WorldWideScience

Sample records for immobilized glucose isomerase

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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 ...

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

    Science.gov (United States)

    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.

  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. 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.

  10. 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

  11. 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.

  12. 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...

  13. 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.

  14. 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...

  15. 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

  16. 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...

  17. Change in blood glucose level in rats after immobilization

    Science.gov (United States)

    Platonov, R. D.; Baskakova, G. M.; Chepurnov, S. A.

    1981-01-01

    Experiments were carried out on male white rats divided into four groups. In group one the blood glucose level was determined immediately after immobilization. In the other three groups, two hours following immobilization, the blood glucose level was determined every 20 minutes for 3 hours 40 minutes by the glucose oxidase method. Preliminary immobilization for 2 hours removed the increase in the blood glucose caused by the stress reaction. By the 2nd hour of immobilization in the presence of continuing stress, the blood glucose level stabilized and varied within 42 + or - 5.5 and 47 + or - 8.1 mg %. Within 2 hours after the immobilization, the differences in the blood glucose level of the rats from the control groups were statistically insignificant.

  18. 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.

  19. 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.

  20. 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.

  1. Bioconversion of D-galactose to D-tagatose: continuous packed bed reaction with an immobilized thermostable L-arabinose isomerase and efficient purification by selective microbial degradation.

    Science.gov (United States)

    Liang, Min; Chen, Min; Liu, Xinying; Zhai, Yafei; Liu, Xian-wei; Zhang, Houcheng; Xiao, Min; Wang, Peng

    2012-02-01

    The continuous enzymatic conversion of D-galactose to D-tagatose with an immobilized thermostable L-arabinose isomerase in packed-bed reactor and a novel method for D-tagatose purification were studied. L-arabinose isomerase from Thermoanaerobacter mathranii (TMAI) was recombinantly overexpressed and immobilized in calcium alginate. The effects of pH and temperature on D-tagatose production reaction catalyzed by free and immobilized TMAI were investigated. The optimal condition for free enzyme was pH 8.0, 60°C, 5 mM MnCl(2). However, that for immobilized enzyme was pH 7.5, 75°C, 5 mM MnCl(2). In addition, the catalytic activity of immobilized enzyme at high temperature and low pH was significantly improved compared with free enzyme. The