at Ph.D. students in AI. The outcome was a thorough, high-quality, and successful event, with all the features one usually finds in the best international conferences: large international committees, comprehensive coverage, published proceedings, renowned speakers and panelists, sub...

  1. The conserved His8 of the Moloney murine leukemia virus Env SU subunit directs the activity of the SU-TM disulphide bond isomerase

    International Nuclear Information System (INIS)

    Li Kejun; Zhang, Shujing; Kronqvist, Malin; Ekstroem, Maria; Wallin, Michael; Garoff, Henrik

    2007-01-01

    Murine leukemia virus (MLV) fusion is controlled by isomerization of the disulphide bond between the receptor-binding surface (SU) and fusion-active transmembrane subunits of the Env-complex. The bond is in SU linked to a CXXC motif. This carries a free thiol that upon receptor binding can be activated (ionized) to attack the disulphide and rearrange it into a disulphide isomer within the motif. To find out whether His8 in the conserved SPHQ sequence of Env directs thiol activation, we analyzed its ionization in MLV vectors with wtEnv and Env with His8 deleted or substituted for Tyr or Arg, which partially or completely arrests fusion. The ionization was monitored by following the pH effect on isomerization in vitro by Ca 2+ depletion or in vivo by receptor binding. We found that wtEnv isomerized optimally at slightly basic pH whereas the partially active mutant required higher and the inactive mutants still higher pH. This suggests that His8 directs the ionization of the CXXC thiol

  2. Triosephosphate isomerase gene promoter variation: -5G/A and -8G/A polymorphisms in clinical malaria groups in two African populations.

    Science.gov (United States)

    Guerra, Mónica; Machado, Patrícia; Manco, Licínio; Fernandes, Natércia; Miranda, Juliana; Arez, Ana Paula

    2015-06-01

    TPI1 promoter polymorphisms occur in high prevalence in individuals from African origin. Malaria-patients from Angola and Mozambique were screened for the TPI1 gene promoter variants rs1800200A>G, (-5G>A), rs1800201G>A, (-8G>A), rs1800202T>G, (-24T>G), and for the intron 5 polymorphism rs2071069G>A, (2262G>A). -5G>A and -8G>A variants occur in 47% and 53% in Angola and Mozambique, respectively while -24T>G was monomorphic for the wild-type T allele. Six haplotypes were identified and -8A occurred in 45% of the individuals, especially associated with the GAG haplotype and more frequent in non-severe malaria groups, although not significantly. The arising and dispersion of -5G>A and -8G>A polymorphisms is controversial. Their age was estimated by analyses of two microsatellite loci, CD4 and ATN1, adjacent to TPI1 gene. The -5G>A is older than -8G>A, with an average estimate of approximately 35,000 years. The -8A variant arose in two different backgrounds, suggesting independent mutational events. The first, on the -5G background, may have occurred in East Africa around 20,800 years ago; the second, on the -5A background, may have occurred in West Africa some 7500 years ago. These estimates are within the period of spread of agriculture and the malaria mosquito vector in Africa, which could has been a possible reason for the selection of -8A polymorphism in malaria endemic countries. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells, Growth, Photosynthetic Capacity, and Biosynthesis of Plastidic Cytokinins in Arabidopsis

    Czech Academy of Sciences Publication Activity Database

    Bahaji, A.; Sanchez-Lopez, A.M.; De Diego, N.; Munoz, F.J.; Humplík, J.F.; Novák, Ondřej; Spíchal, L.; Doležal, K.; Pozueta-Romero, J.

    2015-01-01

    Roč. 10, č. 3 (2015) E-ISSN 1932-6203 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : ADP-GLUCOSE PYROPHOSPHORYLASE * PENTOSE-PHOSPHATE PATHWAY * POSTTRANSLATIONAL REDOX-MODIFICATION Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.057, year: 2015

  4. Mutation of yeast Eug1p CXXS active sites to CXXC results in a dramatic increase in protein disulphide isomerase activity

    DEFF Research Database (Denmark)

    Nørgaard, P; Winther, Jakob R.

    2001-01-01

    to thioredoxin and with CXXC catalytic motifs. EUG1 encodes a yeast protein, Eug1p, that is highly homologous to PDI. However, Eug1p contains CXXS motifs instead of CXXC. In the current model for PDI function both cysteines in this motif are required for PDI-catalysed oxidase activity. To gain more insight...... into the biochemical properties of this unusual variant of PDI we have purified and characterized the protein. We have furthermore generated a number of mutant forms of Eug1p in which either or both of the active sites have been mutated to a CXXC sequence. To determine the catalytic capacity of the wild...

  5. From Drosophila to humans: Reflections on the roles of the prolyl-isomerases and chaperones, cyclophilins, in cell function and disease

    Science.gov (United States)

    Ferreira, Paulo A.; Orry, Andrew

    2013-01-01

    Despite remarkable advances in human genetics and other genetic model systems, the fruit fly, Drosophila melanogaster, remains a powerful experimental tool to probe with ease the inner workings of a myriad of biological and pathological processes, even when evolutionary forces impart apparent divergences to some of such processes. The understanding of such evolutionary differences provides mechanistic insights into genotype-phenotype correlations underpinning biological processes across metazoans. The pioneering work developed by the William Pak laboratory for the past four decades, and the work of others, epitomize the notion of how the Drosophila system breaks new fertile ground or complements research fields of high scientific and medical relevance. Among the three major genetic complementation groups produced by the Pak's laboratory and impairing distinct facets of photoreceptor neuronal function, the nina group (ninaA….J) selectively affects the biogenesis of G protein-coupled receptors (GPCR) mediating the photoconversion and transduction of light-stimuli. Among the nina genes identified, ninaA arguably assumes heightened significance for several reasons. First, it presents unique physiological selectivity toward the biogenesis of a subset of GPCRs, a standalone biological manifestation yet to be discerned for most mammalian homologues of NinaA. Second, NinaA belongs to a family of proteins, immunophilins, which are the primary targets for immunosuppressive drugs at the therapeutic forefront of a multitude of medical conditions. Third, NinaA closest homologue, cyclophilin-B (CyPB/PPIB), is an immunophilin whose loss-of-function was found recently to cause osteogenesis imperfecta in the human. This report highlights advances made by studies on some members of immunophilins, the cyclophilins. Finally, it re-examines critically data and dogmas derived from past and recent genetic, structural, biological and pathological studies on NinaA and few other cyclophilins that support some of such paradigms to be less than definite and advance our understanding of cyclophilins' roles in cell function, disease and therapeutic interventions. PMID:22332926

  6. Using intron sequence comparisons in the triose-phosphate isomerase gene to study the divergence of the fall armyworm host strains

    Science.gov (United States)

    The Noctuid moth, Spodoptera frugiperda (the fall armyworm), is endemic to the Western Hemisphere and appears to be undergoing sympatric speciation to produce two subpopulations that differ in their choice of host plants. The diverging “rice strain” and “corn strain” are morphologically indistinguis...

  7. Polymyxin resistance of Pseudomonas aeruginosa phoQ mutants is dependent on additional two-component regulatory systems

    DEFF Research Database (Denmark)

    Gutu, Alina D; Sgambati, Nicole; Strasbourger, Pnina

    2013-01-01

    Pseudomonas aeruginosa can develop resistance to polymyxin as a consequence of mutations in the PhoPQ regulatory system, mediated by covalent lipid A modification. Transposon mutagenesis of a polymyxin-resistant phoQ mutant defined 41 novel loci required for resistance, including two regulatory s......, indicate that addition of 4-amino-L-arabinose to lipid A is not the only PhoPQ-regulated biochemical mechanism required for resistance, and demonstrate that colRS and cprS mutations can contribute to high-level clinical resistance....... with the known role of this modification in polymyxin resistance. Surprisingly, tandem deletion of colRS or cprRS in the ΔphoQ mutant or individual deletion of cprR or cprS failed to suppress 4-amino-L-arabinose addition to lipid A, indicating that this modification alone is not sufficient for Pho...

  8. Changes in kenaf properties and chemistry as a function of growing time

    Science.gov (United States)

    Roger M. Rowell; James S. Han

    1999-01-01

    Kenaf Tainung 1 cultivar was grown in Madison, WI in 1994. The ratio of core to bast fiber, total plant yield, protein, ash, fiber length, extractives, lignin, and sugar content were determined as a function of growing age. Ash, protein, extractives, L-arabinose, L-rhamnose, D-galactose, and D-mannose contents decreased while lignin, D-glucose and D-xylose content...

  9. 3rd Workshop on "Combinations of Intelligent Methods and Applications"

    CERN Document Server

    Palade, Vasile

    2013-01-01

    The combination of different intelligent methods is a very active research area in Artificial Intelligence (AI). The aim is to create integrated or hybrid methods that benefit from each of their components.  The 3rd Workshop on “Combinations of Intelligent Methods and Applications” (CIMA 2012) was intended to become a forum for exchanging experience and ideas among researchers and practitioners who are dealing with combining intelligent methods either based on first principles or in the context of specific applications. CIMA 2012 was held in conjunction with the 22nd European Conference on Artificial Intelligence (ECAI 2012).This volume includes revised versions of the papers presented at CIMA 2012.  .

  10. Demonstration of glycosomes (microbodies) in the Bodonid flagellate Trypanoplasma borelli (Protozoa, Kinetoplastida)

    NARCIS (Netherlands)

    Opperdoes, Fred R.; Nohynkova, Eva; Schaftingen, Emile Van; Lambeir, Anne-Marie; Veenhuis, Marten; Roy, Joris Van

    1988-01-01

    Homogenates of Trypanoplasma borelli were subjected to subcellular fractionation by sequential differential and isopycnic centrifugation in sucrose. Glycerol-3-phosphate dehydrogenase and the glycolytic enzymes, glucosephosphate isomerase and triosephosphate isomerase, as well as the peroxisomal

  11. Identification and characterization of D-xylulokinase from the D-xylose-fermenting fungus, Mucor circinelloides.

    Science.gov (United States)

    Komeda, Hidenobu; Yamasaki-Yashiki, Shino; Hoshino, Kazuhiro; Asano, Yasuhisa

    2014-11-01

    D-Xylulokinase catalyzes the phosphorylation of D-xylulose in the final step of the pentose catabolic pathway to form d-xylulose-5-phosphate. The D-xylulokinase activity was found to be induced by both D-xylose and L-arabinose, as well as some of the other enzymes involved in the pentose catabolism, in the D-xylose-fermenting zygomycetous fungus, Mucor circinelloides NBRC 4572. The putative gene, xyl3, which may encode D-xylulokinase, was detected in the genome sequence of this strain. The amino acid sequence deduced from the gene was more similar to D-xylulokinases from an animal origin than from other fungi. The recombinant enzyme was purified from the E. coli transformant expressing xyl3 and then characterized. The ATP-dependent phosphorylative activity of the enzyme was the highest toward D-xylulose. Its kinetic parameters were determined as Km (D-xylulose) = 0.29 mM and Km (ATP) = 0.51 mM, indicating that the xyl3 gene encoded D-xylulokinase (McXK). Western blot analysis revealed that McXK was induced by L-arabinose as well as D-xylose and the induction was repressed in the presence of D-glucose, suggesting that the enzyme may be involved in the catabolism of D-xylose and L-arabinose and is subject to carbon catabolite repression in this fungus. This is the first study on D-xylulokinase from zygomycetous fungi. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  12. Some Peculiarities of Growth and Functional Activity of Escherichia coli Strain from Probiotic Formula "ASAP"

    OpenAIRE

    Marine A. Balayan; Susanna S. Mirzabekyan; Marine Isajanyan; Zaven S. Pepoyan; Аrmen H. Trchounian; Аstghik Z. Pepoyan; Helena Bujdakova

    2010-01-01

    It has been shown that pH 7,3 and 37 0C are the optimal condition for the growth of E. coli “ASAP". The cells grow well on Glucose, Lactose, D-Mannitol, D-Sorbitol, (+)-Xylose, L- (+)-Arabinose and Dulcitol. No growth has been observed on Sucrose, Inositol, Phenylalanine, and Tryptophan. The strain is sensitive to a range of antibiotics. The present study has demonstrated that E. coli “ASAP" inhibit the growth of S. enterica ATCC #700931 in vitro. The studies on conjugating activity has revea...

  13. Inhibition of intestinal disaccharidase activity by pentoses

    DEFF Research Database (Denmark)

    Halschou-Jensen, Kia

    on carbohydrate- ingesting enzymes activity in vitro and possible effects on human postprandial blood response. In paper 1 the effects of sugar beet polyphenols from molasses and the potential inhibition of sucrase activity in vitro, was investigated. Two different polyphenol-rich fractions from chromatographic...... separation of molasses from sugar beets and pure ferulic acid were tested. We found no effects of the two fractions of molasses. The pure ferulic acid indicated an inhibition of sucrase in vitr. Both in vitro and in vivo studies have investigated the effects of L-arabinose and D-xylose on carbohydrate...

  14. Chemical analysis of a polysaccharide of unripe (green) tomato (Lycopersicon esculentum).

    Science.gov (United States)

    Chandra, Krishnendu; Ghosh, Kaushik; Ojha, Arnab K; Islam, Syed S

    2009-11-02

    A polysaccharide (PS-I) isolated from the aqueous extract of the unripe (green) tomatoes (Lycopersicon esculentum) consists of D-galactose, D-methyl galacturonate, D-arabinose, L-arabinose, and L-rhamnose. Structural investigation of the polysaccharide was carried out using total acid hydrolysis, methylation analysis, periodate oxidation study, and NMR studies ((1)H, (13)C, DQF-COSY, TOCSY, NOESY, ROESY, HMQC, and HMBC). On the basis of above-mentioned experiments the structure of the repeating unit of the polysaccharide (PS-I) was established as: [structure: see text].

  15. Inhibition by natural dietary substances of gastrointestinal absorption of starch and sucrose in rats and pigs: 1. Acute studies.

    Science.gov (United States)

    Preuss, Harry G; Echard, Bobby; Bagchi, Debasis; Stohs, Sidney

    2007-08-06

    Rapid gastrointestinal absorption of refined carbohydrates (CHO) is linked to perturbed glucose-insulin metabolism that is, in turn, associated with many chronic health disorders. We assessed the ability of various natural substances, commonly referred to as "CHO blockers," to influence starch and sucrose absorption in vivo in ninety-six rats and two pigs. These natural enzyme inhibitors of amylase/sucrase reportedly lessen breakdown of starches and sucrose in the gastrointestinal tract, limiting their absorption. To estimate absorption, groups of nine SD rats were gavaged with water or water plus rice starch and/or sucrose; and circulating glucose was measured at timed intervals thereafter. For each variation in the protocol a total of at least nine different rats were studied with an equal number of internal controls on three different occasions. The pigs rapidly drank CHO and inhibitors in their drinking water. In rats, glucose elevations above baseline over four hours following rice starch challenge as estimated by area-under-curve (AUC) were 40%, 27%, and 85% of their internal control after ingesting bean extract, hibiscus extract, and l-arabinose respectively in addition to the rice starch. The former two were significantly different from control. L-Arabinose virtually eliminated the rising circulating glucose levels after sucrose challenge, whereas hibiscus and bean extracts were associated with lesser decreases than l-arabinose that were still significantly lower than control. The glucose elevations above baseline over four hours in rats receiving sucrose (AUC) were 51%, 43% and 2% of control for bean extract, hibiscus extract, and L-arabinose, respectively. Evidence for dose-response of bean and hibiscus extracts is reported. Giving the natural substances minus CHO challenge caused no significant changes in circulating glucose concentrations, indicating no major effects on overall metabolism. A formula combining these natural products significantly

  16. An arabinoxyloglucan isolated from the midrib of the leaves of Nicotiana tabacum

    Energy Technology Data Exchange (ETDEWEB)

    Eda, S; Kato, K

    1978-01-01

    The structure of an arabinoxyloglucan, separated from the hemicellulosic polysaccharides of the midrib of the leaves of Nicotiana tabacum, was investigated by methylation analyses before and after mild acid hydrolysis, acetolysis and cellulase-degradation. The arabinoxyloglucan consists of L-arabinose, D-xylose and D-glucose in a molar ratio of 13:33:54, and has a backbone of ..beta..-(1..-->..4)-linked D-glucopyranosyl residues. Some of the glucopyranosyl residues are attached at the 6 position by single ..cap alpha..-D-xylopyranosyl and ..cap alpha..-L-arabinofuranosyl-(1..-->..2)-..cap alpha..-D-xylopyranosyl side chains.

  17. Xylitol production from xylose mother liquor: a novel strategy that combines the use of recombinant Bacillus subtilis and Candida maltosa

    OpenAIRE

    Jiang Mingguo; Lv Jiyang; Wang Ben; Cheng Hairong; Lin Shuangjun; Deng Zixin

    2011-01-01

    Abstract Background Xylose mother liquor has high concentrations of xylose (35%-40%) as well as other sugars such as L-arabinose (10%-15%), galactose (8%-10%), glucose (8%-10%), and other minor sugars. Due to the complexity of this mother liquor, further isolation of xylose by simple method is not possible. In China, more than 50,000 metric tons of xylose mother liquor was produced in 2009, and the management of sugars like xylose that present in the low-cost liquor is a problem. Results We d...

  18. High-level expression, purification and antibacterial activity of bovine lactoferricin and lactoferrampin in Photorhabdus luminescens.

    Science.gov (United States)

    Tang, Zhiru; Zhang, Youming; Stewart, Adrian Francis; Geng, Meimei; Tang, Xiangsha; Tu, Qiang; Yin, Yulong

    2010-10-01

    Bovine lactoferricin (LFC) and bovine lactoferrampin (LFA) are two active fragments located in the N(1)-domain of bovine lactoferrin. Recent studies suggested that LFC and LFA have broad-spectrum activity against Gram-positive and Gram-negative bacteria. To date, LFC and LFA have usually been produced from milk. We report here the high-level expression, purification and characterization of LFC and LFA using the Photorhabdus luminescens expression system. After the cipA and cipB genes were deleted by ET recombination, the expression host P. luminescens TZR(001) was constructed. A synthetic LFC-LFA gene containing LFC and LFA was fused with the cipB gene to form a cipB-LFC-LFA gene. To obtain the expression vector pBAD-cipB-LFC-LFA, the cipB-LFC-LFA gene was cloned on the L-arabinose-inducible expression vector pBAD24. pBAD-cipB-LFC-LFA was transformed into P. luminescens TZR(001). The cipB-LFC-LFA fusion protein was expressed under the induction of L-arabinose and its yield reached 12 mg L(-1) bacterial culture. Recombinant LFC-LFA was released from cipB by pepsin. The MIC of recombinant LFC-LFA toward E. coli 0149, 0141 and 020 was 6.25, 12.5 and 3.175 microg ml(-1), respectively. Copyright 2010 Elsevier Inc. All rights reserved.

  19. The beta-1,4-endogalactanase A gene from Aspergillus niger is specifically induced on arabinose and galacturonic acid and plays an important role in the degradation of pectic hairy regions.

    Science.gov (United States)

    De Vries, Ronald P; Parenicová, Lucie; Hinz, Sandra W A; Kester, Harry C M; Beldman, Gerrit; Benen, Jacques A E; Visser, Jaap

    2002-10-01

    The Aspergillus nigerbeta-1,4-endogalactanase encoding gene (galA) was cloned and characterized. The expression of galA in A. niger was only detected in the presence of sugar beet pectin, d-galacturonic acid and l-arabinose, suggesting that galA is coregulated with both the pectinolytic genes as well as the arabinanolytic genes. The corresponding enzyme, endogalactanase A (GALA), contains both active site residues identified previously for the Pseudomonas fluorescensbeta-1,4-endogalactanase. The galA gene was overexpressed to facilitate purification of GALA. The enzyme has a molecular mass of 48.5 kDa and a pH optimum between 4 and 4.5. Incubations of arabinogalactans of potato, onion and soy with GALA resulted initially in the release of d-galactotriose and d-galactotetraose, whereas prolonged incubation resulted in d-galactose and d-galactobiose, predominantly. MALDI-TOF analysis revealed the release of l-arabinose substituted d-galacto-oligosaccharides from soy arabinogalactan. This is the first report of the ability of a beta-1,4-endogalactanase to release substituted d-galacto-oligosaccharides. GALA was not active towards d-galacto-oligosaccharides that were substituted with d-glucose at the reducing end.

  20. Tunable recombinant protein expression with E. coli in a mixed-feed environment.

    Science.gov (United States)

    Sagmeister, Patrick; Schimek, Clemens; Meitz, Andrea; Herwig, Christoph; Spadiut, Oliver

    2014-04-01

    Controlling the recombinant protein production rate in Escherichia coli is of utmost importance to ensure product quality and quantity. Up to now, only the genetic construct, introduced into E. coli, and the specific growth rate of the culture were used to influence and stir the productivity. However, bioprocess technological means to control or even tune the productivity of E. coli are scarce. Here, we present a novel method for the process-technological control over the recombinant protein expression rate in E. coli. A mixed-feed fed-batch bioprocess based on the araBAD promoter expression system using both D-glucose and L-arabinose as assimilable C-sources was designed. Using the model product green fluorescent protein, we show that the specific product formation rate can be efficiently tuned even on the cellular level only via the uptake rate of L-arabinose. This novel approach introduces an additional degree of freedom for the design of recombinant bioprocesses with E. coli. We anticipate that the presented method will result in significant quality and robustness improvement as well as cost and process time reduction for recombinant bacterial bioprocesses in the future.

  1. ORF Alignment: NC_005090 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available ... PEPTIDYL-PROLYL ISOMERASE [Wolinella succinogenes] ... Length = 147 ... Query: 29 ... VVVLETTSGTIELTLFPKAAPKAVENFTTH...VKNGYYDGLIFHRVIKRFMLQXXXXXXXXX 88 ... VVVLETTSGTIELTLFPKAAPKAVENFTTH...VKNGYYDGLIFHRVIKRFMLQ ... Sbjct: 1 ... VVVLETTSGTIELTLFPKAAPKAVENFTTHVKNGYYDGLIFHRVIKRFMLQGGDP

  2. Production of fructose-containing syrup with enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Helwiig-Nielsen, B

    1981-01-01

    A review on enzymic processes used for production of fructose- high syrup from starch including liquefaction by alpha-amylase, saccharification by amyloglucosidase, and isomerization with glucose isomerase.

  3. A Pyranose-2-Phosphate Motif Is Responsible for Both Antibiotic Import and Quorum-Sensing Regulation in Agrobacterium tumefaciens.

    Science.gov (United States)

    El Sahili, Abbas; Li, Si-Zhe; Lang, Julien; Virus, Cornelia; Planamente, Sara; Ahmar, Mohammed; Guimaraes, Beatriz G; Aumont-Nicaise, Magali; Vigouroux, Armelle; Soulère, Laurent; Reader, John; Queneau, Yves; Faure, Denis; Moréra, Solange

    2015-08-01

    Periplasmic binding proteins (PBPs) in association with ABC transporters select and import a wide variety of ligands into bacterial cytoplasm. They can also take up toxic molecules, as observed in the case of the phytopathogen Agrobacterium tumefaciens strain C58. This organism contains a PBP called AccA that mediates the import of the antibiotic agrocin 84, as well as the opine agrocinopine A that acts as both a nutrient and a signalling molecule for the dissemination of virulence genes through quorum-sensing. Here, we characterized the binding mode of AccA using purified agrocin 84 and synthetic agrocinopine A by X-ray crystallography at very high resolution and performed affinity measurements. Structural and affinity analyses revealed that AccA recognizes an uncommon and specific motif, a pyranose-2-phosphate moiety which is present in both imported molecules via the L-arabinopyranose moiety in agrocinopine A and the D-glucopyranose moiety in agrocin 84. We hypothesized that AccA is a gateway allowing the import of any compound possessing a pyranose-2-phosphate motif at one end. This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate. By combining affinity measurements and in vivo assays, we demonstrated that both L-arabinose-2-phosphate and D-glucose-2-phosphate, which are the AccF mediated degradation products of agrocinopine A and agrocin 84 respectively, interact with the master transcriptional regulator AccR and activate the quorum-sensing signal synthesis and Ti plasmid transfer in A. tumefaciens C58. Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif. It also opens future possibilities for the rational design of

  4. A Pyranose-2-Phosphate Motif Is Responsible for Both Antibiotic Import and Quorum-Sensing Regulation in Agrobacterium tumefaciens.

    Directory of Open Access Journals (Sweden)

    Abbas El Sahili

    2015-08-01

    Full Text Available Periplasmic binding proteins (PBPs in association with ABC transporters select and import a wide variety of ligands into bacterial cytoplasm. They can also take up toxic molecules, as observed in the case of the phytopathogen Agrobacterium tumefaciens strain C58. This organism contains a PBP called AccA that mediates the import of the antibiotic agrocin 84, as well as the opine agrocinopine A that acts as both a nutrient and a signalling molecule for the dissemination of virulence genes through quorum-sensing. Here, we characterized the binding mode of AccA using purified agrocin 84 and synthetic agrocinopine A by X-ray crystallography at very high resolution and performed affinity measurements. Structural and affinity analyses revealed that AccA recognizes an uncommon and specific motif, a pyranose-2-phosphate moiety which is present in both imported molecules via the L-arabinopyranose moiety in agrocinopine A and the D-glucopyranose moiety in agrocin 84. We hypothesized that AccA is a gateway allowing the import of any compound possessing a pyranose-2-phosphate motif at one end. This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate. By combining affinity measurements and in vivo assays, we demonstrated that both L-arabinose-2-phosphate and D-glucose-2-phosphate, which are the AccF mediated degradation products of agrocinopine A and agrocin 84 respectively, interact with the master transcriptional regulator AccR and activate the quorum-sensing signal synthesis and Ti plasmid transfer in A. tumefaciens C58. Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif. It also opens future possibilities for the

  5. Purification and characterization of xylitol dehydrogenase with l-arabitol dehydrogenase activity from the newly isolated pentose-fermenting yeast Meyerozyma caribbica 5XY2.

    Science.gov (United States)

    Sukpipat, Wiphat; Komeda, Hidenobu; Prasertsan, Poonsuk; Asano, Yasuhisa

    2017-01-01

    Meyerozyma caribbica strain 5XY2, which was isolated from an alcohol fermentation starter in Thailand, was found to catabolize l-arabinose as well as d-glucose and d-xylose. The highest production amounts of ethanol from d-glucose, xylitol from d-xylose, and l-arabitol from l-arabinose were 0.45 g/g d-glucose, 0.60 g/g d-xylose, and 0.61 g/g l-arabinose with 21.7 g/L ethanol, 20.2 g/L xylitol, and 30.3 g/l l-arabitol, respectively. The enzyme with l-arabitol dehydrogenase (LAD) activity was purified from the strain and found to exhibit broad specificity to polyols, such as xylitol, d-sorbitol, ribitol, and l-arabitol. Xylitol was the preferred substrate with K m =16.1 mM and k cat /K m =67.0 min -1 mM -1 , while l-arabitol was also a substrate for the enzyme with K m =31.1 mM and k cat /K m =6.5 min -1  mM -1 . Therefore, this enzyme from M. caribbica was named xylitol dehydrogenase (McXDH). McXDH had an optimum temperature and pH at 40°C and 9.5, respectively. The McXDH gene included a coding sequence of 1086 bp encoding a putative 362 amino acid protein of 39 kDa with an apparent homopentamer structure. Native McXDH and recombinant McXDH exhibited relative activities toward l-arabitol of approximately 20% that toward xylitol, suggesting the applicability of this enzyme with the functions of XDH and LAD to the development of pentose-fermenting Saccharomyces cerevisiae. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  6. At the Perphery of the Amidohydrolase Superfamily: Bh0493 from Bacillus halodurans Catalyzes the Isomerization of D-Galacturonate to D-Tagaturonate

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen,T.; Brown, S.; Fedorov, A.; Fedorov, E.; Babbitt, P.; Almo, S.; Raushel, F.

    2008-01-01

    The amidohydrolase superfamily is a functionally diverse set of enzymes that catalyzes predominantly hydrolysis reactions involving sugars, nucleic acids, amino acids, and organophosphate esters. One of the most divergent members of this superfamily, uronate isomerase from Escherichia coli, catalyzes the isomerization of d-glucuronate to d-fructuronate and d-galacturonate to d-tagaturonate and is the only uronate isomerase in this organism. A gene encoding a putative uronate isomerase in Bacillus halodurans (Bh0705) was identified based on sequence similarity to uronate isomerases from other organisms. Kinetic evidence indicates that Bh0705 is relatively specific for the isomerization of d-glucuronate to d-fructuronate, confirming this functional assignment. Despite a low sequence identity to all other characterized uronate isomerases, phylogenetic and network-based analysis suggests that a second gene in this organism, Bh0493, is also a uronate isomerase, although it is an outlier in the group, with <20% sequence identity to any other characterized uronate isomerase from another species. The elucidation of the X-ray structure at a resolution of 2.0 Angstroms confirms that Bh0493 is a member of the amidohydrolase superfamily with conserved residues common to other members of the uronate isomerase family. Functional characterization of this protein shows that unlike Bh0705, Bh0493 can utilize both d-glucuronate and d-galacturonate as substrates. In B. halodurans, Bh0705 is found in an operon for the metabolism of d-glucuronate, whereas Bh0493 is in an operon for the metabolism of d-galacturonate. These results provide the first identification of a uronate isomerase that operates in a pathway distinct from that for d-glucuronate. While most organisms that contain this pathway have only one gene for a uronate isomerase, sequence analysis and operon context show that five other organisms also appear to have two genes and one organism appears to have three genes for

  7. Synthesis, spectroscopic and biological studies of transition metal complexes of novel schiff bases derived from cephradine and sugars

    International Nuclear Information System (INIS)

    Naz, N.; Iqbal, M.Z.

    2011-01-01

    Fe(II), Co(II) and Ni(II) metal complexes of novel schiff bases derived from Cephradine and sugars (D-Glucose, L. Arabinose and D-Galactose) were synthesized and characterized by elemental analysis, magnetic susceptibility, thermal analysis, electronic absorption and FT-IR spectral studies. It has been found that schiff bases behave as bi-dentate-ligands forming complexes with 1:2 (metal:ligand) stoichiometry. the neutral nature of the complexes was confirmed by their low conductance values. The biological activities of complexes have been evaluated against two gram negative (Escherichia coli and Pseudomonas aeruginosa) and two gram positive (Bacillus subtilis and staphylococcus aureus) bacteria by Agar diffusion disc method. It has been found that the complexes have higher activity as compared to the pure Cephradine against the same bacteria. (author)

  8. Xylanases and Their Applications in Baking Industry

    Directory of Open Access Journals (Sweden)

    Masood Sadiq Butt

    2008-01-01

    Full Text Available Xylan is the second most abundant polysaccharide and a major component of plant cell wall. Cereal xylans contain large quantities of L-arabinose and are therefore, often referred to as arabinoxylans. Xylanases are hydrolytic enzymes, which randomly cleave the β-1,4 backbone of this complex plant cell wall polysaccharide. Different species of Aspergillus and Trichoderma produce these enzymes. Xylanases are of great value in baking as they have been found to improve the bread volume, crumb structure and reduce stickiness. When xylanases are used at optimum levels, they play a significant role in increasing shelf life of bread and reduce bread staling. There is an increasing trend in baking industry towards the application of xylanases in bread production. This review discusses the application of xylanase in the bakery industry, alone and in combination with other enzymes when it shows synergism in the action with them.

  9. Hydrothermal pentose to furfural conversion and simultaneous extraction with SC-CO2--kinetics and application to biomass hydrolysates.

    Science.gov (United States)

    Gairola, Krishan; Smirnova, Irina

    2012-11-01

    This work explores hydrothermal d-xylose and hemicellulose to furfural conversion coupled with simultaneous furfural extraction by SC-CO(2) and the underlying reaction pathway. A maximum furfural yield of 68% was attained from d-xylose at 230°C and 12MPa. Additionally missing kinetic data for l-arabinose to furfural conversion was provided, showing close similarity to d-xylose. Furfural yields from straw and brewery waste hydrolysates were significantly lower than those obtained from model compounds, indicating side reactions with other hydrolysate components. Simultaneous furfural extraction by SC-CO(2) significantly increased extraction yield in all cases. The results indicate that furfural reacts with intermediates of pentose dehydration. The proposed processing route can be well integrated into existing lignocellulose biorefinery concepts. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. Trapped electrons in irradiated single crystals of polyhydroxy compounds

    International Nuclear Information System (INIS)

    Box, H.C.; Budzinski, E.E.; Freund, H.G.; Potter, W.R.

    1979-01-01

    The intermolecular trapping of electrons has been observed in single crystals of dulcitol and L(+) arabinose x-irradiated at 4.2 0 K. Attribution of a major component of the ESR absorption to trapped electrons is based upon the character of the hyperfine pattern, which arises from multiple anisotropic hyperfine interactions with exchangeable protons, and on the g value of the absorption, which is always less than the free spin value. The removal of the trapped electron absorption upon irradiation with visible light has also been demonstrated. In these experiments all of the electrons are trapped in identical sites. This circumstance provides some important advantages in the study of the factors affecting the stabilization of charge in an environment of polarizable molecules

  11. Base catalysed isomerisation of aldoses of the arabino and lyxo series in the presence of aluminate.

    Science.gov (United States)

    Ekeberg, Dag; Morgenlie, Svein; Stenstrøm, Yngve

    2002-04-30

    Base-catalysed isomerisation of aldoses of the arabino and lyxo series in aluminate solution has been investigated. L-Arabinose and D-galactose give L-erythro-2-pentulose (L-ribulose) and D-lyxo-2-hexulose (D-tagatose), respectively, in good yields, whereas lower reactivity is observed for 6-deoxy-D-galactose (D-fucose). From D-lyxose, D-mannose and 6-deoxy-L-mannose (L-rhamnose) are obtained mixtures of ketoses and C-2 epimeric aldoses. Small amounts of the 3-epimers of the ketoses were also formed. 6-Deoxy-L-arabino-2-hexulose (6-deoxy-L-fructose) and 6-deoxy-L-glucose (L-quinovose) were formed in low yields from 6-deoxy-L-mannose and isolated as their O-isopropylidene derivatives. Explanations of the differences in reactivity and course of the reaction have been suggested on the basis of steric effects.

  12. Development of microbial biosensors for food analysis

    DEFF Research Database (Denmark)

    Lukasiak, Justyna

    in order to fulfill the needs of different fields, from environmental sciences to food industry. Moreover, they can be an answer for the need of novel, less expensive and environmentally neutral methods of analysis particularly in food ingredients assessment. The aim of this PhD thesis was to develop...... heteropolysaccharide commonly used in food industry as a gelling agent and food stabilizer. The chemical analysis of the pectin carbohydrate composition is a significant issue during the study of its function and properties. Arabinoxylan is one of the main non-starch polysaccharide derived from the cell wall of cereal...... grains. It is a dietary fiber, with potential as a functional food ingredient. In this study, reporter strains targeting specifically L-rhamnose, L-arabinose and Dxylose using three different signal transducers: bioluminescence (luxCDABE), fluorescence (gfp) and ice nucleation (inaZ) were developed...

  13. The biosynthesis of polysaccharides. Incorporation of d-[1-14C]glucose and d-[6-14C]glucose into plum-leaf polysaccharides

    Science.gov (United States)

    Andrews, P.; Hough, L.; Picken, J. M.

    1965-01-01

    1. The utilization of specifically labelled d-glucose in the biosynthesis of plum-leaf polysaccharides has been studied. After these precursors had been metabolized in plum leaves, the polysaccharides were isolated from the leaves, and their monosaccharide constituents isolated and purified. 2. Both the specific activities and the distribution of 14C along the carbon chains of the monosaccharides were determined. Significant 14C activity was found in units of d-galactose, d-glucose, d-xylose and l-arabinose, but their specific activities varied widely. The labelling patterns suggest that in the leaves the other monosaccharides all arise directly from d-glucose without any skeletal change in the carbon chain, other than the loss of a terminal carbon atom in the synthesis of pentoses. 3. The results indicated that within the leaf there are various precursor pools for polysaccharide synthesis and that these pools are not in equilibrium with one another. PMID:14342252

  14. Furfural Production from d-Xylose and Xylan by Using Stable Nafion NR50 and NaCl in a Microwave-Assisted Biphasic Reaction

    Directory of Open Access Journals (Sweden)

    Sarah Le Guenic

    2016-08-01

    Full Text Available Pentose dehydration and direct transformation of xylan into furfural were performed in a water-cyclopentyl methyl ether (CPME biphasic system under microwave irradiation. Heated up between 170 and 190 °C in the presence of Nafion NR50 and NaCl, d-xylose, l-arabinose and xylan gave furfural with maximum yields of 80%, 42% and 55%, respectively. The influence of temperature and reaction time on the reaction kinetics was discussed. This study was also completed by the survey of different reactant ratios, such as organic layer-water or catalyst-inorganic salt ratios. The exchange between proton and cation induced by an excess of NaCl was monitored, and a synergetic effect between the remaining protons and the released HCl was also discovered.

  15. Furfural Production from d-Xylose and Xylan by Using Stable Nafion NR50 and NaCl in a Microwave-Assisted Biphasic Reaction.

    Science.gov (United States)

    Le Guenic, Sarah; Gergela, David; Ceballos, Claire; Delbecq, Frederic; Len, Christophe

    2016-08-22

    Pentose dehydration and direct transformation of xylan into furfural were performed in a water-cyclopentyl methyl ether (CPME) biphasic system under microwave irradiation. Heated up between 170 and 190 °C in the presence of Nafion NR50 and NaCl, d-xylose, l-arabinose and xylan gave furfural with maximum yields of 80%, 42% and 55%, respectively. The influence of temperature and reaction time on the reaction kinetics was discussed. This study was also completed by the survey of different reactant ratios, such as organic layer-water or catalyst-inorganic salt ratios. The exchange between proton and cation induced by an excess of NaCl was monitored, and a synergetic effect between the remaining protons and the released HCl was also discovered.

  16. Cathode Assessment for Maximizing Current Generation in Microbial Fuel Cells Utilizing Bioethanol Effluent as Substrate

    DEFF Research Database (Denmark)

    Sun, Guotao; Thygesen, Anders; Meyer, Anne S.

    2016-01-01

    Implementation of microbial fuel cells (MFCs) for electricity production requires effective current generation from waste products via robust cathode reduction. Three cathode types using dissolved oxygen cathodes (DOCs), ferricyanide cathodes (FeCs) and air cathodes (AiCs) were therefore assessed...... to be the most sustainable option since it does not require ferricyanide. The data offer a new add-on option to the straw biorefinery by using bioethanol effluent for microbial electricity production....... using bioethanol effluent, containing 20.5 g/L xylose, 1.8 g/L arabinose and 2.5 g/L propionic acid. In each set-up the anode and cathode had an electrode surface area of 88 cm(2), which was used for calculation of the current density. Electricity generation was evaluated by quantifying current...

  17. Chemical Characteristics and Antioxidant Properties of Crude Water Soluble Polysaccharides from Four Common Edible Mushrooms

    Directory of Open Access Journals (Sweden)

    Pei-Long Sun

    2012-04-01

    Full Text Available Four crude water soluble polysaccharides, CABP, CAAP, CFVP and CLDP, were isolated from common edible mushrooms, including Agaricus bisporus, Auricularia auricula, Flammulina velutipes and Lentinus edodes, and their chemical characteristics and antioxidant properties were determined. Fourier Transform-infrared analysis showed that the four crude polysaccharides were all composed of β-glycoside linkages. The major monosaccharide compositions were D-galactose, D-glucose and D-mannose for CABP, CAAP and CLDP, while CFVP was found to consist of L-arabinose, D-galactose, D-glucose and D-mannose. The main molecular weight distributions of CABP and the other three polysaccharides were 66.0 × 104 Da, respectively. Antioxidant properties of the four polysaccharides were evaluated in in vitro systems and CABP showed the best antioxidant properties. The studied mushroom species could potentially be used in part of well-balanced diets and as a source of antioxidant compounds.

  18. Centrifugal partition chromatography in a biorefinery context: Separation of monosaccharides from hydrolysed sugar beet pulp.

    Science.gov (United States)

    Ward, David P; Cárdenas-Fernández, Max; Hewitson, Peter; Ignatova, Svetlana; Lye, Gary J

    2015-09-11

    A critical step in the bioprocessing of sustainable biomass feedstocks, such as sugar beet pulp (SBP), is the isolation of the component sugars from the hydrolysed polysaccharides. This facilitates their subsequent conversion into higher value chemicals and pharmaceutical intermediates. Separation methodologies such as centrifugal partition chromatography (CPC) offer an alternative to traditional resin-based chromatographic techniques for multicomponent sugar separations. Highly polar two-phase systems containing ethanol and aqueous ammonium sulphate are examined here for the separation of monosaccharides present in hydrolysed SBP pectin: l-rhamnose, l-arabinose, d-galactose and d-galacturonic acid. Dimethyl sulfoxide (DMSO) was selected as an effective phase system modifier improving monosaccharide separation. The best phase system identified was ethanol:DMSO:aqueous ammonium sulphate (300gL(-1)) (0.8:0.1:1.8, v:v:v) which enabled separation of the SBP monosaccharides by CPC (200mL column) in ascending mode (upper phase as mobile phase) with a mobile phase flow rate of 8mLmin(-1). A mixture containing all four monosaccharides (1.08g total sugars) in the proportions found in hydrolysed SBP was separated into three main fractions; a pure l-rhamnose fraction (>90%), a mixed l-arabinose/d-galactose fraction and a pure d-galacturonic acid fraction (>90%). The separation took less than 2h demonstrating that CPC is a promising technique for the separation of these sugars with potential for application within an integrated, whole crop biorefinery. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  19. Extração e caracterização de xilanas de sabugos de milho Extraction and characterization of xylans from corncobs

    Directory of Open Access Journals (Sweden)

    Simone S. Silva

    1998-06-01

    Full Text Available Neste trabalho, duas frações de xilana, denominadas xilana A e xilana B, foram isoladas a partir de sabugos de milho através de três processos diferentes, combinando métodos de extração aquosa, remoção de lipídeos, deslignificação e extração alcalina. Os produtos obtidos durante os processos foram analisados por termogravimetria. A etapa de deslignificação foi responsável por uma acentuada degradação dos polímeros, evidenciada por queda de rendimento e resistência térmica. Os espectros obtidos no infravermelho evidenciaram a ausência de ácidos urônicos na cadeia polimérica. As viscosidades intrínsecas obtidas para a xilana A (56 mL/g e xilana B (75 mL/g associadas aos resultados do infravermelho sugerem um número maior de grupos substituintes, constituídos basicamente por resíduos de L-arabinose, para a xilana B.In this work, two fractions of xylan, named xylan A and xylan B, were isolated from corncobs through three different processes using aqueous extraction, lipid removal, delignification, and alkaline extraction. The products obtained during the processes were analysed by thermogravimetry. The delignification step was responsible for the occurrence of an accentuated polymer degradation, evidenced by yield and thermal resistance decrease. Infrared spectra indicated the absence of uronic acids in the polymeric chains. Intrinsic viscosities obtained for xylan A (56 mL/g and xylan B (75 mL/g, associated to the results from i.r. analysis, suggested a higher number of substituents, basically constituted of L-arabinose residues, in the case of xylan B.

  20. [Cross-sectional analysis of heart failure among patients in the Internal Medicine Service at a third-level hospital. Part I: epidemiologic analysis].

    Science.gov (United States)

    Cinza Sanjurjo, S; Cabarcos Ortiz de Barrón, A; Enrique Nieto Pol, E; Torre Carballada, J A

    2007-06-01

    To observe the epidemiologic characteristics of the patients intake during five years in a internal medicine department, with heart failure. A cross-sectional study of the intake patients in the Internal Medicine Service in the Hospital Clínico Universitario de Santiago de Compostela between 1999 to 2003. The variables analized were: sex, age, days of hospital stay, number of intake by failure cardiac, reason for admission (guide symptom), hypertension, diabetes mellitus, cardiac disease, fibrillation atrium, previous treatment with beta-blockers, blood pressure in the admission moment, to make echocardiography, disfunction systolic, etiology, deceased, treatment at the end. The statistical analysis was performed with qualitative and quantitative measures, chi-cuadrado and t-student, and multivariant analyses. 248 patients were accepted for the study. We observed more women than men (55.2%) and bigger median age (79 years old vs. 73 years old in men, p < 0.001). The mean income was 13.61 days and a median of 11 days. The 41,8% of the patients had hypertension, 30.9% diabetes mellitus and 81,9% had someone heart disease. The aetiologies of heart failure most frequent were ischemic cardiopathy (27.2%) and hypertension (24.2%). The most frequent symptom was the dyspnea (68.9%). It made echocardiography in 20.9% of patients and 45.1% showed systolic disfunction. The only factor related with this small percentage of echocardiographies was the incoming time. The most frequent etiology was respiratory infections (39.5%). The 8.6% of patients was deceased. The pharmacologic treatment more prescribed were the diuretics (86.9%) and transcutaneous nitrates (49.5%). It was indicated ECAI or AAR-II in the 86.9% of patients and beta-blockers in 0.9%. The number of echocardiograms practiced to the patients is smaller that the number advised by international associations and smaller to the cardiologist registers. The beta-blockers and ECAI use is smaller too.

  1. [Cross-sectional study of heart failure of patients intaked in an internal medicine service in the third level hospital in mixed area. Part II: prevalence and hypertension control].

    Science.gov (United States)

    Cinza Sanjurjo, S; Cabarcos Ortiz de Barrón, A; Nieto Pol, E; Torre Carballada, J A

    2007-07-01

    To know the arterial hypertension prevalencia and hypertension control in the patients income by heart failure. A cross-sectional study of the intaked patients in the Internal Medicine Service in the Hospital Clínico Universitario de Santiago de Compostela between 1999 to 2003. The variables analysed were: sex, age, days of hospital stay, number of intaked by failure cardiac, reason for admission (guide symptom), hypertension, diabetes mellitus, cardiac disease, fibrillation atrium, previous treatment with beta-blockers, blood pressure in the admission moment, to make echocardiography, disfunction systolic, etiology, deceased, treatment at the end. The statistical analysis was performed with qualitative and quantitative measures, chi-cuadrado and t-student, and multivariant analyses. 248 patients were accepted for the study, and 100 were hypertensive patients (41.8%). We observed more women than men in hypertensive group (63.0%) and in non hypertensive group (51.1%). The median age was 77 years old in both groups. The median income was 11 days. The number of patients with diabetes mellitus and ischemic cardiopathy was bigger in hypertension group (43.0 vs. 22.3%), p < 0.001; (38 vs. 21.6%), p = 0.005. The most frequent symptom was the dyspnea (66,9%), in both groups, p = 0.62. The 62.6% of the patients were bad control of blood pressures. The prevalence of bad control in hypertensive patients was bigger tha non-hypertensive patients (76.9 vs. 59.4%, p = 0.01). The pharmacologic treatment more prescribed in hypertensive patients ECAI or AAR-II (62.6 vs. 26.8%, p < 0.001). And the diuretics wee more prescribed in non-hypertensive patients (91.1 vs. 81.1%, p = 0.03). The prevalence of diabetes mellitus is associated with hypertension in the patients. The ECAI prescription was acceptable. The number of echocardiograms practiced to the patients is smaller that the number advised by international associations and smaller to the cardiologist registers. The beta

  2. Strains for the production of flavonoids from glucose

    Energy Technology Data Exchange (ETDEWEB)

    Stephanopoulos, Gregory; Santos, Christine; Koffas, Mattheos

    2015-11-13

    The invention relates to the production of flavonoids and flavonoid precursors in cells through recombinant expression of tyrosine ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI).

  3. JB_054_Supplementary_Tables.docx

    Indian Academy of Sciences (India)

    LENOVO

    TRI 10032, T. aestivum L. var. aestivum, Mexico, Spring, Q4 ... TRI 10311, T. aestivum L. var. aestivum, Japan, Spring, Q2 ..... precursor (sucrose export defective 1), dihydrolipoamide dehydrogenase precursor, disulfide-isomerase precursor.

  4. Fulltext PDF

    Indian Academy of Sciences (India)

    Unknown

    deoxyribonucleic acid. M GOPALa*, M S ... base pairs of DNA and interference with normal functioning of the enzyme topo- isomerase II which ... Data from the fluorescence titrations were used to determine the binding constant of. MPTQ with ...

  5. FKBP immunophilins and Alzheimer's disease: A chaperoned affair

    Indian Academy of Sciences (India)

    All FKBPs contain a domain with prolyl cis/trans isomerase (PPIase) activity. ... FKBP proteins are expressed ubiquitously but show relatively high levels of expression in the nervous .... evolution, higher organisms like humans, who normally.

  6. Process for assembly and transformation into Saccharomyces cerevisiae of a synthetic yeast artificial chromosome containing a multigene cassette to express enzymes that enhance xylose utilization designed for an automated pla

    Science.gov (United States)

    A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system ...

  7. Main: 1QRR [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available Molecule: Sulfolipid Biosynthesis (Sqd1) Protein; Chain: A; Engineered: Yes Isomerase A.M.Mulichak, M.J.Thei...IASIHDRISRWKALTGKSIELYVGDICDFEFLAESFKSFEPDSVVHFGEQRSAPYSMIDRSRAVYTQHNNVIGTLNVLFAIKEFGEECHLVKLGTMGEYGTPNIDIEE...LNRFCVQAAVGHPLTVYGKGGQTRGYLDIRDTVQCVEIAIANPAKAGEFRVFNQFTEQFSVNELASLVTKAGSKLGLDVKKMTVPNPRVEAEEHYYNAKHTKLMELGLEPHYLSDSLLDSLLNFAVQFKDRVDTKQIMPSVSWKKIGVKTKSMTT arabi_1QRR.jpg ...

  8. Materials Biotechnology Symposium Proceedings Held in Natick, Massachusetts on June 23 and 24, 1987

    Science.gov (United States)

    1987-11-05

    21. O.J. Lantaro, New immobilized whole cell glucose isomerase for fructose syrup production, Eng. Foundations Conf., White Haven, PA, Sept. 25-30...1983. 22. Miles Laboratories, Inc., Taka-Sweets- Immobilized glucose isomerase for high fructose syrup production, Elkhart, IN. 203...for poly(L-valine) and poly(L-isoleucine) in water differ markedly from each other, primarily because of the different degrees of hydration of the

  9. Pnp gene modification for improved xylose utilization in Zymomonas

    Science.gov (United States)

    Caimi, Perry G G; Qi, Min; Tao, Luan; Viitanen, Paul V; Yang, Jianjun

    2014-12-16

    The endogenous pnp gene encoding polynucleotide phosphorylase in the Zymomonas genome was identified as a target for modification to provide improved xylose utilizing cells for ethanol production. The cells are in addition genetically modified to have increased expression of ribose-5-phosphate isomerase (RPI) activity, as compared to cells without this genetic modification, and are not limited in xylose isomerase activity in the absence of the pnp modification.

  10. Competition between pentoses and glucose during uptake and catabolism in recombinant Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Subtil Thorsten

    2012-03-01

    Full Text Available Abstract Background In mixed sugar fermentations with recombinant Saccharomyces cerevisiae strains able to ferment D-xylose and L-arabinose the pentose sugars are normally only utilized after depletion of D-glucose. This has been attributed to competitive inhibition of pentose uptake by D-glucose as pentose sugars are taken up into yeast cells by individual members of the yeast hexose transporter family. We wanted to investigate whether D-glucose inhibits pentose utilization only by blocking its uptake or also by interfering with its further metabolism. Results To distinguish between inhibitory effects of D-glucose on pentose uptake and pentose catabolism, maltose was used as an alternative carbon source in maltose-pentose co-consumption experiments. Maltose is taken up by a specific maltose transport system and hydrolyzed only intracellularly into two D-glucose molecules. Pentose consumption decreased by about 20 - 30% during the simultaneous utilization of maltose indicating that hexose catabolism can impede pentose utilization. To test whether intracellular D-glucose might impair pentose utilization, hexo-/glucokinase deletion mutants were constructed. Those mutants are known to accumulate intracellular D-glucose when incubated with maltose. However, pentose utilization was not effected in the presence of maltose. Addition of increasing concentrations of D-glucose to the hexo-/glucokinase mutants finally completely blocked D-xylose as well as L-arabinose consumption, indicating a pronounced inhibitory effect of D-glucose on pentose uptake. Nevertheless, constitutive overexpression of pentose-transporting hexose transporters like Hxt7 and Gal2 could improve pentose consumption in the presence of D-glucose. Conclusion Our results confirm that D-glucose impairs the simultaneous utilization of pentoses mainly due to inhibition of pentose uptake. Whereas intracellular D-glucose does not seem to have an inhibitory effect on pentose utilization

  11. Combinatorial control of gene expression in Aspergillus niger grown on sugar beet pectin.

    Science.gov (United States)

    Kowalczyk, Joanna E; Lubbers, Ronnie J M; Peng, Mao; Battaglia, Evy; Visser, Jaap; de Vries, Ronald P

    2017-09-27

    Aspergillus niger produces an arsenal of extracellular enzymes that allow synergistic degradation of plant biomass found in its environment. Pectin is a heteropolymer abundantly present in the primary cell wall of plants. The complex structure of pectin requires multiple enzymes to act together. Production of pectinolytic enzymes in A. niger is highly regulated, which allows flexible and efficient capture of nutrients. So far, three transcriptional activators have been linked to regulation of pectin degradation in A. niger. The L-rhamnose-responsive regulator RhaR controls the production of enzymes that degrade rhamnogalacturonan-I. The L-arabinose-responsive regulator AraR controls the production of enzymes that decompose the arabinan and arabinogalactan side chains of rhamnogalacturonan-II. The D-galacturonic acid-responsive regulator GaaR controls the production of enzymes that act on the polygalacturonic acid backbone of pectin. This project aims to better understand how RhaR, AraR and GaaR co-regulate pectin degradation. For that reason, we constructed single, double and triple disruptant strains of these regulators and analyzed their growth phenotype and pectinolytic gene expression in A. niger grown on sugar beet pectin.

  12. High resolution visualization and exo-proteomics reveal the physiological role of XlnR and AraR in plant biomass colonization and degradation by Aspergillus niger.

    Science.gov (United States)

    Kowalczyk, Joanna E; Khosravi, Claire; Purvine, Samuel; Dohnalkova, Alice; Chrisler, William B; Orr, Galya; Robinson, Errol; Zink, Erika; Wiebenga, Ad; Peng, Mao; Battaglia, Evy; Baker, Scott; de Vries, Ronald P

    2017-11-01

    In A. niger, two transcription factors, AraR and XlnR, regulate the production of enzymes involved in degradation of arabinoxylan and catabolism of the released l-arabinose and d-xylose. Deletion of both araR and xlnR in leads to reduced production of (hemi)cellulolytic enzymes and reduced growth on arabinan, arabinogalactan and xylan. In this study, we investigated the colonization and degradation of wheat bran by the A. niger reference strain CBS 137562 and araR/xlnR regulatory mutants using high-resolution microscopy and exo-proteomics. We discovered that wheat bran flakes have a 'rough' and 'smooth' surface with substantially different affinity towards fungal hyphae. While colonization of the rough side was possible for all strains, the xlnR mutants struggled to survive on the smooth side of the wheat bran particles after 20 and 40 h post inoculation. Impaired colonization ability of the smooth surface of wheat bran was linked to reduced potential of ΔxlnR to secrete arabinoxylan and cellulose-degrading enzymes and indicates that XlnR is the major regulator that drives colonization of wheat bran in A. niger. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  13. Physicochemical properties and antidiabetic effects of a polysaccharide from corn silk in high-fat diet and streptozotocin-induced diabetic mice.

    Science.gov (United States)

    Pan, Yuxiang; Wang, Cong; Chen, Zhongqin; Li, Weiwei; Yuan, Guoqi; Chen, Haixia

    2017-05-15

    This study aimed to investigate the physicochemical properties and antidiabetic effects of a polysaccharide obtained from corn silk (PCS2). PCS2 was isolated and the physicochemical properties were characterized. The hypoglycemic effects were determined using the high-fat diet and streptozocin induced type 2 diabetic mellitus (T2DM) insulin resistance mice. The results showed that PCS2 was a heteropolysaccharide with the average molecular weight of 45.5kDa. PCS2 was composed of d-galactose, d-mannose, d-(+)-glucose, d-(+)-xylose, l-arabinose and l-rhamnose. PCS2 treatment significantly reduced the body weight loss, decreased blood glucose and serum insulin levels, and improved glucose intolerance (P<0.05). The levels of serum lipid profile were regulated and the levels of glycated serum protein, non-esterified fatty acid were decreased significantly (P<0.01). The activities of superoxide dismutase, glutathione peroxidase and catalase were notably improved (P<0.05). PCS2 also exerted cytoprotective action from histopathological observation. These results suggested that PCS2 could be a good candidate of functional food or medicine for T2DM treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Anti-tumoral effect of the mitochondrial target domain of Noxa delivered by an engineered Salmonella typhimurium.

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    Jae-Ho Jeong

    Full Text Available Bacterial cancer therapy relies on the fact that several bacterial species are capable of targeting tumor tissue and that bacteria can be genetically engineered to selectively deliver therapeutic proteins of interest to the targeted tumors. However, the challenge of bacterial cancer therapy is the release of the therapeutic proteins from the bacteria and entry of the proteins into tumor cells. This study employed an attenuated Salmonella typhimurium to selectively deliver the mitochondrial targeting domain of Noxa (MTD as a potential therapeutic cargo protein, and examined its anti-cancer effect. To release MTD from the bacteria, a novel bacterial lysis system of phage origin was deployed. To facilitate the entry of MTD into the tumor cells, the MTD was fused to DS4.3, a novel cell-penetrating peptide (CPP derived from a voltage-gated potassium channel (Kv2.1. The gene encoding DS4.3-MTD and the phage lysis genes were placed under the control of PBAD , a promoter activated by L-arabinose. We demonstrated that DS4.3-MTD chimeric molecules expressed by the Salmonellae were anti-tumoral in cultured tumor cells and in mice with CT26 colon carcinoma.

  15. Arabinoxylan from finger millet (Eleusine coracana, v. Indaf 15) bran: purification and characterization.

    Science.gov (United States)

    Savitha Prashanth, M R; Muralikrishna, G

    2014-01-01

    Water unextractable portion from finger millet bran was sequentially extracted with saturated barium hydroxide (BE) and 1M potassium hydroxide (KE) solutions. They consisted preponderantly of arabinose and xylose in different ratios. Ferulic, caffeic, coumaric and vanillic acids were identified as major bound phenolic acids. BE and KE were purified on DEAE-cellulose column by eluting successively with different eluants. The major fractions (0.1 M ammonium carbonate) were resolved into one (BE) and two subfractions (KE1 and KE2) respectively on Sephacryl S-400 gel filtration chromatography and their homogeneity was ascertained by gel filtration, cellulose acetate membrane electrophoresis and capillary electrophoresis. The average molecular weight of BE, KE1 and KE2 were found to be 430, 1028 and 40 kDa respectively. The structural elucidation of the purified polysaccharides by (1)H and (13)C NMR analysis indicated the backbone to be 1,4-β-D-linked xylan with substitution mainly at O-2 or O-3 and/or both by α-l-arabinose residues. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Biomolecular Mechanisms of Pseudomonas aeruginosa and Escherichia coli Biofilm Formation

    Science.gov (United States)

    Laverty, Garry; Gorman, Sean P.; Gilmore, Brendan F.

    2014-01-01

    Pseudomonas aeruginosa and Escherichia coli are the most prevalent Gram-negative biofilm forming medical device associated pathogens, particularly with respect to catheter associated urinary tract infections. In a similar manner to Gram-positive bacteria, Gram-negative biofilm formation is fundamentally determined by a series of steps outlined more fully in this review, namely adhesion, cellular aggregation, and the production of an extracellular polymeric matrix. More specifically this review will explore the biosynthesis and role of pili and flagella in Gram-negative adhesion and accumulation on surfaces in Pseudomonas aeruginosa and Escherichia coli. The process of biofilm maturation is compared and contrasted in both species, namely the production of the exopolysaccharides via the polysaccharide synthesis locus (Psl), pellicle Formation (Pel) and alginic acid synthesis in Pseudomonas aeruginosa, and UDP-4-amino-4-deoxy-l-arabinose and colonic acid synthesis in Escherichia coli. An emphasis is placed on the importance of the LuxR homologue sdiA; the luxS/autoinducer-II; an autoinducer-III/epinephrine/norepinephrine and indole mediated Quorum sensing systems in enabling Gram-negative bacteria to adapt to their environments. The majority of Gram-negative biofilms consist of polysaccharides of a simple sugar structure (either homo- or heteropolysaccharides) that provide an optimum environment for the survival and maturation of bacteria, allowing them to display increased resistance to antibiotics and predation. PMID:25438014

  17. Protein enrichment of an Opuntia ficus-indica cladode hydrolysate by cultivation of Candida utilis and Kluyveromyces marxianus.

    Science.gov (United States)

    Akanni, Gabriel B; du Preez, James C; Steyn, Laurinda; Kilian, Stephanus G

    2015-03-30

    The cladodes of Opuntia ficus-indica (prickly pear cactus) have a low protein content; for use as a balanced feed, supplementation with other protein sources is therefore desirable. We investigated protein enrichment by cultivation of the yeasts Candida utilis and Kluyveromyces marxianus in an enzymatic hydrolysate of the cladode biomass. Dilute acid pretreatment and enzymatic hydrolysis of sun-dried cladodes resulted in a hydrolysate containing (per litre) 45.5 g glucose, 6.3 g xylose, 9.1 g galactose, 10.8 g arabinose and 9.6 g fructose. Even though K. marxianus had a much higher growth rate and utilized l-arabinose and d-galactose more completely than C. utilis, its biomass yield coefficient was lower due to ethanol and ethyl acetate production despite aerobic cultivation. Yeast cultivation more than doubled the protein content of the hydrolysate, with an essential amino acid profile superior to sorghum and millet grains. This K. marxianus strain was weakly Crabtree positive. Despite its low biomass yield, its performance compared well with C. utilis. This is the first report showing that the protein content and quality of O. ficus-indica cladode biomass could substantially be improved by yeast cultivation, including a comparative evaluation of C. utilis and K. marxianus. © 2014 The Authors. Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

  18. Computer simulation of protein—carbohydrate complexes: application to arabinose-binding protein and pea lectin

    Science.gov (United States)

    Rao, V. S. R.; Biswas, Margaret; Mukhopadhyay, Chaitali; Balaji, P. V.

    1989-03-01

    The CCEM method (Contact Criteria and Energy Minimisation) has been developed and applied to study protein-carbohydrate interactions. The method uses available X-ray data even on the native protein at low resolution (above 2.4 Å) to generate realistic models of a variety of proteins with various ligands. The two examples discussed in this paper are arabinose-binding protein (ABP) and pea lectin. The X-ray crystal structure data reported on ABP-β- L-arabinose complex at 2.8, 2.4 and 1.7 Å resolution differ drastically in predicting the nature of the interactions between the protein and ligand. It is shown that, using the data at 2.4 Å resolution, the CCEM method generates complexes which are as good as the higher (1.7 Å) resolution data. The CCEM method predicts some of the important hydrogen bonds between the ligand and the protein which are missing in the interpretation of the X-ray data at 2.4 Å resolution. The theoretically predicted hydrogen bonds are in good agreement with those reported at 1.7 Å resolution. Pea lectin has been solved only in the native form at 3 Å resolution. Application of the CCEM method also enables us to generate complexes of pea lectin with methyl-α- D-glucopyranoside and methyl-2,3-dimethyl-α- D-glucopyranoside which explain well the available experimental data in solution.

  19. ISOLATION AND CHARACTERIZATION OF A MOLYBDENUM-REDUCING AND GLYPHOSATE-DEGRADING Klebsiella oxytoca STRAIN SAW-5 IN SOILS FROM SARAWAK

    Directory of Open Access Journals (Sweden)

    M.K. Sabullah

    2016-02-01

    Full Text Available Bioremediation of pollutants including heavy metals and xenobiotics is an economic and environmentally friendly process. A novel molybdenum-reducing bacterium with the ability to utilize the pesticide glyphosate as a carbon source is reported. The characterization works were carried out utilizing bacterial resting cells in a microplate format. The bacterium reduces molybdate to Mo-blue optimally between pH 6.3 and 6.8 and at 34oC. Glucose was the best electron donor for supporting molybdate reduction followed by lactose, maltose, melibiose, raffinose, d-mannitol, d-xylose, l-rhamnose, l-arabinose, dulcitol, myo-inositol and glycerol in descending order. Other requirements include a phosphate concentration at 5.0 mM and a molybdate concentration between 20 and 30 mM. The molybdenum blue exhibited an absorption spectrum resembling a reduced phosphomolybdate. Molybdenum reduction was inhibited by mercury, silver, cadmium and copper at 2 ppm by 45.5, 26.0, 18.5 and 16.3%, respectively. Biochemical analysis identified the bacterium as Klebsiella oxytoca strain Saw-5. To conclude, the capacity of this bacterium to reduce molybdenum into a less toxic form and to grow on glyphosate is novel and makes the bacterium an important instrument for bioremediation of these pollutants.

  20. The production of arabitol by a novel plant yeast isolate Candida parapsilosis 27RL-4

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    Kordowska-Wiater Monika

    2017-10-01

    Full Text Available Polyalcohol arabitol can be used in the food and pharmaceutical industries as a natural sweetener, a dental caries reducer, and texturing agent. Environmental samples were screened to isolate effective yeast producers of arabitol. The most promising isolate 27RL-4, obtained from raspberry leaves, was identified genetically and biochemically as Candida parapsilosis. It secreted 10.42– 10.72 g l-1 of product from 20 g l-1 of L-arabinose with a yield of 0.51 - 0.53 g g-1 at 28°C and a rotational speed of 150 rpm. Batch cultures showed that optimal pH value for arabitol production was 5.5. High yields and productivities of arabitol were obtained during incubation of the yeast at 200 rpm, or at 32°C, but the concentrations of the polyol did not exceed 10 g l-1. In modified medium, with reduced amounts of nitrogen compounds and pH 5.5-6.5, lower yeast biomass produced a similar concentration of arabitol, suggesting higher efficiency of yeast cells. This strain also produced arabitol from glucose, with much lower yields. The search for new strains able to successfully produce arabitol is important for allowing the utilization of sugars abundant in plant biomass.

  1. Physical, chemical, and metabolic state sensors expand the synthetic biology toolbox for Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Immethun, Cheryl M; DeLorenzo, Drew M; Focht, Caroline M; Gupta, Dinesh; Johnson, Charles B; Moon, Tae Seok

    2017-07-01

    Many under-developed organisms possess important traits that can boost the effectiveness and sustainability of microbial biotechnology. Photoautotrophic cyanobacteria can utilize the energy captured from light to fix carbon dioxide for their metabolic needs while living in environments not suited for growing crops. Various value-added compounds have been produced by cyanobacteria in the laboratory; yet, the products' titers and yields are often not industrially relevant and lag behind what have been accomplished in heterotrophic microbes. Genetic tools for biological process control are needed to take advantage of cyanobacteria's beneficial qualities, as tool development also lags behind what has been created in common heterotrophic hosts. To address this problem, we developed a suite of sensors that regulate transcription in the model cyanobacterium Synechocystis sp. PCC 6803 in response to metabolically relevant signals, including light and the cell's nitrogen status, and a family of sensors that respond to the inexpensive chemical, l-arabinose. Increasing the number of available tools enables more complex and precise control of gene expression. Expanding the synthetic biology toolbox for this cyanobacterium also improves our ability to utilize this important under-developed organism in biotechnology. Biotechnol. Bioeng. 2017;114: 1561-1569. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  2. Some unique features of alkaliphilic anaerobes

    Science.gov (United States)

    Roof, Erin; Pikuta, Elena; Otto, Christopher; Williams, George; Hoover, Richard

    2013-09-01

    This article explores two topics involving the examination of four strains of alkaliphilic anaerobes. The first topic was dedicated to detection of the ability of microorganisms to metabolize alternative chirality substrates. Two saccharolytic anaerobic bacteria were chosen for the first experiment: Anaerovirgula multivorans strain SCAT, which is gram positive and spore-forming; and Spirochaeta dissipatitropha, strain ASpC2T, which is gram negative. It was found that both checked sugarlytics were able to use L-ribose and L-arabinose, as growth substrates. The second part was concerned of study a chemolithotrophy in two halo-alkaliphilic sulfate reducing bacteria: Desulfonatornum thiodismutans strain MLF1T and Desulfonatronum lacustre strain Z-7951T. The experiments with lithotrophs had demonstrated that strain MLF1T was capable to grow without any organic source of carbon, while strain Z-7951T had required at least 2 mM sodium acetate for growth. Anaerobic technique was used for preparation of the growth media and maintenance of these bacterial cultures. Standard methods for Gram, spore, and flagella staining were applied for characterization of cytomorphology. In this article, the results of the experiments performed on cytological, physiological, and biochemical levels are presented and discussed.

  3. Vibrio rotiferianus sp. nov., isolated from cultures of the rotifer Brachionus plicatilis.

    Science.gov (United States)

    Gomez-Gil, B; Thompson, F L; Thompson, C C; Swings, J

    2003-01-01

    Five Gram-negative bacterial strains, oxidase-positive, motile by means of more than one polar flagella, facultative anaerobe, arginine dihydrolase-negative, lysine- and omithine decarboxylase-positive, sensitive to the vibriostatic agent O/129, were isolated from a flow-through rotifer culture system in Gent, Belgium, and previously characterized by fluorescent amplified fragment length polymorphism. Comparison of the 16S rDNA sequence of strain LMG 21460T indicated close relationships (approximately 99% similarity) to Vibrio campbellii, Vibrio harveyi, Vibrio alginolyticus and Vibrio parahaemolyticus. However, DNA hybridization experiments revealed similarity values below 70% with its closest species V. campbellii and V. harveyi. Additionally, the analysed strains differ from related Vibrio species by the utilization of melibiose and production of acid from L-arabinose and amygdalin. Among the strains analysed, differences were observed in some phenotypic characters, particularly susceptibility to ampicillin, polymyxin B and amikacin, and urease activity. The major fatty acids identified were 16:0, 18:1 omega7c, 14:0, 12:0 3-OH and 18:0. Vibrio rotiferianus sp. nov. is proposed, with type strain LMG 21460T (=CAIM 577T); it has a DNA G+C content of 44.5 +/- 0.01 mol%.

  4. Lactobacillus arizonensis sp. nov., isolated from jojoba meal.

    Science.gov (United States)

    Swezey, J L; Nakamura, L K; Abbott, T P; Peterson, R E

    2000-09-01

    Five strains of simmondsin-degrading, lactic-acid-producing bacteria were isolated from fermented jojoba meal. These isolates were facultatively anaerobic, gram-positive, non-motile, non-spore-forming, homofermentative, rod-shaped organisms. They grew singly and in short chains, produced lactic acid but no gas from glucose, and did not exhibit catalase activity. Growth occurred at 15 and 45 degrees C. All strains fermented cellobiose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannitol, D-mannose, melibiose, D-ribose, salicin, D-sorbitol, sucrose and trehalose. Some strains fermented L-(-)-arabinose and L-rhamnose. D-Xylose was not fermented and starch was not hydrolysed. The mean G+C content of the DNA was 48 mol%. Phylogenetic analyses of 16S rDNA established that the isolates were members of the genus Lactobacillus. DNA reassociation of 45% or less was obtained between the new isolates and the reference strains of species with G+C contents of about 48 mol%. The isolates were differentiated from other homofermentative Lactobacillus spp. on the basis of 16S rDNA sequence divergence, DNA relatedness, stereoisomerism of the lactic acid produced, growth temperature and carbohydrate fermentation. The data support the conclusion that these organisms represent strains of a new species, for which the name Lactobacillus arizonensis is proposed. The type strain of L. arizonensis is NRRL B-14768T (= DSM 13273T).

  5. Purification and Structural Characterization of a Novel Water-Soluble Neutral Polysaccharide from Cantharellus cibarius and Its Immunostimulating Activity in RAW264.7 Cells

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    Long Chen

    2017-01-01

    Full Text Available Polysaccharide is one of the important active ingredients of Cantharellus cibarius. The aims of this work were to analyze preliminary characterization and to investigate immunostimulating activity of a novel water-soluble neutral polysaccharide named JP1, which was purified from the fruiting body of Cantharellus cibarius using DEAE-FF chromatography and Sephadex G-100 chromatography. The characteristics of JP1 were determined by HPGPC, FT-IR spectra, gas chromatography, and Congo Red Method. Immunostimulating activity of JP1 was investigated in RAW264.7 cells. Results indicated that JP1 consisted of L-Arabinose, D-Mannose, D-Glucose, and D-Galactose in a molar ratio of 1 : 1.06 : 1.95 : 1.17 with a molecular weight of 336 kDa. JP1 is nontoxic to RAW264.7 cells at this concentration range (62.5–1000 μg/mL. Furthermore, JP1 can promote mouse peritoneal macrophages to secrete NO and enhance the secretion of macrophages’ cytokines IL-6 in RAW264.7 cells. These results suggested that JP1 could have potential immunostimulating activity applications as medicine or functional food.

  6. Characterization and Bioactivity of Polysaccharides Obtained from Pine Cones of Pinus koraiensis by Graded Ethanol Precipitation

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    Xin Yang

    2013-08-01

    Full Text Available Pinus koraiensis polysaccharides (PKP were extracted by hot water from P. koraiensis pine cones. Five polysaccharide fractions named PKP-A, PKP-B, PKP-C, PKP-D and PKP-E were successfully separated at final ethanol concentrations of 30%, 50%, 60%, 70% and 80%, respectively. HPLC, FT-IR, GC-MS and automatic amino-acid analysis were applied to investigate their chemical characteristics. Monosaccharide component analysis indicated that the five fractions were all composed of d-ribose, l-rhamnose, l-arabinose, d-xylose, d-mannose, d-glucose and d-galactose, but their molar ratios were quite different. HPLC results revealed that the polysaccharides precipitated by higher concentrations of ethanol solution had lower molecular masses. Moreover, the antioxidant activities of the five fractions were studied on the basis of hydroxyl radical and ABTS radical scavenging tests. The five graded polysaccharide fractions exhibited good inhibitory power, and MTT tests in vitro showed the IC50 of PKP-A and PKP-E were 1,072.5 and 2,070.0 μg·mL−1, respectively. These results demonstrated that the PKP could be a potential source of natural antioxidants or dietary supplements.

  7. Phenotypic variation in Lactococcus lactis subsp. lactis isolates derived from intestinal tracts of marine and freshwater fish.

    Science.gov (United States)

    Itoi, S; Yuasa, K; Washio, S; Abe, T; Ikuno, E; Sugita, H

    2009-09-01

    We compared phenotypic characteristics of Lactococcus lactis subsp. lactis derived from different sources including the intestinal tract of marine fish and freshwater fish, and cheese starter culture. In the phylogenetic analysis based on partial 16S rRNA gene nucleotide sequences (1371 bp), freshwater fish-, marine fish- and cheese starter culture-derived strains were identical to that of L. lactis subsp. lactis previously reported. Fermentation profiles determined using the API 50 CH system were similar except for fermentation of several sugars including l-arabinose, mannitol, amygdalin, saccharose, trehalose, inulin and gluconate. The strains did have distinct levels of halotolerance: marine fish-derived strains > cheese starter-derived strain > freshwater fish-derived isolate. Lactococcus lactis subsp. lactis showed extensive diversity in phenotypic adaptation to various environments. The phenotypic properties of these strains suggested that L. lactis subsp. lactis strains from fish intestine have additional functions compared with the cheese starter-derived strain that has previously described. The unique phenotypic traits of the fish intestinal tract-derived L. lactis subsp. lactis might make them useful as a probiotics in aquaculture, and contribute to the development of functional foods and novel food additives, since the strains derived from fish intestines might have additional functions such as antibacterial activity.

  8. Reclassification of Lactobacillus kimchii and Lactobacillus bobalius as later subjective synonyms of Lactobacillus paralimentarius.

    Science.gov (United States)

    Pang, Huili; Kitahara, Maki; Tan, Zhongfang; Wang, Yanping; Qin, Guangyong; Ohkuma, Moriya; Cai, Yimin

    2012-10-01

    Characterization and identification of strain CW 1 ( = JCM 17161) isolated from corn silage were performed. Strain CW 1 was a Gram-positive, catalase-negative and homofermentative rod that produced the DL-form of lactic acid. This strain exhibited more than 99.6% 16S rRNA gene sequence similarity and greater than 82% DNA-DNA reassociation with type strains of Lactobacillus kimchii, L. bobalius and L. paralimentarius. To clarify the taxonomic positions of these type strains, phenotypic characterization, 16S rRNA gene sequencing, ribotyping and DNA-DNA relatedness were examined. The three type strains displayed different L-arabinose, lactose, melibiose, melezitose, raffinose and N-acetyl-β-glucosaminidase fermentation patterns. Phylogenetic analysis showed that L. paralimentarius is a closer neighbour of L. kimchii and L. bobalius, sharing 99.5-99.9% 16S rRNA gene sequence similarity, which was confirmed by the high DNA-DNA relatedness (≥82%) between L. paralimentarius JCM 10415(T), L. bobalius JCM 16180(T) and L. kimchii JCM 10707(T). Therefore, it is proposed that L. kimchii and L. bobalius should be reclassified as later synonyms of L. paralimentarius.

  9. A single amino acid change (Y318F) in the L-arabitol dehydrogenase (LadA) from Aspergillus niger results in a significant increase in affinity for D-sorbitol

    Science.gov (United States)

    2009-01-01

    Background L-arabitol dehydrogenase (LAD) and xylitol dehydrogenase (XDH) are involved in the degradation of L-arabinose and D-xylose, which are among the most abundant monosaccharides on earth. Previous data demonstrated that LAD and XDH not only differ in the activity on their biological substrate, but also that only XDH has significant activity on D-sorbitol and may therefore be more closely related to D-sorbitol dehydrogenases (SDH). In this study we aimed to identify residues involved in the difference in substrate specificity. Results Phylogenetic analysis demonstrated that LAD, XDH and SDH form 3 distinct groups of the family of dehydrogenases containing an Alcohol dehydrogenase GroES-like domain (pfam08240) and likely have evolved from a common ancestor. Modelling of LadA and XdhA of the saprobic fungus Aspergillus niger on human SDH identified two residues in LadA (M70 and Y318), that may explain the absence of activity on D-sorbitol. While introduction of the mutation M70F in LadA of A. niger resulted in a nearly complete enzyme inactivation, the Y318F resulted in increased activity for L-arabitol and xylitol. Moreover, the affinity for D-sorbitol was increased in this mutant. Conclusion These data demonstrates that Y318 of LadA contributes significantly to the substrate specificity difference between LAD and XDH/SDH. PMID:19674460

  10. Conversion of acid hydrolysate of oil palm empty fruit bunch to L-lactic acid by newly isolated Bacillus coagulans JI12.

    Science.gov (United States)

    Ye, Lidan; Hudari, Mohammad Sufian Bin; Zhou, Xingding; Zhang, Dongxu; Li, Zhi; Wu, Jin Chuan

    2013-06-01

    Cost-effective conversion of lignocellulose hydrolysate to optically pure lactic acid is commercially attractive but very challenging. Bacillus coagulans JI12 was isolated from natural environment and used to produce L-lactic acid (optical purity > 99.5 %) from lignocellulose sugars and acid hydrolysate of oil palm empty fruit bunch (EFB) at 50 °C and pH 6.0 without sterilization of the medium. In fed-batch fermentation with 85 g/L initial xylose and 55 g/L xylose added after 7.5 h, 137.5 g/L lactic acid was produced with a yield of 98 % and a productivity of 4.4 g/L h. In batch fermentation of a sugar mixture containing 8.5 % xylose, 1 % glucose, and 1 % L-arabinose, the lactic acid yield and productivity reached 98 % and 4.8 g/L h, respectively. When EFB hydrolysate was used, 59.2 g/L of lactic acid was produced within 9.5 h at a yield of 97 % and a productivity of 6.2 g/L h, which are the highest among those ever reported from lignocellulose hydrolysates. These results indicate that B. coagulans JI12 is a promising strain for industrial production of L-lactic acid from lignocellulose hydrolysate.

  11. Characteristics of a pink-pigmented bacterium isolated from biofilm in a cooling tower in Tokyo, Japan.

    Science.gov (United States)

    Furuhata, Katsunori; Goto, Keiichi; Kato, Yuko; Saitou, Keiko; Sugiyama, Jun-ichi; Hara, Motonobu; Yoshida, Shin-ichi; Fukuyama, Masafumi

    2007-01-01

    Strain K-20, a Gram-negative, non-motile, non-spore-forming and strictly aerobic rod, which produces a pale pink pigment, was isolated from biofilm in a cooling tower in Tokyo, Japan. The taxonomic feature of the strain was studied using phenotypic tests and phylogenetic analysis. Phylogenetic analysis of 16S rRNA gene sequences showed that the strain was related to Roseomonas gilardii subsp. rosea, Roseomonas gilardii subsp. gilardii, Roseomonas cervicalis and Roseomonas mucosa at 94.3-94.6 sequence similarities. Growth occurred at 25-40 C and pH 5.0-10.0, optimal at 35 C and pH 7.0. Growth did not occur in the presence of >or=2% NaCl. The API 20NE identification system gave a positive result for urease, L-arabinose, potassium gluconate, adipic acid, malic acid and trisodium citrate (API code number 0201465). The predominant fatty acids of strain K-20 were C18:1Delta11 (50.8%) and C16:1 (17.2%). Cells contained ubiquinone 10 (Q-10) as the major quinone and the G+C content was 72.0 mol%. Based on phenotypic, chemotaxonomic and phylogenetic data, it was assumed that strain K-20 (=JCM 14634) is a novel species of the genus Roseomonas.

  12. Roseomonas, a new genus associated with bacteremia and other human infections.

    Science.gov (United States)

    Rihs, J D; Brenner, D J; Weaver, R E; Steigerwalt, A G; Hollis, D G; Yu, V L

    1993-01-01

    In the 1980s, a pink bacterium different from species of the genus Methylobacterium was implicated in human infection. Using biochemical tests and DNA hybridization, we examined 42 strains of pink-pigmented, gram-negative bacteria that were not members of the genus Methylobacterium. The isolates included 6 strains each of CDC "pink coccoid" groups I, II, III, and IV; 10 isolates from Gilardi's "unnamed taxon"; and 8 blood isolates from ill, debilitated, or immunosuppressed patients. The DNA hybridization studies supported the creation of six genomospecies encompassing the 42 strains. Reactions for esculin hydrolysis, glycerol oxidation, and D-mannose oxidation enabled separation of genomospecies 1 through 4. These tests, as well as motility, nitrate reduction, citrate utilization, and oxidation of L-arabinose, D-galactose, and D-xylose, differentiated genomospecies 5 and 6 from each other and from genomospecies 1 through 4. These organisms were susceptible in vitro to the aminoglycosides, tetracycline, and imipenem and generally susceptible to the quinolones. We propose the new genus, Roseomonas, for these bacteria to include three named species, Roseomonas gilardii sp. nov., Roseomonas cervicalis sp. nov., and Roseomonas fauriae sp. nov., and three unnamed genomospecies. Images PMID:8308122

  13. Some physico-chemical properties of Prunus armeniaca L. gum exudates.

    Science.gov (United States)

    Fathi, Morteza; Mohebbi, Mohebbat; Koocheki, Arash

    2016-01-01

    The objectives of this paper were to investigate some physicochemical properties of Prunus armeniaca L. gum exudates (PAGE). PAGE had, on average, 66.89% carbohydrate, 10.47% uronic acids, 6.9% moisture (w.b.), 2.91% protein, 4% ash and 1.59% fat. PAGE was composed of monosaccharides including l-arabinose, d-galactose, xylose, mannose and rhamnose in molar percentages of 41.52%, 23.72%, 17.82%, 14.40% and 2.54%, respectively. Elemental analysis showed that PAGE had high values of nutrients. FTIR analysis demonstrated the presence of carboxyl, hydroxyl and methyl groups and glycoside bonds. The weight average molecular weight, number average molecular weight and polydispersity index were found to be approximately 5.69 × 10(5)g/mol, 4.33 g/mol and 1.31, respectively. Rheological measurement of PAGE solutions as a function of concentration (8, 10 and 12% (w/w)) and temperature (10, 20, 30 and 40°C) demonstrated that the gum solutions had a non Newtonian shear thinning behaviour. Intrinsic viscosity for PAGE in deionized water was 3.438 dl/g based on Kramer equation. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Polysaccharide peptide isolated from grass-cultured Ganoderma lucidum induces anti-proliferative and pro-apoptotic effects in the human U251 glioma cell line.

    Science.gov (United States)

    Wang, Chunhua; Lin, Dongmei; Chen, Quan; Lin, Shuqian; Shi, Songsheng; Chen, Chunmei

    2018-04-01

    The Ganoderma lucidum ( G. lucidum ) mushroom is one of the most extensively studied functional foods, known for its numerous health benefits, including the inhibition of tumor cell growth. The present study assessed the anti-proliferative and pro-apoptotic activity of a novel G. lucidum polysaccharide peptide (GL-PP) in human glioma U251 cells, which was purified from grass-cultured G. lucidum . GL-PP is a glycopeptide with an average molecular weight of 42,635 Da and a polysaccharide-to-peptide ratio of 88.70:11.30. The polysaccharides were composed of l-arabinose, d-mannose and d-glucose at a molar ratio of 1.329:0.372:2.953 and a total of 17 amino acids were detected. The results of the current study demonstrated that GL-PP significantly inhibited U251 cellular proliferation. The proportion of G 0 /G 1 phase cells and sub-G 1 phase cells significantly increased as the concentration of GL-PP increased, as did the activity of caspase-3. These results indicate that GL-PP directly inhibited human glioma U251 proliferation by inducing cell cycle arrest and promoting apoptosis.

  15. Design and synthesis of biotin analogues reversibly binding with streptavidin.

    Science.gov (United States)

    Yamamoto, Tomohiro; Aoki, Kiyoshi; Sugiyama, Akira; Doi, Hirofumi; Kodama, Tatsuhiko; Shimizu, Yohei; Kanai, Motomu

    2015-04-01

    Two new biotin analogues, biotin carbonate 5 and biotin carbamate 6, have been synthesized. These molecules were designed to reversibly bind with streptavidin by replacing the hydrogen-bond donor NH group(s) of biotin's cyclic urea moiety with oxygen. Biotin carbonate 5 was synthesized from L-arabinose (7), which furnishes the desired stereochemistry at the 3,4-cis-dihydroxy groups, in 11% overall yield (over 10 steps). Synthesis of biotin carbamate 6 was accomplished from L-cysteine-derived chiral aldehyde 33 in 11% overall yield (over 7 steps). Surface plasmon resonance analysis of water-soluble biotin carbonate analogue 46 and biotin carbamate analogue 47 revealed that KD values of these compounds for binding to streptavidin were 6.7×10(-6)  M and 1.7×10(-10)  M, respectively. These values were remarkably greater than that of biotin (KD =10(-15)  M), and thus indicate the importance of the nitrogen atoms for the strong binding between biotin and streptavidin. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Effect of carbon source on the accumulation of cytochrome P-450 in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Kärenlampi, S O; Marin, E; Hänninen, O O

    1981-02-15

    The appearance of cytochrome P-450 in the yeast Saccharomyces cerevisiae depended on the substrate supporting growth. Cytochrome P-450 was apparent in yeast cells grown on a strongly fermentable sugar such as D-glucose, D-fructose or sucrose. When yeast was grown on D-galactose, D-mannose or maltose, where fermentation and respiration occurred concomitantly, cytochrome P-450 was also formed. The cytochrome P-450 concentration was maximal at the beginning of the stationary phase of the culture. Thereafter the concentration decreased, reaching zero at a late-stationary phase. When the yeast was grown on a medium that contained lactose or pentoses (L-arabinose, L-rhamnose, D-ribose and D-xylose), cytochrome P-450 did not occur. When a non-fermentable energy source (glycerol, lactate or ethanol) was used, no cytochrome P-450 was detectable. Transfer of cells from D-glucose medium to ethanol medium caused a slow disappearance of cytochrome P-450, although the amount of the haemoprotein still continued to increase in the control cultures. Cytochrome P-450 appeared thus to accumulate in conditions where the rate of growth was fast and fermentation occurred. Occurrence of this haemoprotein is not necessarily linked, however, with the repression of mitochondrial haemoprotein synthesis.

  17. Reactivities of some aldoses towards iodine in alkaline

    International Nuclear Information System (INIS)

    Azmat, R.; Nizami, S.S.

    2005-01-01

    The kinetics studies of oxidation of some reducing sugars by aqueous alkaline solution of iodine investigated. Results demonstrated that iodine in the presence of alkali converted into hypoiodous acid which was effective oxidizing species. Reaction between iodine and sugars was slowest reaction. The rate of oxidation of sugars was affected by change in pH of the medium and maximum at pH 11.4 where the concentration of hypoiodous acid was maximum which oxidized the sugars into respective acids. The rate of oxidation followed first order kinetics with respect to substrate and obeyed zero order kinetics with that of iodine. Change in ionic strength of the medium showed no effect on the rate of oxidation indicating that reaction occurred between molecular species and there was no ionic species present in the rate determining step. Reaction was affected by the change in temperature and value of energy of activation corresponding to glucose, galactose, D-mannose and L-arabinose were 10.16 kj/mol, 12.17 kj/mol, 14.00 kj/mol and 20.22 kj/mol respectively. (author)

  18. Biomolecular Mechanisms of Pseudomonas aeruginosa and Escherichia coli Biofilm Formation

    Directory of Open Access Journals (Sweden)

    Garry Laverty

    2014-07-01

    Full Text Available Pseudomonas aeruginosa and Escherichia coli are the most prevalent Gram-negative biofilm forming medical device associated pathogens, particularly with respect to catheter associated urinary tract infections. In a similar manner to Gram-positive bacteria, Gram-negative biofilm formation is fundamentally determined by a series of steps outlined more fully in this review, namely adhesion, cellular aggregation, and the production of an extracellular polymeric matrix. More specifically this review will explore the biosynthesis and role of pili and flagella in Gram-negative adhesion and accumulation on surfaces in Pseudomonas aeruginosa and Escherichia coli. The process of biofilm maturation is compared and contrasted in both species, namely the production of the exopolysaccharides via the polysaccharide synthesis locus (Psl, pellicle Formation (Pel and alginic acid synthesis in Pseudomonas aeruginosa, and UDP-4-amino-4-deoxy-l-arabinose and colonic acid synthesis in Escherichia coli. An emphasis is placed on the importance of the LuxR homologue sdiA; the luxS/autoinducer-II; an autoinducer-III/epinephrine/norepinephrine and indole mediated Quorum sensing systems in enabling Gram-negative bacteria to adapt to their environments. The majority of Gram-negative biofilms consist of polysaccharides of a simple sugar structure (either homo- or heteropolysaccharides that provide an optimum environment for the survival and maturation of bacteria, allowing them to display increased resistance to antibiotics and predation.

  19. Structure analysis of a novel heteroxylan from the stem of Dendrobium officinale and anti-angiogenesis activities of its sulfated derivative.

    Science.gov (United States)

    Yue, Han; Liu, Yanqiu; Qu, Huanhuan; Ding, Kan

    2017-10-01

    Dendrobium officinale Kimura et Migo (Tie-Pi-Shi-Hu), a precious folk medicine exhibiting multiple bioactivities, including antitumor, immune-enhancing and so on. Although evidences showed polysaccharide is one of the major bioactive substances from this herb, several of them were homogenous with fine structures elucidated. In this study, we showed a novel homogeneous heteroxylan obtained from alkali-extracted crude polysaccharide. It composed of arabinose, xylose, glucose and 4-O-methylglucuronic acid (4-MGA) as well as trace amount of rhamnose and galactose in a ratio of 8.9:62.7:8.5:12.3:3.9:3.7. We further showed that it contained a backbone of 1,4-linked β-d-xylan, with branches of 1,4-linked α-d-glucose, 1,3-linked α-l-rhamnose, and terminal-linked α-l-arabinose, β-d-galactose, 4-MGA, and β-d-xylose directly or indirectly attached to C-2 position of glycosyl residues on backbone. The sulfated derivative with substitution degree about 0.9 was prepared according to the chlorosulfonic acid (CSA)-pyridine method. Bioactivity tests suggested that the sulfated polysaccharide could significantly disrupt tube formation and inhibit the migration of human microvascular endothelial cells (HMEC-1) at a low concentration (0.29μM) in a dose-dependent way without significant cytotoxity. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Monosaccharide composition of acidic gum exudates from Indian Acacia tortilis ssp. raddiana (Savi) Brenan.

    Science.gov (United States)

    Lakhera, Ajeet Kumar; Kumar, Vineet

    2017-01-01

    Acacia tortilis ssp. raddiana (Savi) Brenan commonly known as Israeli Babool has contributed immensely for sand dunes management in Indian desert leading to wind erosion control and increased biological productivity. The species is extensively used in traditional medicine system for a number of therapeutic applications and as nutraceutical. The polysaccharide was isolated in 43.6% yield from gum exudates. The monosaccharides, L-arabinose, D-galactose D-glucose, L-rhamnose and D-mannose were determined in molar ratio of 78.1%, 18.64%, 0.60%, 1.71% and 0.74% respectively. The molar ratio of uronic acids was studied using diverse spectrophotometric methods and compared with GLC. The content of D-galacturonic acid and D-glucuronic was determined as 3.88% and 4.35% respectively by GLC. The results were compared with the spectrophotometric methods. The results using DMP as chromogenic reagent are closer to that obtained by GLC. Structural analysis of the polysaccharide may provide scientific basis for nutraceutical, pharmaceutical and biological applications of gum exudates from A. tortilis, which is extensively planted in India. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Preparation of hydrolytic liquid from dried distiller's grains with solubles and fumaric acid fermentation by Rhizopus arrhizus RH 7-13.

    Science.gov (United States)

    Liu, Huan; Yue, Xuemin; Jin, Yuhan; Wang, Meng; Deng, Li; Wang, Fang; Tan, Tianwei

    2017-10-01

    Fumaric acid production from lignocellulosic materials is an alternative chemicals production system. This work investigated the suitable conditions for hydrolysis of dried distiller's grains with solubles (DDGS). The hydrolytic liquid was subsequently used for the production of fumaric acid. After optimizing the hydrolysis conditions, the most suitable concentration of H 2 SO 4 (2%), hydrolysis temperature (120 °C), hydrolysis time (100min) and solid/liquid ratio (1:10) were obtained. The yield of monosaccharides reached 258 mg/g DDGS and 15.88 g/L glucose, 7.53 g/L xylose and 2.35 g/L arabinose were obtained in unprocessed hydrolytic liquid. The furfural inhibitor in the hydrolytic liquid was also detected and the yield of it was reducing progressively in the pretreatment process. The ferment ability of the hydrolytic liquid from DDGS was tested through the process of fumaric acid production by Rhizopus arrhizus RH 7-13. The unprocessed hydrolytic liquid was not appropriate for the fermentation process. The yield of fumaric acid from the concentrated processed hydrolytic liquid reached 18.93 g/L, which was close to the yield of fermenting 80 g/L glucose. This result indicated that the commonly used carbon resource glucose could to some extent be replaced by processed hydrolytic liquid. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Polymyxin susceptibility testing, interpretative breakpoints and resistance mechanisms: An update.

    Science.gov (United States)

    Bakthavatchalam, Yamuna Devi; Pragasam, Agila Kumari; Biswas, Indranil; Veeraraghavan, Balaji

    2018-03-01

    Emerging multidrug-resistant (MDR) nosocomial pathogens are a great threat. Polymyxins, an old class of cationic polypeptide antibiotic, are considered as last-resort drugs in treating infections caused by MDR Gram-negative bacteria. Increased use of polymyxins in treating critically ill patients necessitates routine polymyxin susceptibility testing. However, susceptibility testing both of colistin and polymyxin B (PMB) is challenging. In this review, currently available susceptibility testing methods are briefly discussed. The multicomponent composition of colistin and PMB significantly influences susceptibility testing. In addition, poor diffusion in the agar medium, adsorption to microtitre plates and the synergistic effect of the surfactant polysorbate 80 with polymyxins have a great impact on the performance of susceptibility testing methods This review also describes recently identified chromosomal resistance mechanisms, including modification of lipopolysaccharide (LPS) with 4-amino-4-deoxy-l-arabinose (L-Ara4-N) and phosphoethanolamine (pEtN) resulting in alteration of the negative charge, as well as the plasmid-mediated colistin resistance determinants mcr-1, mcr-1.2, mcr-2 and mcr-3. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

  3. Development of a Multimarker Urine Test for Prostate Cancer

    Science.gov (United States)

    2017-10-01

    connected to a chemically etched 20 μm i.d. fused-silica emitter via a Valco stainless steel union. Four μL of individual peptide fractions (total...isomerase family A, member 17 (PD1A17 or member 17); or secreted cement gland protein XAG-2 homolog, AGR2 belongs to the protein disulfide 5 isomerase...silica emitter via a Valco stainless steel union. Four microliters of individual peptide fractions (total volume 20 µL) following PRISM were

  4. Glycogen metabolism in the liver of Indian desert gerbils (Meriones hurrianae, Jerdon) exposed to internal beta irradiation

    International Nuclear Information System (INIS)

    Gupta, N.K.

    1996-01-01

    Glycogen content and the activities of phosphorylase, glycogen synthetase, phosphohexose isomerase, glucose-6-phosphatase, succinate dehydrogenase, alanine and aspartate aminotransferases have been biochemically determined in the liver of Indian desert gerbils following radiocalcium internal irradiation. Decline in glycogen, phosphohexose isomerase, with a concomitant increase in phosphorylase, succinate dehydrogenase reveals a switch over from glycolytic to oxidative metabolism in liver. Activities of aminotransferases indicate the utilization of transamination products of alanine and aspartate in oxidative pathway during early periods. Transiently increased glucose-6-phosphatase seems to restrict glycogenolytic and glycolytic metabolism and thereby pave way for the acceleration of oxidative metabolism. (author). 52 refs., 2 tabs

  5. Mutations in iron-sulfur cluster proteins that improve xylose utilization

    Science.gov (United States)

    Froehlich, Allan; Henningsen, Brooks; Covalla, Sean; Zelle, Rintze M.

    2018-03-20

    There is provided an engineered host cells comprising (a) one or more mutations in one or more endogenous genes encoding a protein associated with iron metabolism; and (b) at least one gene encoding a polypeptide having xylose isomerase activity, and methods of their use thereof.

  6. Genetically modified yeast species and fermentation processes using genetically modified yeast

    Science.gov (United States)

    Rajgarhia, Vineet [Kingsport, TN; Koivuranta, Kari [Helsinki, FI; Penttila, Merja [Helsinki, FI; Ilmen, Marja [Helsinki, FI; Suominen, Pirkko [Maple Grove, MN; Aristidou, Aristos [Maple Grove, MN; Miller, Christopher Kenneth [Cottage Grove, MN; Olson, Stacey [St. Bonifacius, MN; Ruohonen, Laura [Helsinki, FI

    2011-05-17

    Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications', include deletion of non-specific or specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.

  7. Incorporating the effect of ionic strength in free energy calculations using explicit ions

    NARCIS (Netherlands)

    Donnini, S; Mark, AE; Juffer, AH; Villa, Alessandra

    2005-01-01

    The incorporation of explicit ions to mimic the effect of ionic strength or to neutralize the overall charge on a system in free energy calculations using molecular dynamics simulations is investigated. The difference in the free energy of hydration between two triosephosphate isomerase inhibitors

  8. Protamine 3 expressions in crossbred bull spermatozoa may not be ...

    African Journals Online (AJOL)

    The mRNA expression of PRM3 gene among two groups was evaluated by real time quantitative polymerase chain reaction (PCR) using TaqMan chemistry, where peptidylprolyl isomerase A (PPIA) was used as an internal control. Our finding revealed that expression of PRM3 was down regulated in poor quality semen ...

  9. Genetically modified yeast species, and fermentation processes using genetically modified yeast

    Energy Technology Data Exchange (ETDEWEB)

    Rajgarhia, Vineet [Kingsport, TN; Koivuranta, Kari [Helsinki, FI; Penttila, Merja [Helsinki, FI; Ilmen, Marja [Helsinki, FI; Suominen, Pirkko [Maple Grove, MN; Aristidou, Aristos [Maple Grove, MN; Miller, Christopher Kenneth [Cottage Grove, MN; Olson, Stacey [St. Bonifacius, MN; Ruohonen, Laura [Helsinki, FI

    2014-01-07

    Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications include deletion of non-specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.

  10. Genetically modified yeast species, and fermentation processes using genetically modified yeast

    Energy Technology Data Exchange (ETDEWEB)

    Rajgarhia, Vineet; Koivuranta, Kari; Penttila, Merja; Ilmen, Marja; Suominen, Pirkko; Aristidou, Aristos; Miller, Christopher Kenneth; Olson, Stacey; Ruohonen, Laura

    2017-09-12

    Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications include deletion of non-specific or specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.

  11. Genetically modified yeast species, and fermentation processes using genetically modified yeast

    Energy Technology Data Exchange (ETDEWEB)

    Rajgarhia, Vineet; Koivuranta, Kari; Penttila, Merja; Ilmen, Marja; Suominen, Pirkko; Aristidou, Aristos; Miller, Christopher Kenneth; Olson, Stacey; Ruohonen, Laura

    2016-08-09

    Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications include deletion of non-specific or specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.

  12. Genetically modified yeast species and fermentation processes using genetically modified yeast

    Energy Technology Data Exchange (ETDEWEB)

    Rajgarhia, Vineet [Kingsport, TN; Koivuranta, Kari [Helsinki, FI; Penttila, Merja [Helsinki, FI; Ilmen, Marja [Helsinki, FI; Suominen, Pirkko [Maple Grove, MN; Aristidou, Aristos [Maple Grove, MN; Miller, Christopher Kenneth [Cottage Grove, MN; Olson, Stacey [St. Bonifacius, MN; Ruohonen, Laura [Helsinki, FI

    2011-05-17

    Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications', include deletion of non-specific or specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.

  13. The effects of mutating Tyr9 and Arg15 on the structure, stability, conformational dynamics and mechanism of GSTA3-3

    CSIR Research Space (South Africa)

    Robertson, GJ

    2017-03-01

    Full Text Available activity of the steroid isomerase reaction; however, Arg15 is more important for lowering the pKa of GSH. Lowering the pKa of GSH being how GSTs catalyse their reactions. Additionally, there is evidence to suggest that Arg15 is integral to allowingGSTA3...

  14. Development of a strain of saccharomyces cereviase to utilize hemicellulosic biomass

    International Nuclear Information System (INIS)

    Batt, C.A.

    1991-01-01

    The current status of yeast conversion to utilize pentose sugar is discussed in this paper. The development of processes for the production of ethanol from agricultural wastes provides both a beneficial utilization of the resources presently available and an alternate source of liquid transportation fuel. The efficient conversion of agricultural bio mass is in part dependent on utilization of all the potential sugars, including the pentoses in the hemicellulosic fraction. A number of approaches have been investigated, including the engineering of strain of S. cerevisiae which express a xylose isomerase activity. Despite the apparent lack of success with respect to expressing an active xylose isomerase, a great deal of knowledge has been gained on the metabolism of pentoses by yeast and the genetics, structure/function of the enzyme xylose isomerase. Hopefully this cumulative knowledge base will lead to the design of a xylose isomerase with the appropriate structure to allow it retain activity in S. cerevisiae. This coupled with the elegant efforts in a number of laboratories to develop cellulose utilizing strains of S. cerevisiae might yield a single yeast capable of fermenting all of the major carbon substrates in agricultural to fuel grade ethanol. (Orig./A.B.)

  15. Genetically modified yeast species, and fermentation processes using genetically modified yeast

    Science.gov (United States)

    Rajgarhia, Vineet; Koivuranta, Kari; Penttila, Merja; Ilmen, Marja; Suominen, Pirkko; Aristidou, Aristos; Miller, Christopher Kenneth; Olson, Stacey; Ruohonen, Laura

    2013-05-14

    Yeast cells are transformed with an exogenous xylose isomerase gene. Additional genetic modifications enhance the ability of the transformed cells to ferment xylose to ethanol or other desired fermentation products. Those modifications include deletion of non-specific or specific aldose reductase gene(s), deletion of xylitol dehydrogenase gene(s) and/or overexpression of xylulokinase.

  16. Dicty_cDB: SLA264 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available -propyl cis-trans isomerase (CYP1) gene, complete cds. 52 3e-22 4 AJ537762 |AJ537762.1 Timarcha... balearica EST, clone Timarcha3B3. 88 5e-22 3 AJ537662 |AJ537662.1 Timarcha balearica EST, clone Timarcha

  17. An overview of the non-mevalonate pathway for terpenoid

    Indian Academy of Sciences (India)

    Unknown

    troversial role of isomerase via non-MVA route in which both IPP and DMAPP are reported to be synthe- ... chemical scheme was proposed with a head-to-head con- densation of ..... berry exocarp and mesocarp; Phytochemistry 60 451–459.

  18. 21 CFR 173.357 - Materials used as fixing agents in the immobilization of enzyme preparations.

    Science.gov (United States)

    2010-04-01

    ... glucose isomerase enzyme preparations for use in the manufacture of high fructose corn syrup, in... manufacture of high fructose corn syrup, in accordance with § 184.1372 of this chapter. Cellulose triacetate... enzyme preparations for use in the manufacture of high fructose corn syrup, in accordance with § 184.1372...

  19. Production of high fructose corn syrup Streptomyces sp

    Energy Technology Data Exchange (ETDEWEB)

    Bhatia, M; Prabhu, K A

    1978-01-01

    A Streptomyces strain exhibiting considerable glucose isomerase activity was isolated from soil. The cell free extract of the culture was able to convert glucose to fructose in a period of 48 ha and gave 40% conversion. With acid hydrolyzates of corn and bagasse as substrates, the cell-free extract gave glucose to fructose conversions of 39.8 and 29%, respectively.

  20. 21 CFR 184.1866 - High fructose corn syrup.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true High fructose corn syrup. 184.1866 Section 184.1866... Listing of Specific Substances Affirmed as GRAS § 184.1866 High fructose corn syrup. (a) High fructose... partial enzymatic conversion of glucose (dextrose) to fructose using an insoluble glucose isomerase enzyme...

  1. Proč axony nezabloudí

    Czech Academy of Sciences Publication Activity Database

    Balaštík, Martin

    2017-01-01

    Roč. 96, č. 2 (2017), s. 78-79 ISSN 0042-4544 R&D Projects: GA ČR(CZ) GA16-15915S Institutional support: RVO:67985823 Keywords : axon guidance * prolyl isomerase * neural development Subject RIV: FH - Neurology OBOR OECD: Developmental biology

  2. Regulation of carbohydrate metabolism during Giardia encystment

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Jarroll, E.L.; Macechko, P.T.; Steimle, P.A.; Bulik, D.; Karr, C.D.; Keulen, Harry van; Paget, P.A.

    2001-01-01

    Giardia intestinalis trophozoites encyst when they are exposed to bile. During encystment, events related to the inducible synthesis of a novel N-acetyl-d-galactosamine (GalNAc) homopolymer, occur. Within the first 6 h of encystment, mRNA for glucosamine 6-P isomerase (GPI), the first inducible

  3. GenBank blastx search result: AK061794 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061794 001-039-F11 AF031161.1 Pseudomonas sp. VLB120 styrene degradation genes in...cluding histidine kinase (stdSc) gene, partial cds; and transcriptional activator (stdR), styrene monooxygen...ase large component (stdA), styrene monooxygenase small component (stdB), styrene oxide isomerase (stdC), an

  4. A critical role for ethylene in hydrogen peroxide release during programmed cell death in tomato suspension cells

    NARCIS (Netherlands)

    Jong, de A.J.; Yakimova, E.T.; Kapchina, V.M.; Woltering, E.J.

    2002-01-01

    Camptothecin, a topo isomerase-I inhibitor used in cancer therapy, induces apoptosis in animal cells. In tomato (Lycopersicon esculentum Mill.) suspension cells, camptothecin induces cell death that is accompanied by the characteristic nuclear morphological changes such as chromatin condensation and

  5. Xylose utilization in recombinant zymomonas

    Science.gov (United States)

    Caimi, Perry G; McCole, Laura; Tao, Luan; Tomb, Jean-Francois; Viitanen, Paul V

    2014-03-25

    Xylose-utilizing Zymomonas strains studied were found to accumulate ribulose when grown in xylose-containing media. Engineering these strains to increase ribose-5-phosphate isomerase activity led to reduced ribulose accumulation, improved growth, improved xylose utilization, and increased ethanol production.

  6. Structure-specificity relationships in Abp, a GH27 β-L-arabinopyranosidase from Geobacillus stearothermophilus T6.

    Science.gov (United States)

    Lansky, Shifra; Salama, Rachel; Solomon, Hodaya V; Feinberg, Hadar; Belrhali, Hassan; Shoham, Yuval; Shoham, Gil

    2014-11-01

    L-Arabinose sugar residues are relatively abundant in plants and are found mainly in arabinan polysaccharides and in other arabinose-containing polysaccharides such as arabinoxylans and pectic arabinogalactans. The majority of the arabinose units in plants are present in the furanose form and only a small fraction of them are present in the pyranose form. The L-arabinan-utilization system in Geobacillus stearothermophilus T6, a Gram-positive thermophilic soil bacterium, has recently been characterized, and one of the key enzymes was found to be an intracellular β-L-arabinopyranosidase (Abp). Abp, a GH27 enzyme, was shown to remove β-L-arabinopyranose residues from synthetic substrates and from the native substrates sugar beet arabinan and larch arabinogalactan. The Abp monomer is made up of 448 amino acids, and based on sequence homology it was suggested that Asp197 is the catalytic nucleophile and Asp255 is the catalytic acid/base. In the current study, the detailed three-dimensional structure of wild-type Abp (at 2.28 Å resolution) and its catalytic mutant Abp-D197A with (at 2.20 Å resolution) and without (at 2.30 Å resolution) a bound L-arabinose product are reported as determined by X-ray crystallography. These structures demonstrate that the three-dimensional structure of the Abp monomer correlates with the general fold observed for GH27 proteins, consisting of two main domains: an N-terminal TIM-barrel domain and a C-terminal all-β domain. The two catalytic residues are located in the TIM-barrel domain, such that their carboxylic functional groups are about 5.9 Å from each other, consistent with a retaining mechanism. An isoleucine residue (Ile67) located at a key position in the active site is shown to play a critical role in the substrate specificity of Abp, providing a structural basis for the high preference of the enzyme towards arabinopyranoside over galactopyranoside substrates. The crystal structure demonstrates that Abp is a tetramer

  7. Purification and characterization of endo-xylanases from aspergillus Niger. II. An enzyme of PL 45

    Energy Technology Data Exchange (ETDEWEB)

    Shei, J.C.; Fratzke, A.R.; Frederick, M.M.; Frederick, J.R.; Reilly, P.J.

    1985-04-01

    A homogeneous endo-xylanase (1,4-..beta..-D-xylan xylano-hydrolase, EC 3.2.1.8) was obtained from a crude Aspergillus niger pentosanase by chromatography with Ultrogel AcA 54, SP-Sephadex C-25 at pH 4.5, DEAE-Sephadex A-25 at pH 5.4, Sephadex G-50, and SP-Sephadex C-25 with a gradient from pH 2.8 to pH 4.6. It was much more active on soluble than on insoluble xylan yielding large amounts of unreacted xylan and a mixture of oligosaccharides with chain lengths from two to six. No xylose or L-arabinose was produced. There was high activity on a xylopentaose through xylononaose mixture, but not on xylobiose, xylotriose, or xylotetraose. The enzyme had slight activity on untreated cellulose, carboxymethylcellulose, and pectin. Molecular weight was ca. 1.4 x 10/sup 4/, with an isoelectric point of 4.5 and an amino acid profile high in acidic but low in sulfur-containing residues. In a 25-min assay at pH 4.7, this endo-xylanase was most active at 45 degrees C, with an activation energy from 5 to 35 degrees C of 33.3 kJ/mol. The optimum pH for activity was 4.9. Decay in buffer was first order, with an activation energy at pH 4.7 from 48 to 53 degrees C of 460 kJ/mol. Optimum pH for stability was about 5.6, where the half-life at 48 degrees C in buffer was ca. 40 h.

  8. Isolation, characterization and modification of citrus pectins

    Directory of Open Access Journals (Sweden)

    MARIA KRATCHANOVA

    2012-01-01

    Full Text Available Orange and lemon peels were used for obtaining pectic polysaccharides. Citrus peels were previously treated with 96% ethanol, and the obtained alcohol-insoluble solids (AIS were subjected to a sequential extraction with hot distilled water and hot 0.5% HCl. Water- and acid-extracted orange (WEOP and AEOP and lemon (WELP and AELP pectins were obtained. Acid extraction gave higher yields of pectin than water extraction and lemon peels were richer in pectin. Comparative investigations were carried out with chromatographically purified commercial citrus pectin (CPCP. Chemical and physicochemical characterization of all pectins was accomplished. It was found that pectins were similar in anhydrouronic acid content (AUАC, 69-81%, but differed in their degree of methylesterification (DM, 55-81%. Generally water-extracted pectins were with higher DM. Both orange pectins were with higher DM and degree of acetylation (DA, 2%, in comparison with the corresponding lemon pectins. Water-extracted pectins were with higher degree of feruloylation (DF, 0.12-0.34%. To our knowledge this is the first report on the estimation of ester-linked ferulic acid in orange and lemon peel pectins. Pectic polysaccharides differed in molecular weight and homogeneity. WELP was with the highest molecular weight and homogeneity. The pectins contained D-galacturonic and D-glucuronic acids, L-arabinose, D-galactose, L-fucose, L-rhamnose and D-xylose. All investigated pectins showed immunostimulating activity by complement activation in the classical pathway at 1.25 and 2.5 mg/mL. Pectic polysaccharides were modified with endopolygalacturonase. Enzyme-modified CPCP and WEOP had higher anti-complementary activity than the corresponding initial pectins.

  9. Spot 42 Small RNA Regulates Arabinose-Inducible araBAD Promoter Activity by Repressing Synthesis of the High-Affinity Low-Capacity Arabinose Transporter

    Science.gov (United States)

    Chen, Jiandong

    2016-01-01

    ABSTRACT The l-arabinose-inducible araBAD promoter (PBAD) enables tightly controlled and tunable expression of genes of interest in a broad range of bacterial species. It has been used successfully to study bacterial sRNA regulation, where PBAD drives expression of target mRNA translational fusions. Here we report that in Escherichia coli, Spot 42 sRNA regulates PBAD promoter activity by affecting arabinose uptake. We demonstrate that Spot 42 sRNA represses araF, a gene encoding the AraF subunit of the high-affinity low-capacity arabinose transporter AraFGH, through direct base-pairing interactions. We further show that endogenous Spot 42 sRNA is sufficient to repress araF expression under various growth conditions. Finally, we demonstrate this posttranscriptional repression has a biological consequence, decreasing the induction of PBAD at low levels of arabinose. This problem can be circumvented using strategies reported previously for avoiding all-or-none induction behavior, such as through constitutive expression of the low-affinity high-capacity arabinose transporter AraE or induction with a higher concentration of inducers. This work adds araF to the set of Spot 42-regulated genes, in agreement with previous studies suggesting that Spot 42, itself negatively regulated by the cyclic AMP (cAMP) receptor protein-cAMP complex, reinforces the catabolite repression network. IMPORTANCE The bacterial arabinose-inducible system is widely used for titratable control of gene expression. We demonstrate here that a posttranscriptional mechanism mediated by Spot 42 sRNA contributes to the functionality of the PBAD system at subsaturating inducer concentrations by affecting inducer uptake. Our finding extends the inputs into the known transcriptional control for the PBAD system and has implications for improving its usage for tunable gene expression. PMID:27849174

  10. Evaluation of wheat stillage for ethanol production by recombinant Zymomonas mobilis

    Energy Technology Data Exchange (ETDEWEB)

    Davis, L.; Peiris, P. [University of Western Sydney, Penrith (Australia). School of Science, Food and Horticulture; Young-Jae Jeon; Svenson, C.; Rogers, P. [University of New South Wales, Sydney (Australia). School of Biotechnology and Biomolecular Sciences; Pearce, J. [Manildra Group, Bomaderry (Australia)

    2005-07-01

    Stillage is the main residue from the starch-to-ethanol fermentation process.Carbohydrates (hemicellulose and cellulose) comprise approximately 50% (w/w)of the total components of stillage. Conversion of the hemicellulose and cellulose to fermentable sugars and then to ethanol has the potential to significantly increase the efficiency of the process. The hydrolysis of stillage to fermentable sugars was optimised using 2% (v/v) H{sub 2}SO{sub 4} at 100{sup o}C for 5.5 h and produced 18 g/L xylose, 11.5 g/L arabinose and 6.5 g/L glucose from 120 g/L stillage. Further hydrolysis using enzymes increased the release of glucose by 61%. Furfural, acetate and lactate were the main inhibitors present in the acid hydrolysate of stillage. The lignin-derived inhibitors hydroxymethylfuraldehyde, hydroxybenzaldehyde, vanillin and syringaldehyde were not detected. Neutralisation of the hydrolysate with lime to pH 5 decreased the concentration of furfural by 50%. Fermentation of hydrolysate supplemented with glucose 10 g/L, by recombinant Zymomonas mobilis ZM4(pZB5), produced 11 g/L of ethanol after 70 h, with residual xylose 12 g/L. Supplementation of the hydrolysate with 5 g/L yeast extract and 40 g/L glucose produced 28 g/L ethanol with 2.6 g/L residual xylose after 18 h. Arabinose was not utilised by this particular recombinant strain. From the results, Z. mobilis ZM4(pZB5) may be a suitable candidate for the fermentation of both glucose and xylose in stillage acid hydrolysates. (author)

  11. An integrated biorefinery concept for conversion of sugar beet pulp into value-added chemicals and pharmaceutical intermediates.

    Science.gov (United States)

    Cárdenas-Fernández, Max; Bawn, Maria; Hamley-Bennett, Charlotte; Bharat, Penumathsa K V; Subrizi, Fabiana; Suhaili, Nurashikin; Ward, David P; Bourdin, Sarah; Dalby, Paul A; Hailes, Helen C; Hewitson, Peter; Ignatova, Svetlana; Kontoravdi, Cleo; Leak, David J; Shah, Nilay; Sheppard, Tom D; Ward, John M; Lye, Gary J

    2017-09-21

    Over 8 million tonnes of sugar beet are grown annually in the UK. Sugar beet pulp (SBP) is the main by-product of sugar beet processing which is currently dried and sold as a low value animal feed. SBP is a rich source of carbohydrates, mainly in the form of cellulose and pectin, including d-glucose (Glu), l-arabinose (Ara) and d-galacturonic acid (GalAc). This work describes the technical feasibility of an integrated biorefinery concept for the fractionation of SBP and conversion of these monosaccharides into value-added products. SBP fractionation is initially carried out by steam explosion under mild conditions to yield soluble pectin and insoluble cellulose fractions. The cellulose is readily hydrolysed by cellulases to release Glu that can then be fermented by a commercial yeast strain to produce bioethanol at a high yield. The pectin fraction can be either fully hydrolysed, using physico-chemical methods, or selectively hydrolysed, using cloned arabinases and galacturonases, to yield Ara-rich and GalAc-rich streams. These monomers can be separated using either Centrifugal Partition Chromatography (CPC) or ultrafiltration into streams suitable for subsequent enzymatic upgrading. Building on our previous experience with transketolase (TK) and transaminase (TAm) enzymes, the conversion of Ara and GalAc into higher value products was explored. In particular the conversion of Ara into l-gluco-heptulose (GluHep), that has potential therapeutic applications in hypoglycaemia and cancer, using a mutant TK is described. Preliminary studies with TAm also suggest GluHep can be selectively aminated to the corresponding chiral aminopolyol. The current work is addressing the upgrading of the remaining SBP monomer, GalAc, and the modelling of the biorefinery concept to enable economic and Life Cycle Analysis (LCA).

  12. L-lactic acid production from apple pomace by sequential hydrolysis and fermentation.

    Science.gov (United States)

    Gullón, Beatriz; Yáñez, Remedios; Alonso, José Luis; Parajó, J C

    2008-01-01

    The potential of apple pomace (a solid waste from cider and apple juice making factories) as a source of sugars and other compounds for fermentation was evaluated. The effect of the cellulase-to-solid ratio (CSR) and the liquor-to-solid ratio (LSR) on the kinetics of glucose and total monosaccharide generation was studied. Mathematical models suitable for reproducing and predicting the hydrolyzate composition were developed. When samples of apple pomace were subjected to enzymatic hydrolysis, the glucose and fructose present in the raw material as free monosaccharides were extracted at the beginning of the process. Using low cellulase and cellobiase charges (8.5 FPU/g-solid and 8.5 IU/g-solid, respectively), 79% of total glucan was saccharified after 12 h, leading to solutions containing up to 43.8 g monosaccharides/L (glucose, 22.8 g/L; fructose, 14.8 g/L; xylose+mannose+galactose, 2.5 g/L; arabinose+rhamnose, 2.8g/L). These results correspond to a monosaccharide/cellulase ratio of 0.06 g/FPU and to a volumetric productivity of 3.65 g of monosaccharides/L h. Liquors obtained under these conditions were used for fermentative lactic acid production with Lactobacillus rhamnosus CECT-288, leading to media containing up to 32.5 g/L of L-lactic acid after 6 h (volumetric productivity=5.41 g/L h, product yield=0.88 g/g).

  13. Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae.

    Science.gov (United States)

    Madhavan, Anjali; Srivastava, Aradhana; Kondo, Akihiko; Bisaria, Virendra S

    2012-03-01

    Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.

  14. Differential blood-brain barrier permeabilities to [14C]sucrose and [3H]inulin after osmotic opening in the rat

    International Nuclear Information System (INIS)

    Ziylan, Y.Z.; Robinson, P.J.; Rapoport, S.I.

    1983-01-01

    The blood-brain barrier (B-BB) in 3-month-old rats was opened unilaterally by infusing 1.8 m L(+)arabinose in water into the internal carotid artery through a catheter in the external carotid. Two poorly penetrating uncharged test radiotracers of differing molecular weight and size, [ 14 C]sucrose (340 daltons, radius 5 A) and [ 3 H]inulin (5500 daltons, radius 15 A), were simultaneously injected i.v. in untreated rats, or rats at 1, 30, or 50 min after infusion of hypertonic arabinose solution. Evans-blue solution was injected 5 min prior to osmotic treatment as a visual indicator of barrier integrity. In regions of uninfused control brains, the [ 14 C]sucrose permeability-surface area (PA) product approximated 10(-5) s-1, whereas PA was not measurable for [ 3 H]inulin. In arabinose-infused animals, PA products on the ipsilateral hemisphere for both [ 14 C]sucrose and [ 3 H]inulin were markedly elevated 6 min after infusion, but decreased by 35 and 55 min. In nearly all regions, statistically significant differences were not found between 6-min [ 14 C]sucrose- and [ 3 H]inulin-PA values (P greater than 0.05). However, at 35 and 55 min in most regions, the PA for [ 3 H]inulin was significantly lower (P less than 0.05) than PA for [ 14 C]sucrose. The results indicated that the B-BB closed more rapidly to larger than to smaller molecules after osmotic treatment and were consistent with a pore model for osmotic B-BB opening

  15. Diversity of Melissococcus plutonius from Honeybee Larvae in Japan and Experimental Reproduction of European Foulbrood with Cultured Atypical Isolates

    Science.gov (United States)

    Arai, Rie; Tominaga, Kiyoshi; Wu, Meihua; Okura, Masatoshi; Ito, Kazutomo; Okamura, Naomi; Onishi, Hidetaka; Osaki, Makoto; Sugimura, Yuya; Yoshiyama, Mikio; Takamatsu, Daisuke

    2012-01-01

    European foulbrood (EFB) is an important infectious disease of honeybee larvae, but its pathogenic mechanisms are still poorly understood. The causative agent, Melissococcus plutonius, is a fastidious organism, and microaerophilic to anaerobic conditions and the addition of potassium phosphate to culture media are required for growth. Although M. plutonius is believed to be remarkably homologous, in addition to M. plutonius isolates with typical cultural characteristics, M. plutonius-like organisms, with characteristics seemingly different from those of typical M. plutonius, have often been isolated from diseased larvae with clinical signs of EFB in Japan. Cultural and biochemical characterization of 14 M. plutonius and 19 M. plutonius-like strain/isolates revealed that, unlike typical M. plutonius strain/isolates, M. plutonius-like isolates were not fastidious, and the addition of potassium phosphate was not required for normal growth. Moreover, only M. plutonius-like isolates, but not typical M. plutonius strain/isolates, grew anaerobically on sodium phosphate-supplemented medium and aerobically on some potassium salt-supplemented media, were positive for β-glucosidase activity, hydrolyzed esculin, and produced acid from L-arabinose, D-cellobiose, and salicin. Despite the phenotypic differences, 16S rRNA gene sequence analysis and DNA-DNA hybridization demonstrated that M. plutonius-like organisms were taxonomically identical to M. plutonius. However, by pulsed-field gel electrophoresis analysis, these typical and atypical (M. plutonius-like) isolates were separately grouped into two genetically distinct clusters. Although M. plutonius is known to lose virulence quickly when cultured artificially, experimental infection of representative isolates showed that atypical M. plutonius maintained the ability to cause EFB in honeybee larvae even after cultured in vitro in laboratory media. Because the rapid decrease of virulence in cultured M. plutonius was a major

  16. Raman and infrared spectroscopy of carbohydrates: A review

    Science.gov (United States)

    Wiercigroch, Ewelina; Szafraniec, Ewelina; Czamara, Krzysztof; Pacia, Marta Z.; Majzner, Katarzyna; Kochan, Kamila; Kaczor, Agnieszka; Baranska, Malgorzata; Malek, Kamilla

    2017-10-01

    Carbohydrates are widespread and naturally occurring compounds, and essential constituents for living organisms. They are quite often reported when biological systems are studied and their role is discussed. However surprisingly, up till now there is no database collecting vibrational spectra of carbohydrates and their assignment, as has been done already for other biomolecules. So, this paper serves as a comprehensive review, where for selected 14 carbohydrates in the solid state both FT-Raman and ATR FT-IR spectra were collected and assigned. Carbohydrates can be divided into four chemical groups and in the same way is organized this review. First, the smallest molecules are discussed, i.e. monosaccharides (D-(-)-ribose, 2-deoxy-D-ribose, L-(-)-arabinose, D-(+)-xylose, D-(+)-glucose, D-(+)-galactose and D-(-)-fructose) and disaccharides (D-(+)-sucrose, D-(+)-maltose and D-(+)-lactose), and then more complex ones, i.e. trisaccharides (D-(+)-raffinose) and polysaccharides (amylopectin, amylose, glycogen). Both Raman and IR spectra were collected in the whole spectral range and discussed looking at the specific regions, i.e. region V (3600-3050 cm- 1), IV (3050-2800 cm- 1) and II (1200-800 cm- 1) assigned to the stretching vibrations of the OH, CH/CH2 and C-O/C-C groups, respectively, and region III (1500-1200 cm- 1) and I (800-100 cm- 1) dominated by deformational modes of the CH/CH2 and CCO groups, respectively. In spite of the fact that vibrational spectra of saccharides are significantly less specific than spectra of other biomolecules (e.g. lipids or proteins), marker bands of the studied molecules can be identified and correlated with their structure.

  17. Lectin activity in mycelial extracts of Fusarium species.

    Science.gov (United States)

    Bhari, Ranjeeta; Kaur, Bhawanpreet; Singh, Ram S

    2016-01-01

    Lectins are non-immunogenic carbohydrate-recognizing proteins that bind to glycoproteins, glycolipids, or polysaccharides with high affinity and exhibit remarkable ability to agglutinate erythrocytes and other cells. In the present study, ten Fusarium species previously not explored for lectins were screened for the presence of lectin activity. Mycelial extracts of F. fujikuroi, F. beomiformii, F. begoniae, F. nisikadoi, F. anthophilum, F. incarnatum, and F. tabacinum manifested agglutination of rabbit erythrocytes. Neuraminidase treatment of rabbit erythrocytes increased lectin titers of F. nisikadoi and F. tabacinum extracts, whereas the protease treatment resulted in a significant decline in agglutination by most of the lectins. Results of hapten inhibition studies demonstrated unique carbohydrate specificity of Fusarium lectins toward O-acetyl sialic acids. Activity of the majority of Fusarium lectins exhibited binding affinity to d-ribose, l-fucose, d-glucose, l-arabinose, d-mannitol, d-galactosamine hydrochloride, d-galacturonic acid, N-acetyl-d-galactosamine, N-acetyl-neuraminic acid, 2-deoxy-d-ribose, fetuin, asialofetuin, and bovine submaxillary mucin. Melibiose and N-glycolyl neuraminic acid did not inhibit the activity of any of the Fusarium lectins. Mycelial extracts of F. begoniae, F. nisikadoi, F. anthophilum, and F. incarnatum interacted with most of the carbohydrates tested. F. fujikuroi and F. anthophilum extracts displayed strong interaction with starch. The expression of lectin activity as a function of culture age was investigated. Most species displayed lectin activity on the 7th day of cultivation, and it varied with progressing of culture age. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  18. Expression capable library for studies of Neisseria gonorrhoeae, version 1.0

    Directory of Open Access Journals (Sweden)

    Wachocki Susi

    2005-09-01

    Full Text Available Abstract Background The sexually transmitted disease, gonorrhea, is a serious health problem in developed as well as in developing countries, for which treatment continues to be a challenge. The recent completion of the genome sequence of the causative agent, Neisseria gonorrhoeae, opens up an entirely new set of approaches for studying this organism and the diseases it causes. Here, we describe the initial phases of the construction of an expression-capable clone set representing the protein-coding ORFs of the gonococcal genome using a recombination-based cloning system. Results The clone set thus far includes 1672 of the 2250 predicted ORFs of the N. gonorrhoeae genome, of which 1393 (83% are sequence-validated. Included in this set are 48 of the 61 ORFs of the gonococcal genetic island of strain MS11, not present in the sequenced genome of strain FA1090. L-arabinose-inducible glutathione-S-transferase (GST-fusions were constructed from random clones and each was shown to express a fusion protein of the predicted size following induction, demonstrating the use of the recombination cloning system. PCR amplicons of each ORF used in the cloning reactions were spotted onto glass slides to produce DNA microarrays representing 2035 genes of the gonococcal genome. Pilot experiments indicate that these arrays are suitable for the analysis of global gene expression in gonococci. Conclusion This archived set of Gateway® entry clones will facilitate high-throughput genomic and proteomic studies of gonococcal genes using a variety of expression and analysis systems. In addition, the DNA arrays produced will allow us to generate gene expression profiles of gonococci grown in a wide variety of conditions. Together, the resources produced in this work will facilitate experiments to dissect the molecular mechanisms of gonococcal pathogenesis on a global scale, and ultimately lead to the determination of the functions of unknown genes in the genome.

  19. Serratia glossinae sp. nov., isolated from the midgut of the tsetse fly Glossina palpalis gambiensis.

    Science.gov (United States)

    Geiger, A; Fardeau, M-L; Falsen, E; Ollivier, B; Cuny, G

    2010-06-01

    We report the isolation of a novel bacterium, strain C1(T), from the midgut of the tsetse fly Glossina palpalis gambiensis, one of the vector insects responsible for transmission of the trypanosomes that cause sleeping sickness in sub-Saharan African countries. Strain C1(T) is a motile, facultatively anaerobic, rod-like bacterium (0.8-1.0 microm in diameter; 2-6 microm long) that grows as single cells or in chains. Optimum growth occurred at 25-35 degrees C, at pH 6.7-8.4 and in medium containing 5-20 g NaCl l(-1). The bacterium hydrolysed urea and used L-lysine, L-ornithine, citrate, pyruvate, D-glucose, D-mannitol, inositol, D-sorbitol, melibiose, amygdalin, L-arabinose, arbutin, aesculin, D-fructose, D-galactose, glycerol, maltose, D-mannose, raffinose, trehalose and d-xylose; it produced acetoin, reduced nitrate to nitrite and was positive for beta-galactosidase and catalase. The DNA G+C content was 53.6 mol%. It was related phylogenetically to members of the genus Serratia, family Enterobacteriaceae, the type strain of Serratia fonticola being its closest relative (99 % similarity between 16S rRNA gene sequences). However, DNA-DNA relatedness between strain C1(T) and S. fonticola DSM 4576(T) was only 37.15 %. Therefore, on the basis of morphological, nutritional, physiological and fatty acid analysis and genetic criteria, strain C1(T) is proposed to be assigned to a novel Serratia species, Serratia glossinae sp. nov. (type strain C1(T) =DSM 22080(T) =CCUG 57457(T)).

  20. Crystal structure of metagenomic β-xylosidase/ α-l-arabinofuranosidase activated by calcium.

    Science.gov (United States)

    Matsuzawa, Tomohiko; Kaneko, Satoshi; Kishine, Naomi; Fujimoto, Zui; Yaoi, Katsuro

    2017-09-01

    The crystal structure of metagenomic β-xylosidase/α-l-arabinofuranosidase CoXyl43, activated by calcium ions, was determined in its apo and complexed forms with xylotriose or l-arabinose in the presence and absence of calcium. The presence of calcium ions dramatically increases the kcat of CoXyl43 for p-nitrophenyl β-d-xylopyranoside and reduces the Michaelis constant for p-nitrophenyl α-l-arabinofuranoside. CoXyl43 consists of a single catalytic domain comprised of a five-bladed β-propeller. In the presence of calcium, a single calcium ion was observed at the centre of this catalytic domain, behind the catalytic pocket. In the absence of calcium, the calcium ion was replaced with one sodium ion and one water molecule, and the positions of these cations were shifted by 1.3 Å. The histidine-319 side chain, which coordinates to the 2-hydroxyl oxygen atom of the bound xylose molecule in the catalytic pocket, also coordinates to the calcium ion, but not to the sodium ion. The calcium-dependent increase in activity appears to be caused by the structural change in the catalytic pocket induced by the tightly bound calcium ion and coordinating water molecules, and by the protonation state of glutamic acid-268, the catalytic acid of the enzyme. Our findings further elucidate the complex relationship between metal ions and glycosidases. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  1. Purification of extensin from cell walls of tomato (hybrid of Lycopersicon esculentum and L. peruvianum) cells in suspension culture.

    Science.gov (United States)

    Brownleader, M D; Dey, P M

    1993-01-01

    Extensin, a hydroxyproline-rich glycoprotein comprising substantial amounts of beta-L-arabinose-hydroxyproline glycosidic linkages is believed to be insolubilized in the cell wall during host-pathogen interaction by a peroxidase/hydroperoxide-mediated cross-linking process. Both extensin precursor and extensin peroxidase were ionically eluted from intact water-washed tomato (hybrid of Lycopersicon esculentum Mill. and L. peruvianum L. (Mill.) cells in suspension cultures and purified to homogeneity by a rapid and simple procedure under mild and non-destructive experimental conditions. The molecular weight of native extensin precursor was estimated to be greater than 240-300 kDa by Superose-12 gel-filtration chromatography. Extensin monomers have previously been designated a molecular weight of approximately 80 kDa. Our results indicate that salt-eluted extensin precursor is not monomeric. Agarose-gel electrophoresis, Superose-12-gel-filtration, extensin-peroxidase-catalysed cross-linking, Mono-S ion-exchange fast protein liquid chromatography (FPLC), and peptide-sequencing data confirmed the homogeneity of the extensin preparation. Evidence that the purified protein was extensin is attributed to the presence of the putative sequence motif--Ser (Hyp)4--within the N-terminal end of the protein. Treatment of extensin with trifluoroacetic acid demonstrated that arabinose was the principal carbohydrate. The amino-acid composition of the purified extensin was similar to those reported in the literature. The cross-linking of extensin in vitro upon incubation with extensin peroxidase and exogenous H2O2 was characteristic of other reported extensins. Furthermore, Mono-S ion-exchange FPLC of native extensin precursor resolved it into two isoforms, A (90%) and B (10%). The amino-acid compositions of extensin A and extensin B were found to be similar to each other and both extensins were cross-linked in vitro by extensin peroxidase.

  2. [Construction of a recombinant HVT virus expressing the HA gene of avian influenza virus H5N1 via Rde/ET recombination system].

    Science.gov (United States)

    Lan, Desong; Shi, Xingming; Wang, Yunfeng; Liu, Changjun; Wang, Mei; Cui, Hongyu; Tian, Guobin; Li, Jisong; Tong, Guangzhi

    2009-01-01

    In recent years,manipulation of large herpesvirus genomes has been facilitated by using bacterial artificial chromosome (BAC) vectors. We have previously reported the construction of the BAC clones (HVT BACs) of herpesvirus of turkey (HVT). With these BAC clones in hand,we manipulated the genome of HVT by utilizing Red/ET recombination system, and developed a biologically safe live vaccine based on the HVT BACs. In this two-step approach, we first transformed the plasmid pRedET into the DH10B competent cells that carried the HVT BACs,and added inducer L-arabinose into the cells. We prepared the cells into competent cells and electroporated the linear rpsL-neo counter-selection/selection cassette flanked by the 50 bp long homology arms into the cells. So the functional cassette was inserted into the U(S)2 locus. Only colonies carrying the modified BAC would survive Kanamycin selection on the agar plates. The successful integration of the rpsL-neo cassette was monitored by PCR and Streptomycin selection, for the insertion of rpsL-neo cassette cells will become Streptomycin sensitive. Secondly, in the same way, we replaced the rpsL-neo cassette with the hemagglutinin (HA) gene of (HPAIV) A/Goose/ Guangdong/1/96(H5N1) flanked by the same homology arms. Only colonies which lost the rpsL-neo cassette will grow on Streptomycin containing plates. Finally, we obtained many colonies of which the HA gene of the AIV was inserted into the U(S)2 locus to be modified of HVT. And we reconstituted one recombinant virus from transfecting one of these BAC clones DNA into chick embryo fibroblasts (CEFs). We achieved one rescued recombinant virus which designated as rHVT-HA3. The H5 subtype HA gene expression in this recombinant virus rHVT-HA3 was confirmed by immunofluorescence assay.

  3. Structural insight into mechanism and diverse substrate selection strategy of L-ribulokinase

    Energy Technology Data Exchange (ETDEWEB)

    Agarwal R.; Swaminathan S.; Burley, S. K.

    2012-01-01

    The araBAD operon encodes three different enzymes required for catabolism of L-arabinose, which is one of the most abundant monosaccharides in nature. L-ribulokinase, encoded by the araB gene, catalyzes conversion of L-ribulose to L-ribulose-5-phosphate, the second step in the catabolic pathway. Unlike other kinases, ribulokinase exhibits diversity in substrate selectivity and catalyzes phosphorylation of all four 2-ketopentose sugars with comparable k{sub cat} values. To understand ribulokinase recognition and phosphorylation of a diverse set of substrates, we have determined the X-ray structure of ribulokinase from Bacillus halodurans bound to L-ribulose and investigated its substrate and ATP co-factor binding properties. The polypeptide chain is folded into two domains, one small and the other large, with a deep cleft in between. By analogy with related sugar kinases, we identified {sup 447}{und GG}LPQ{und K}{sup 452} as the ATP-binding motif within the smaller domain. L-ribulose binds in the cleft between the two domains via hydrogen bonds with the side chains of highly conserved Trp126, Lys208, Asp274, and Glu329 and the main chain nitrogen of Ala96. The interaction of L-ribulokinase with L-ribulose reveals versatile structural features that help explain recognition of various 2-ketopentose substrates and competitive inhibition by L-erythrulose. Comparison of our structure to that of the structures of other sugar kinases revealed conformational variations that suggest domain-domain closure movements are responsible for establishing the observed active site environment.

  4. The influence of different cultivation conditions on the metabolome of Fusarium oxysporum.

    Science.gov (United States)

    Panagiotou, Gianni; Christakopoulos, Paul; Olsson, Lisbeth

    2005-08-22

    The two most widespread pentose sugars found in the biosphere are d-xylose and l-arabinose. They are both potential substrates for ethanol production. The purpose of this study was to better understand the redox constraints imposed to Fusarium oxysporum during utilization of pentoses. In order to increase ethanol yield and decrease by-product formation, nitrate was used as nitrogen source. The use of NADH, the cofactor in denitrification process when using nitrate as a nitrogen source, improved the ethanol yield on xylose to 0.89 mol mol(-1) compared to the ethanol yield achieved using ammonium as nitrogen source 0.44 mol mol(-1). The improved ethanol yield was followed by a 28% decrease in yield of the by-product xylitol. In order to investigate the metabolic pathway of arabinose and the metabolic limitations for the efficient ethanol production from this sugar, the extracellular and intracellular metabolite profiles were determined under aerobic and anaerobic cultivation conditions. The results of this study clearly show difficulties in channelling of glucose-1-P (G1P) to pentose phosphate pathway (PPP) and reduced NADPH regeneration, suggesting that NADPH becomes a limiting factor for arabinose conversion, resulting in excessive acetate production. Variations of the fungus intracellular amino and non-amino acid pool, under different culture conditions, were evaluated using principal component analysis (PCA). PCA projection of the metabolome data collected from F. oxysporum subjected to environmental perturbations succeeded to visualize different physiological states and the conclusions of this study were that the metabolite profile is unique according to: (1) the carbon source and (2) the oxygen supply, and to a lesser extent to the cultivation phase.

  5. Lambda Red-mediated mutagenesis and efficient large scale affinity purification of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I).

    Science.gov (United States)

    Pohl, Thomas; Uhlmann, Mareike; Kaufenstein, Miriam; Friedrich, Thorsten

    2007-09-18

    The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. The Escherichia coli complex I consists of 13 different subunits named NuoA-N (from NADH:ubiquinone oxidoreductase), that are coded by the genes of the nuo-operon. Genetic manipulation of the operon is difficult due to its enormous size. The enzymatic activity of variants is obscured by an alternative NADH dehydrogenase, and purification of the variants is hampered by their instability. To overcome these problems the entire E. coli nuo-operon was cloned and placed under control of the l-arabinose inducible promoter ParaBAD. The exposed N-terminus of subunit NuoF was chosen for engineering the complex with a hexahistidine-tag by lambda-Red-mediated recombineering. Overproduction of the complex from this construct in a strain which is devoid of any membrane-bound NADH dehydrogenase led to the assembly of a catalytically active complex causing the entire NADH oxidase activity of the cytoplasmic membranes. After solubilization with dodecyl maltoside the engineered complex binds to a Ni2+-iminodiacetic acid matrix allowing the purification of approximately 11 mg of complex I from 25 g of cells. The preparation is pure and monodisperse and comprises all known subunits and cofactors. It contains more lipids than earlier preparations due to the gentle and fast purification procedure. After reconstitution in proteoliposomes it couples the electron transfer with proton translocation in an inhibitor sensitive manner, thus meeting all prerequisites for structural and functional studies.

  6. Modulation of intestinal absorption of calcium

    Energy Technology Data Exchange (ETDEWEB)

    Fournier, P; Dupuis, Y [Ecole Pratique des Hautes Etudes, 75 - Paris (France); Paris-11 Univ., 92 - Chatenay-Malabry (France))

    1975-01-01

    Absorption of ingested calcium (2ml of a 10mM CaCl/sub 2/ solution + /sup 45/Ca) by the adult rat was shown to be facilitated by the simultaneous ingestion of an active carbohydrate, L-arabinose. As the carbohydrate concentration is increased from 10 to 200mM, the absorption of calcium is maximised at a level corresponding to about twice the control absorption level. A similar doubling of calcium absorption is obtained when a 100mM concentration of any one of a number of other carbohydrates is ingested simultaneously with a 10mM CaCl/sub 2/ solution. Conversely, the simultaneous ingestion of increasing doses (10 to 100mM) of phosphate (NaH/sub 2/PO/sub 4/) with a 10mM CaCl/sub 2/ solution results in decreased /sup 45/Ca absorption and retention by the adult rat. The maximum inhibition of calcium absorption by phosphate is independent of the concentration of the ingested calcium solution (from 5 to 50mM CaCl/sub 2/). The simultaneous ingestion of CaCl/sub 2/ (10mM) with lactose and sodium phosphate (50 and 10mM respectively) shows that the activation effect of lactose upon /sup 45/Ca absorption may be partly dissimulated by the presence of phosphate. These various observations indicate that, within a large concentration range (2 to 50mM CaCl/sub 2/) calcium absorption appears to be a precisely modulated diffusion process. Calcium absorption varies (between minimum and maximum levels) as a function of the state of saturation by the activators (carbohydrates) and inhibitors (phosphate) of the calcium transport system.

  7. Genes regulated by AoXlnR, the xylanolytic and cellulolytic transcriptional regulator, in Aspergillus oryzae.

    Science.gov (United States)

    Noguchi, Yuji; Sano, Motoaki; Kanamaru, Kyoko; Ko, Taro; Takeuchi, Michio; Kato, Masashi; Kobayashi, Tetsuo

    2009-11-01

    XlnR is a Zn(II)2Cys6 transcriptional activator of xylanolytic and cellulolytic genes in Aspergillus. Overexpression of the aoxlnR gene in Aspergillus oryzae (A. oryzae xlnR gene) resulted in elevated xylanolytic and cellulolytic activities in the culture supernatant, in which nearly 40 secreted proteins were detected by two-dimensional electrophoresis. DNA microarray analysis to identify the transcriptional targets of AoXlnR led to the identification of 75 genes that showed more than fivefold increase in their expression in the AoXlnR overproducer than in the disruptant. Of these, 32 genes were predicted to encode a glycoside hydrolase, highlighting the biotechnological importance of AoXlnR in biomass degradation. The 75 genes included the genes previously identified as AoXlnR targets (xynF1, xynF3, xynG2, xylA, celA, celB, celC, and celD). Thirty-six genes were predicted to be extracellular, which was consistent with the number of proteins secreted, and 61 genes possessed putative XlnR-binding sites (5'-GGCTAA-3', 5'-GGCTAG-3', and 5'-GGCTGA-3') in their promoter regions. Functional annotation of the genes revealed that AoXlnR regulated the expression of hydrolytic genes for degradation of beta-1,4-xylan, arabinoxylan, cellulose, and xyloglucan and of catabolic genes for the conversion of D-xylose to xylulose-5-phosphate. In addition, genes encoding glucose-6-phosphate 1-dehydrogenase and L-arabinitol-4- dehydrogenase involved in D-glucose and L-arabinose catabolism also appeared to be targets of AoXlnR.

  8. The use of triphenyltetrazolium chloride in the study of dehydrogenase activity of Brucellae O emprêgo do cloreto de trifeniltetrazólio no estudo da atividade dehidrogenásica de brucelas

    Directory of Open Access Journals (Sweden)

    Milton Thiago de Mello

    1955-05-01

    Full Text Available Experiments for the investigation of dehydrogenase activity of washed cells of a strains of Br. abortus and another of Br. suis in presence of different single added substrates are reported. The activity was measured as the amount of formazan produced by the reduction of 2, 3, 5-triphenyltetrazolum chloride acting as a hydrogen ions acceptor, at pH 7.0. In a general manner the dehydrogenase activity of Br. suis was much more intense than that of Br. abortus (fig. 5. In the conditions of the experiments Br. abortus oxidized L-arabinose, D-galactose, D-glucose, glycerol, D-xylose, DL-alanine, D-fructose, and D-sorbitol. Brucella suis oxidized D-xylose, L-arabinose, D-glucose, D-galactose, DL-alanine, sodium acetate, maltose, glycine, D-fructose, and D-sorbitol. Glycerol was oxidized by Br. abortus but its oxidation by Br. suir was very slight. Sodium acetate and maltose were intensely oxidized by Br. suir but not by Br. abortus. The sites of more intense enzymatic acitivity were seen as small red colored round granules located in one pole of the cells.Com a finalidade de observar a atividade dhidrogenásica de brucelas, em presença de diversos substratos isolados, empregamos o cloreto de trifeniltetrazólio (em solução aquosa a 0,1% como receptor de hidrogênio. Os substratos (em solução aquosa M 50 foram os seguintes: Hidratos de carbono: L-arabinose, D-frutose, D-galactose, D-glucose, D-lactose, matose e D-xilose; alcoóis: glicerol, L-inositol, D-manitol e D-sobitol; ácidos aminados: ácido D-glutâmico, D-arginina, DL-alanina, L-asparagina e glicina; acetato de sódio. Empregamos suspensões de culturas de 48 horas de duas amostras típicas: Brucella abortus (aeróbica, nº 1 868, amostra B-99, Weybridge e Br. suis (nº 1 568, amostra SIG do Dr. S. S. Elberg, da Universidade de Califórnia. As culturas em agar, lavadas 5 vêzes em solução de cloreto de sódio a 0,9% ("resting cells" foram suspensas nessas solução salina de maneira a

  9. In-house zinc SAD phasing at Cu Kα edge.

    Science.gov (United States)

    Kim, Min-Kyu; Lee, Sangmin; An, Young Jun; Jeong, Chang-Sook; Ji, Chang-Jun; Lee, Jin-Won; Cha, Sun-Shin

    2013-07-01

    De novo zinc single-wavelength anomalous dispersion (Zn-SAD) phasing has been demonstrated with the 1.9 Å resolution data of glucose isomerase and 2.6 Å resolution data of Staphylococcus aureus Fur (SaFur) collected using in-house Cu Kα X-ray source. The successful in-house Zn-SAD phasing of glucose isomerase, based on the anomalous signals of both zinc ions introduced to crystals by soaking and native sulfur atoms, drove us to determine the structure of SaFur, a zinc-containing transcription factor, by Zn-SAD phasing using in-house X-ray source. The abundance of zinc-containing proteins in nature, the easy zinc derivatization of the protein surface, no need of synchrotron access, and the successful experimental phasing with the modest 2.6 Å resolution SAD data indicate that inhouse Zn-SAD phasing can be widely applicable to structure determination.

  10. Effect of combined gamma-irradiation and storage on biochemical changes in sweet potato

    International Nuclear Information System (INIS)

    Ajlouni, S.; Handy, M.K.

    1992-01-01

    Sucrose of uncured red jewel sweet potato increased from 3.8% to 10.7% after a combined treatment of a 300 Krad dose and 4 days storage at 24 C o post-irradiation (PI). Starch decreased from 16.8% to 6.1% after 16 days following a 500 Krad treatment. Phosphorylase, phosphoglucomutase and sucrose phosphate synthase enzyme specific activities increased 2.4-, 1.8- and 1.3-fold, respectively, after 3 days PI following 200 Krad exposures compared to nonirradiated roots. The beta-Amylase, phospho glucose isomerase and sucrose synthase specific activities increased 1.2-fold. Sucrose synthesis in the irradiated sweet potato was attributed to beta-amylase, phosphorylase, phosphoglucomutase, phospho glucose isomerase and sucrose synthase. (author). 28 refs., 3 figs., 2 tabs

  11. Effect of combined gamma-irradiation and storage on biochemical changes in sweet potato

    International Nuclear Information System (INIS)

    Ajlouni, S.; Hamdy, M.K.

    1988-01-01

    Sucrose of uncured Red Jewel sweet potato increased from 3.8% to 10.7% after a combined treatment of a 300 Krad dose ( 60 Co) and 4 days storage at 24 0 C post-irradiation (PI). Starch decreased from 16.8% to 6.1% after 16 days following a 500 Krad treatment. Phosphorylase, phosphoglucomutase and sucrose phosphate synthase enzyme specific activities increase 2.4-, 1.8- and 1.3-fold, respectively, after 3 days PI following 200 Krad exposures compared to nonirradiated roots. The beta-Amylase, phosphoglucose isomerase and sucrose synthase specific activities increased 1.2-fold. Sucrose synthesis in the irradiated sweet potato was attributed to beta-amylase, phosphorylase, phosphoglucomutase, phosphoglucose isomerase and sucrose synthase

  12. Lack of cyclophilin B in osteogenesis imperfecta with normal collagen folding.

    Science.gov (United States)

    Barnes, Aileen M; Carter, Erin M; Cabral, Wayne A; Weis, MaryAnn; Chang, Weizhong; Makareeva, Elena; Leikin, Sergey; Rotimi, Charles N; Eyre, David R; Raggio, Cathleen L; Marini, Joan C

    2010-02-11

    Osteogenesis imperfecta is a heritable disorder that causes bone fragility. Mutations in type I collagen result in autosomal dominant osteogenesis imperfecta, whereas mutations in either of two components of the collagen prolyl 3-hydroxylation complex (cartilage-associated protein [CRTAP] and prolyl 3-hydroxylase 1 [P3H1]) cause autosomal recessive osteogenesis imperfecta with rhizomelia (shortening of proximal segments of upper and lower limbs) and delayed collagen folding. We identified two siblings who had recessive osteogenesis imperfecta without rhizomelia. They had a homozygous start-codon mutation in the peptidyl-prolyl isomerase B gene (PPIB), which results in a lack of cyclophilin B (CyPB), the third component of the complex. The proband's collagen had normal collagen folding and normal prolyl 3-hydroxylation, suggesting that CyPB is not the exclusive peptidyl-prolyl cis-trans isomerase that catalyzes the rate-limiting step in collagen folding, as is currently thought. 2010 Massachusetts Medical Society

  13. Improved production of isomaltulose by a newly isolated mutant of Serratia sp. cells immobilized in calcium alginate.

    Science.gov (United States)

    Kim, Yonghwan; Koo, Bong-Seong; Lee, Hyeon-Cheol; Yoon, Youngdae

    2015-03-01

    Isomaltulose, also known as palatinose, is produced by sucrose isomerase and has been highlighted as a sugar substitute due to a number of advantageous properties. For the massive production of isomaltulose, high resistance to sucrose and stability of sucrose isomerase as well as sucrose conversion yields would be critical factors. We describe a series of screening procedures to isolate the mutant strain of Serratia sp. possessing enhanced isomaltulose production with improved stability. The new Serratia sp. isolated from a series of screening procedures allowed us to produce isomaltulose from 60% sucrose solution, with over 90% conversion yield. Moreover, when this strain was immobilized in calcium alginate beads and placed in a medium containing 60% sucrose, it showed over 70% sucrose conversion yields for 30 cycles of repeated-batch reactions. Thus, improved conversion activity and stability of the newly isolated Serratia sp. strain in the present study would be highly valuable for industries related to isomaltulose production.

  14. Association of a multi-synthetase complex with translating ribosomes in the archaeon Thermococcus kodakarensis

    DEFF Research Database (Denmark)

    Raina, Medha; Elgamal, Sara; Santangelo, Thomas J

    2012-01-01

    -dependent methyltransferase 144, GTP cyclohydrolase 398, DNA topoisomerase VI subunit A 209, DNA topoisomerase VI subunit B 192, Type A Flavoprotein 911, NAD(P)H:rubredoxin oxidoreductase (Fatty acid metabolism) 120, NAD(P)H:rubredoxin oxidoreductase 120, cofactor-independent phosphoglycerate mutase 909, bis(5'-adenosyl...... subunit 2 255, glycerol kinase 257, phosphomannomutase-related protein 321, ribose-5-phosphate isomerase A 107, phosphate transport regulator 193, isopentenyl pyrophosphate isomerase (mevanolate Pathway) 500, amino acid kinase 203, NADH:polysulfide oxidoreductase 203, 5'-methylthioadenosine phosphorylase......, cysteine desulfurase 521, hydrogenase maturation protein HypF 235, iron-molybdenum cofactor-binding protein 192, ATPase 260, 4Fe-4S cluster-binding protein 254, phosphopyruvate hydratase 650, fructose-1,6-bisphosphatase 140, aspartate carbamoyltransferase catalytic subunit 158, Bipolar DNA helicase 448...

  15. First insights into the mode of action of a "lachrymatory factor synthase"--implications for the mechanism of lachrymator formation in Petiveria alliacea, Allium cepa and Nectaroscordum species.

    Science.gov (United States)

    He, Quan; Kubec, Roman; Jadhav, Abhijit P; Musah, Rabi A

    2011-11-01

    A study of an enzyme that reacts with the sulfenic acid produced by the alliinase in Petiveria alliacea L. (Phytolaccaceae) to yield the P. alliacea lachrymator (phenylmethanethial S-oxide) showed the protein to be a dehydrogenase. It functions by abstracting hydride from sulfenic acids of appropriate structure to form their corresponding sulfines. Successful hydride abstraction is dependent upon the presence of a benzyl group on the sulfur to stabilize the intermediate formed on abstraction of hydride. This dehydrogenase activity contrasts with that of the lachrymatory factor synthase (LFS) found in onion, which catalyzes the rearrangement of 1-propenesulfenic acid to (Z)-propanethial S-oxide, the onion lachrymator. Based on the type of reaction it catalyzes, the onion LFS should be classified as an isomerase and would be called a "sulfenic acid isomerase", whereas the P. alliacea LFS would be termed a "sulfenic acid dehydrogenase". Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. Short and long-term effects of internal irradiation on the murine hepatic glycogen and its metabolizing enzymes

    International Nuclear Information System (INIS)

    Gupta, N.K.

    1990-01-01

    Glycogen content and the activities of phosphorylase, phosphorhexose isomerase, glucose 6-phosphatase, glycogen synthesis' phosphorylase and succinate dehydrogenase have been biochemically determined in the liver of Swiss albino mice after radiocalcium internal irradiation up to 225 days posttreatment. Increase in the glycogen content and glycogen synthesis phosphorylase with a concomitant decrease in the activities of phosphorylase, glucose 6-phosphatase, phosphohexose isomerase and succinate dehydrogenase reveals inhibited glycolysis in the presence of normal glyogenesis and inhibited Kreb's cycle in the liver during early intervals. Decrease in the glycogen content at later stages along with decrease in the activities of all these enzymes is probably because of an inhibited glycogen biosynthesis and its catabolism through HMP shunt. (orig.)

  17. Gibberellic Acid-Induced Aleurone Layers Responding to Heat Shock or Tunicamycin Provide Insight into the N-Glycoproteome, Protein Secretion, and Endoplasmic Reticulum Stress

    DEFF Research Database (Denmark)

    Barba Espin, Gregorio; Dedvisitsakul, Plaipol; Hägglund, Per

    2014-01-01

    respond to gibberellic acid by secreting an array of proteins and provide a unique system for the analysis of plant protein secretion. Perturbation of protein secretion in gibberellic acid-induced aleurone layers by two independent mechanisms, heat shock and tunicamycin treatment, demonstrated overlapping...... and secretion, such as calreticulin, protein disulfide isomerase, proteasome subunits, and isopentenyl diphosphate isomerase. Sixteen heat shock proteins in 29 spots showed diverse responses to the treatments, with only a minority increasing in response to heat shock. The majority, all of which were small heat...... shock proteins, decreased in heat-shocked aleurone layers. Additionally, glycopeptide enrichment and N-glycosylation analysis identified 73 glycosylation sites in 65 aleurone layer proteins, with 53 of the glycoproteins found in extracellular fractions and 36 found in intracellular fractions...

  18. Subterranean Microhabitat Dependent Intra Versus Extracellular Enzyme Secretion Capabilities of Deinococcus radiodurans

    Directory of Open Access Journals (Sweden)

    Jayant Biswas

    2015-04-01

    Full Text Available Deinococcus radiodurans is one of the most yet discovered extremophilic microbe, the isolation of which from the various habitats of Kotumsar cave is always a matter of enticement to discover its ecological economics. In the present work we studied the intra versus extracellular alkaline protease and glucose isomerase secretion capabilities of Deinococcus radiodurans; KCB21, KCB50, KCB93 isolated from three distinct subterranean niches of Kotumsar cave. The selected niches/zones were the entrance zone, transient zone and the deep inner zone from where the soil sediments were collected to isolate the bacterial strains. The results revealed high extracellular alkaline protease activity from the Deinococcus radiodurans strain which was isolated from the deeper zones of the cave, whereas no such phenomenon was revealed for glucose isomerase. The possible reason for the obtained results has been discussed.

  19. Effects of 45Ca on murine skeletal muscle. 1

    International Nuclear Information System (INIS)

    Asotra, K.; Katoch, S.S.; Krishan, K.; Malhotra, R.K.

    1983-01-01

    Adult Swiss albino mice weighing 16+-1 g were injected with 3.7x10 4 Bq and 7.4x10 4 Bq/g body weight of 45 Ca. Mice of both dose groups were autopsied on days 1, 3, 5, 7, 14 and 28 after 45 Ca administration. Diaphragm and gastrocnemius in the 45 Ca-treated and normal mice were analyzed for quantitation of glycogen as well as bioassay of phosphorylase and phosphohexose isomerase activities. Internal irradiation with the two doses of 45 Ca resulted in glycogen accumulation in both the muscles. 45 Ca-treated diaphragm showed greater radioresponse but a slower recovery than gastrocnemius with respect to glycogen accumulation. A decline in the rates of glycogenolysis and glycolysis indicated by decreased phosphorylase and phosphohexose isomerase activities appeared to be responsible for glycogen accumulation in skeletal muscle on account of 45 Ca treatment. (author)

  20. Lecithin:Retinol Acyltransferase: A Key Enzyme Involved in the Retinoid (visual) Cycle

    OpenAIRE

    Sears, Avery E.; Palczewski, Krzysztof

    2016-01-01

    Lecithin:retinol acyltransferase (LRAT) catalyzes the acyl transfer from the sn-1 position of phosphatidylcholine (PC) to all-trans-retinol, creating fatty acid retinyl esters (palmitoyl, stearoyl, and some unsaturated derivatives). In the eye, these retinyl esters are substrates for the 65 kDa retinoid isomerase (RPE65). LRAT is well characterized biochemically, and recent structural data from closely related family members of the NlpC/P60 superfamily and a chimeric protein have established ...

  1. Nucleotide sequence analysis of the Legionella micdadei mip gene, encoding a 30-kilodalton analog of the Legionella pneumophila Mip protein

    DEFF Research Database (Denmark)

    Bangsborg, Jette Marie; Cianciotto, N P; Hindersson, P

    1991-01-01

    After the demonstration of analogs of the Legionella pneumophila macrophage infectivity potentiator (Mip) protein in other Legionella species, the Legionella micdadei mip gene was cloned and expressed in Escherichia coli. DNA sequence analysis of the L. micdadei mip gene contained in the plasmid p...... homology with the mip-like genes of several Legionella species. Furthermore, amino acid sequence comparisons revealed significant homology to two eukaryotic proteins with isomerase activity (FK506-binding proteins)....

  2. Catalytic Enzyme-Based Methods for Water Treatment and Water Distribution System Decontamination. 1. Literature Survey

    Science.gov (United States)

    2006-06-01

    best examples of this is glucose isomerase, which has been used in the commercial production of high fructose corn syrup (HFCS) since 1967.230 Most...EDGEWOOD CHEMICAL BIOLOGICAL CENTER U.S. ARMY RESEARCH, DEVELOPMENT AND ENGINEERING COMMAND ECBC-TR-489 CATALYTIC ENZYME-BASED METHODS FOR WATER ...TREATMENT AND WATER DISTRIBUTION SYSTEM DECONTAMINATION 1. LITERATURE SURVEY Joseph J. DeFrank RESEARCH AND TECHNOLOGY DIRECTORATE June 2006 Approved for

  3. European Congress on Biotechnology (4th) Held in Amsterdam, The Netherlands, on June 1987

    Science.gov (United States)

    1988-02-19

    car be used and thus higher Nmaraso Fumarate enzyme loadings can be achieved with a Glucose isomerase High- fructose corn syrup large effectiveness...is only after most of the the following reactions: glucose + oxy- glucose has been metabolized that aerobic gen + glucose oxidasa + water - gluconic...are obtained by mixing water solutions of two water -soluble * Biocatalysis polymers. Both phases have a high water * Animal cell cultures content and do

  4. A Proteomic Screen Identified Stress-Induced Chaperone Proteins as Targets of Akt Phosphorylation in Mesangial Cells

    OpenAIRE

    Barati, Michelle T.; Rane, Madhavi J.; Klein, Jon B.; McLeish, Kenneth R.

    2006-01-01

    The serine-threonine kinase Akt regulates mesangial cell apoptosis, proliferation, and hypertrophy. To define Akt signaling pathways in mesangial cells, we performed a functional proteomic screen for rat mesangial cell proteins phosphorylated by Akt. A group of chaperone proteins, heat shock protein (Hsp) 70, Hsp90α, Hsp90β, Glucose-regulated protein (Grp) Grp78, Grp94, and protein disulfide isomerase (PDI) were identified as potential Akt substrates by two techniques: (a) in vitro phosphoryl...

  5. Etude par génétique inverse du gène codant la protéine TARGET OF RAPAMYCIN d'Arabidopsis thaliana (AtTOR), l'homologue d'une kinase contrôlant la croissance cellulaire chez les eucaryotes

    OpenAIRE

    Menand, Benoit

    2002-01-01

    TOR (target of rapamycin) protein kinases were identified in yeast, mammals and Drosophila as central controllers of cell growth. Thu, G1 to S phases progression through the cell cycle is blocked by rapamycin, a drug which specifically inhibits TOR activity by forming a ternary complex with the peptidyl-prolyl isomerase FKBP12 (FK506 and rapamycin binding protein), and the FKBP-rapamycin binding domain (FRB) of TOR proteins. This work presents the study, the Arabidopsis homologue of yeast and...

  6. Cytokinin metabolism of pathogenic fungus Leptosphaeria maculans involves isopentenyltransferase, adenosine kinase and cytokinin oxidase/dehydrogenase

    Czech Academy of Sciences Publication Activity Database

    Trdá, Lucie; Barešová, Monika; Šašek, Vladimír; Nováková, Miroslava; Zahajská, Lenka; Dobrev, Petre; Motyka, Václav; Burketová, Lenka

    2017-01-01

    Roč. 8, JUL 21 (2017), č. článku 1374. ISSN 1664-302X R&D Projects: GA ČR GA13-26798S; GA ČR(CZ) GA16-14649S Institutional support: RVO:61389030 Keywords : Adenosine kinase * Cytokinin * Cytokinin oxidase/dehydrogenase * Isopentenyltransferase * Leptosphaeria maculans * Zeatin cis/trans isomerase Subject RIV: GF - Plant Pathology, Vermin, Weed, Plant Protection OBOR OECD: Microbiology Impact factor: 4.076, year: 2016

  7. Incorporation of rapid thermodynamic data in fragment-based drug discovery.

    Science.gov (United States)

    Kobe, Akihiro; Caaveiro, Jose M M; Tashiro, Shinya; Kajihara, Daisuke; Kikkawa, Masato; Mitani, Tomoya; Tsumoto, Kouhei

    2013-03-14

    Fragment-based drug discovery (FBDD) has enjoyed increasing popularity in recent years. We introduce SITE (single-injection thermal extinction), a novel thermodynamic methodology that selects high-quality hits early in FBDD. SITE is a fast calorimetric competitive assay suitable for automation that captures the essence of isothermal titration calorimetry but using significantly fewer resources. We describe the principles of SITE and identify a novel family of fragment inhibitors of the enzyme ketosteroid isomerase displaying high values of enthalpic efficiency.

  8. Cyclophilin B facilitates the replication of Orf virus

    OpenAIRE

    Zhao, Kui; Li, Jida; He, Wenqi; Song, Deguang; Zhang, Ximu; Zhang, Di; Zhou, Yanlong; Gao, Feng

    2017-01-01

    Background Viruses interact with host cellular factors to construct a more favourable environment for their efficient replication. Expression of cyclophilin B (CypB), a cellular peptidyl-prolyl cis-trans isomerase (PPIase), was found to be significantly up-regulated. Recently, a number of studies have shown that CypB is important in the replication of several viruses, including Japanese encephalitis virus (JEV), hepatitis C virus (HCV) and human papillomavirus type 16 (HPV 16). However, the f...

  9. Mutations in PPIB (cyclophilin B) delay type I procollagen chain association and result in perinatal lethal to moderate osteogenesis imperfecta phenotypes.

    Science.gov (United States)

    Pyott, Shawna M; Schwarze, Ulrike; Christiansen, Helena E; Pepin, Melanie G; Leistritz, Dru F; Dineen, Richard; Harris, Catharine; Burton, Barbara K; Angle, Brad; Kim, Katherine; Sussman, Michael D; Weis, Maryann; Eyre, David R; Russell, David W; McCarthy, Kevin J; Steiner, Robert D; Byers, Peter H

    2011-04-15

    Recessive mutations in the cartilage-associated protein (CRTAP), leucine proline-enriched proteoglycan 1 (LEPRE1) and peptidyl prolyl cis-trans isomerase B (PPIB) genes result in phenotypes that range from lethal in the perinatal period to severe deforming osteogenesis imperfecta (OI). These genes encode CRTAP (encoded by CRTAP), prolyl 3-hydroxylase 1 (P3H1; encoded by LEPRE1) and cyclophilin B (CYPB; encoded by PPIB), which reside in the rough endoplasmic reticulum (RER) and can form a complex involved in prolyl 3-hydroxylation in type I procollagen. CYPB, a prolyl cis-trans isomerase, has been thought to drive the prolyl-containing peptide bonds to the trans configuration needed for triple helix formation. Here, we describe mutations in PPIB identified in cells from three individuals with OI. Cultured dermal fibroblasts from the most severely affected infant make some overmodified type I procollagen molecules. Proα1(I) chains are slow to assemble into trimers, and abnormal procollagen molecules concentrate in the RER, and bind to protein disulfide isomerase (PDI) and prolyl 4-hydroxylase 1 (P4H1). These findings suggest that although CYPB plays a role in helix formation another effect is on folding of the C-terminal propeptide and trimer formation. The extent of procollagen accumulation and PDI/P4H1 binding differs among cells with mutations in PPIB, CRTAP and LEPRE1 with the greatest amount in PPIB-deficient cells and the least in LEPRE1-deficient cells. These findings suggest that prolyl cis-trans isomerase may be required to effectively fold the proline-rich regions of the C-terminal propeptide to allow proα chain association and suggest an order of action for CRTAP, P3H1 and CYPB in procollagen biosynthesis and pathogenesis of OI.

  10. Renewable Hydrogen Carrier - Carbohydrate: Constructing the Carbon-Neutral Carbohydrate Economy

    Science.gov (United States)

    2011-01-31

    combinations have been investigated for the production of hydrogen from biomass carbohydrate. Chemical catalysis approaches include pyrolysis [19...temperature. High fructose corn syrup, low-cost sucrose replacement, is made by stabilized glucose isomerase, which can work at ~60 °C for even about two...gasoline, vegetable oil vs. biodiesel, corn kernels vs. ethanol [31,109]. Given a price of $0.18/kg carbohydrate (i.e., $10.6/GJ) [2,44], the hydrogen

  11. Immobilized enzymes and cells

    Energy Technology Data Exchange (ETDEWEB)

    Bucke, C; Wiseman, A

    1981-04-04

    This article reviews the current state of the art of enzyme and cell immobilization and suggests advances which might be made during the 1980's. Current uses of immobilized enzymes include the use of glucoamylase in the production of glucose syrups from starch and glucose isomerase in the production of high fructose corn syrup. Possibilities for future uses of immobilized enzymes and cells include the utilization of whey and the production of ethanol.

  12. In Silico Prediction of T and B Cell Epitopes of Der f 25 in Dermatophagoides farinae

    Directory of Open Access Journals (Sweden)

    Xiaohong Li

    2014-01-01

    Full Text Available The house dust mites are major sources of indoor allergens for humans, which induce asthma, rhinitis, dermatitis, and other allergic diseases. Der f 25 is a triosephosphate isomerase, representing the major allergen identified in Dermatophagoides farinae. The objective of this study was to predict the B and T cell epitopes of Der f 25. In the present study, we analyzed the physiochemical properties, function motifs and domains, and structural-based detailed features of Der f 25 and predicted the B cell linear epitopes of Der f 25 by DNAStar protean system, BPAP, and BepiPred 1.0 server and the T cell epitopes by NetMHCIIpan-3.0 and NetMHCII-2.2. As a result, the sequence and structure analysis identified that Der f 25 belongs to the triosephosphate isomerase family and exhibited a triosephosphate isomerase pattern (PS001371. Eight B cell epitopes (11–18, 30–35, 71–77, 99–107, 132–138, 173–187, 193–197, and 211–224 and five T cell epitopes including 26–34, 38–54, 66–74, 142–151, and 239–247 were predicted in this study. These results can be used to benefit allergen immunotherapies and reduce the frequency of mite allergic reactions.

  13. Biochemical characterization of the prolyl 3-hydroxylase 1.cartilage-associated protein.cyclophilin B complex.

    Science.gov (United States)

    Ishikawa, Yoshihiro; Wirz, Jackie; Vranka, Janice A; Nagata, Kazuhiro; Bächinger, Hans Peter

    2009-06-26

    The rough endoplasmic reticulum-resident protein complex consisting of prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP), and cyclophilin B (CypB) can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. Prolyl 3-hydroxylase 1 modifies a single proline residue in the alpha chains of type I, II, and III collagens to (3S)-hydroxyproline. The peptidyl-prolyl cis-trans isomerase activity of cyclophilin B was shown previously to catalyze the rate of triple helix formation. Here we show that cyclophilin B in the complex shows peptidyl-prolyl cis-trans isomerase activity and that the P3H1.CRTAP.CypB complex has another important function: it acts as a chaperone molecule when tested with two classical chaperone assays. The P3H1.CRTAP.CypB complex inhibited the thermal aggregation of citrate synthase and was active in the denatured rhodanese refolding and aggregation assay. The chaperone activity of the complex was higher than that of protein-disulfide isomerase, a well characterized chaperone. The P3H1.CRTAP.CypB complex also delayed the in vitro fibril formation of type I collagen, indicating that this complex is also able to interact with triple helical collagen and acts as a collagen chaperone.

  14. Biochemical Characterization of the Prolyl 3-Hydroxylase 1·Cartilage-associated Protein·Cyclophilin B Complex*

    Science.gov (United States)

    Ishikawa, Yoshihiro; Wirz, Jackie; Vranka, Janice A.; Nagata, Kazuhiro; Bächinger, Hans Peter

    2009-01-01

    The rough endoplasmic reticulum-resident protein complex consisting of prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP), and cyclophilin B (CypB) can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. Prolyl 3-hydroxylase 1 modifies a single proline residue in the α chains of type I, II, and III collagens to (3S)-hydroxyproline. The peptidyl-prolyl cis-trans isomerase activity of cyclophilin B was shown previously to catalyze the rate of triple helix formation. Here we show that cyclophilin B in the complex shows peptidyl-prolyl cis-trans isomerase activity and that the P3H1·CRTAP·CypB complex has another important function: it acts as a chaperone molecule when tested with two classical chaperone assays. The P3H1·CRTAP·CypB complex inhibited the thermal aggregation of citrate synthase and was active in the denatured rhodanese refolding and aggregation assay. The chaperone activity of the complex was higher than that of protein-disulfide isomerase, a well characterized chaperone. The P3H1·CRTAP·CypB complex also delayed the in vitro fibril formation of type I collagen, indicating that this complex is also able to interact with triple helical collagen and acts as a collagen chaperone. PMID:19419969

  15. Integrative Gene Cloning and Expression System for Streptomyces sp. US 24 and Streptomyces sp. TN 58 Bioactive Molecule Producing Strains

    Directory of Open Access Journals (Sweden)

    Samiha Sioud

    2009-01-01

    Full Text Available Streptomyces sp. US 24 and Streptomyces sp. TN 58, two strains producing interesting bioactive molecules, were successfully transformed using E. coli ET12567 (pUZ8002, as a conjugal donor, carrying the integrative plasmid pSET152. For the Streptomyces sp. US 24 strain, two copies of this plasmid were tandemly integrated in the chromosome, whereas for Streptomyces sp. TN 58, the integration was in single copy at the attB site. Plasmid pSET152 was inherited every time for all analysed Streptomyces sp. US 24 and Streptomyces sp. TN 58 exconjugants under nonselective conditions. The growth, morphological differentiation, and active molecules production of all studied pSET152 integrated exconjugants were identical to those of wild type strains. Consequently, conjugal transfer using pSET152 integration system is a suitable means of genes transfer and expression for both studied strains. To validate the above gene transfer system, the glucose isomerase gene (xylA from Streptomyces sp. SK was expressed in strain Streptomyces sp. TN 58. Obtained results indicated that heterologous glucose isomerase could be expressed and folded effectively. Glucose isomerase activity of the constructed TN 58 recombinant strain is of about eighteenfold higher than that of the Streptomyces sp. SK strain. Such results are certainly of importance due to the potential use of improved strains in biotechnological process for the production of high-fructose syrup from starch.

  16. Lactic Acid Bacteria Combinations for Wheat Sourdough Preparation and Their Influence on Wheat Bread Quality and Acrylamide Formation.

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    Bartkiene, Elena; Bartkevics, Vadims; Krungleviciute, Vita; Pugajeva, Iveta; Zadeike, Daiva; Juodeikiene, Grazina

    2017-10-01

    Different lactic acid bacteria (LAB) from spontaneous wheat sourdough were isolated, identified, and characterized by their growth, acidification rate, and carbohydrate metabolism. The combinations of isolated LAB (Pediococcus pentosaceus LUHS183 and Leuconostoc mesenteroides LUHS242, P. pentosaceus LUHS183 and Lactobacillus brevis LUHS173, P. pentosaceus LUHS183 and Enterococcus pseudoavium LUHS 234, P. pentosaceus LUHS183 and Lactobacillus curvatus LUHS51, Lactobacillus plantarum LUHS135 and L. curvatus LUHS51, L. plantarum LUHS135 and P. pentosaceus LUHS183) were used for wheat sourdough production, and the effects of LAB fermentation in sourdoughs on wheat bread quality parameters and acrylamide formation were evaluated. All of the tested strains (except E. pseudoavium LUHS 234) were able to ferment l-arabinose, d-ribose, d-galactose, d-fructose, and d-maltose and showed high tolerance to acidic conditions. The highest overall acceptability (135.8 ± 5.5 mm) was found in the bread produced with L. plantarum and P. pentosaceus sourdough. This group of bread also showed the highest shape coefficient (2.59 ± 0.02), the highest specific volume (3.40 ± 0.03 cm 3 /g), the highest porosity (76.6 ± 0.3%), and the highest moisture content (33.7%). Selected sourdoughs reduced acrylamide content in bread samples by 29.5% (sourdough prepared with P. pentosaceus and L. mesenteroides) to 67.2% (sourdough prepared with P. pentosaceus and L. curvatus). These cultures potentially can be used to reduce acrylamide in breads. The data of this study have practical applications. L. plantarum and P. pentosaceus sourdoughs increases overall acceptability, specific volume, and porosity of wheat bread. Besides the fact that sourdoughs produced by using combinations of selected LAB strains improved the quality parameters of bread, fermentation with prepared sourdoughs also reduced the acrylamide content in wheat bread samples by 29.5% (sourdough prepared with P. pentosaceus

  17. Differential gene expression in tomato fruit and Colletotrichum gloeosporioides during colonization of the RNAi-SlPH tomato line with reduced fruit acidity and higher pH.

    Science.gov (United States)

    Barad, Shiri; Sela, Noa; Dubey, Amit K; Kumar, Dilip; Luria, Neta; Ment, Dana; Cohen, Shahar; Schaffer, Arthur A; Prusky, Dov

    2017-08-04

    The destructive phytopathogen Colletotrichum gloeosporioides causes anthracnose disease in fruit. During host colonization, it secretes ammonia, which modulates environmental pH and regulates gene expression, contributing to pathogenicity. However, the effect of host pH environment on pathogen colonization has never been evaluated. Development of an isogenic tomato line with reduced expression of the gene for acidity, SlPH (Solyc10g074790.1.1), enabled this analysis. Total RNA from C. gloeosporioides colonizing wild-type (WT) and RNAi-SlPH tomato lines was sequenced and gene-expression patterns were compared. C. gloeosporioides inoculation of the RNAi-SlPH line with pH 5.96 compared to the WT line with pH 4.2 showed 30% higher colonization and reduced ammonia accumulation. Large-scale comparative transcriptome analysis of the colonized RNAi-SlPH and WT lines revealed their different mechanisms of colonization-pattern activation: whereas the WT tomato upregulated 13-LOX (lipoxygenase), jasmonic acid and glutamate biosynthesis pathways, it downregulated processes related to chlorogenic acid biosynthesis II, phenylpropanoid biosynthesis and hydroxycinnamic acid tyramine amide biosynthesis; the RNAi-SlPH line upregulated UDP-D-galacturonate biosynthesis I and free phenylpropanoid acid biosynthesis, but mainly downregulated pathways related to sugar metabolism, such as the glyoxylate cycle and L-arabinose degradation II. Comparison of C. gloeosporioides gene expression during colonization of the WT and RNAi-SlPH lines showed that the fungus upregulates ammonia and nitrogen transport and the gamma-aminobutyric acid metabolic process during colonization of the WT, while on the RNAi-SlPH tomato, it mainly upregulates the nitrate metabolic process. Modulation of tomato acidity and pH had significant phenotypic effects on C. gloeosporioides development. The fungus showed increased colonization on the neutral RNAi-SlPH fruit, and limited colonization on the WT acidic fruit

  18. Xylitol production from waste xylose mother liquor containing miscellaneous sugars and inhibitors: one-pot biotransformation by Candida tropicalis and recombinant Bacillus subtilis.

    Science.gov (United States)

    Wang, Hengwei; Li, Lijuan; Zhang, Lebin; An, Jin; Cheng, Hairong; Deng, Zixin

    2016-05-16

    The process of industrial xylitol production is a massive source of organic pollutants, such as waste xylose mother liquor (WXML), a viscous reddish-brown liquid. Currently, WXML is difficult to reuse due to its miscellaneous low-cost sugars, high content of inhibitors and complex composition. WXML, as an organic pollutant of hemicellulosic hydrolysates, accumulates and has become an issue of industrial concern in China. Previous studies have focused only on the catalysis of xylose in the hydrolysates into xylitol using one strain, without considering the removal of other miscellaneous sugars, thus creating an obstacle to subsequent large-scale purification. In the present study, we aimed to develop a simple one-pot biotransformation to produce high-purity xylitol from WXML to improve its economic value. In the present study, we developed a procedure to produce xylitol from WXML, which combines detoxification, biotransformation and removal of by-product sugars (purification) in one bioreactor using two complementary strains, Candida tropicalis X828 and Bacillus subtilis Bs12. At the first stage of micro-aerobic biotransformation, the yeast cells were allowed to grow and metabolized glucose and the inhibitors furfural and hydroxymethyl furfural (HMF), and converted xylose into xylitol. At the second stage of aerobic biotransformation, B. subtilis Bs12 was activated and depleted the by-product sugars. The one-pot process was successfully scaled up from shake flasks to 5, 150 L and 30 m(3) bioreactors. Approximately 95 g/L of pure xylitol could be obtained from the medium containing 400 g/L of WXML at a yield of 0.75 g/g xylose consumed, and the by-product sugars glucose, L-arabinose and galactose were depleted simultaneously. Our results demonstrate that the one-pot procedure is a viable option for the industrial application of WXML to produce value-added chemicals. The integration of complementary strains in the biotransformation of hemicellulosic hydrolysates is

  19. Capacity for absorption of water-soluble secondary metabolites greater in birds than in rodents.

    Science.gov (United States)

    Karasov, William H; Caviedes-Vidal, Enrique; Bakken, Bradley Hartman; Izhaki, Ido; Samuni-Blank, Michal; Arad, Zeev

    2012-01-01

    Plant secondary metabolites (SMs) are pervasive in animal foods and potentially influence feeding behavior, interspecies interactions, and the distribution and abundance of animals. Some of the major classes of naturally occurring SMs in plants include many water-soluble compounds in the molecular size range that could cross the intestinal epithelium via the paracellular space by diffusion or solvent drag. There are differences among species in paracellular permeability. Using Middle Eastern rodent and avian consumers of fruits containing SMs, we tested the hypothesis that avian species would have significantly higher paracellular permeability than rodent species. Permeability in intact animals was assessed using standard pharmacological methodology to measure absorption of two radiolabeled, inert, neutral water-soluble probes that do not interact with intestinal nutrient transporters, L-arabinose (M(r) = 150.1 Da) and lactulose (M(r) = 342.3 Da). We also measured absorption of labeled 3-O-methyl-D-glucose (3OMD-glucose; M(r) = 194.2 Da), which is a nonmetabolized analogue of D-glucose that is passively absorbed through the paracellular space but also transported across the enterocyte membranes. Most glucose was absorbed by all species, but arabinose fractional absorption (f) was nearly three times higher in birds (1.03±0.17, n = 15 in two species) compared to rodents (0.37±0.06, n = 10 in two species) (Pbirds of arabinose exceeded those of 3OMD-glucose. Our findings are in agreement with previous work showing that the paracellular pathway is more prominent in birds relative to nonflying mammals, and suggests that birds may be challenged by greater absorption of water-soluble, dietary SMs. The increased expression of the paracellular pathway in birds hints at a tradeoff: the free energy birds gain by absorbing water-soluble nutrients passively may be offset by the metabolic demands placed on them to eliminate concomitantly absorbed SMs.

  20. Sugar recognition by human galactokinase

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    Timson David J

    2003-11-01

    Full Text Available Abstract Background Galactokinase catalyses the first committed step of galactose catabolism in which the sugar is phosphorylated at the expense of MgATP. Recent structural studies suggest that the enzyme makes several contacts with galactose – five side chain and two main chain hydrogen bonds. Furthermore, it has been suggested that inhibition of galactokinase may help sufferers of the genetic disease classical galactosemia which is caused by defects in another enzyme of the pathway galactose-1-phosphate uridyl transferase. Galactokinases from different sources have a range of substrate specificities and a diversity of kinetic mechanisms. Therefore only studies on the human enzyme are likely to be of value in the design of therapeutically useful inhibitors. Results Using recombinant human galactokinase expressed in and purified from E. coli we have investigated the sugar specificity of the enzyme and the kinetic consequences of mutating residues in the sugar-binding site in order to improve our understanding of substrate recognition by this enzyme. D-galactose and 2-deoxy-D-galactose are substrates for the enzyme, but N-acetyl-D-galactosamine, L-arabinose, D-fucose and D-glucose are all not phosphorylated. Mutation of glutamate-43 (which forms a hydrogen bond to the hydroxyl group attached to carbon 6 of galactose to alanine results in only minor changes in the kinetic parameters of the enzyme. Mutation of this residue to glycine causes a ten-fold drop in the turnover number. In contrast, mutation of histidine 44 to either alanine or isoleucine results in insoluble protein following expression in E. coli. Alteration of the residue that makes hydrogen bonds to the hydroxyl attached to carbons 3 and 4 (aspartate 46 results in an enzyme that although soluble is essentially inactive. Conclusions The enzyme is tolerant to small changes at position 2 of the sugar ring, but not at positions 4 and 6. The results from site directed mutagenesis could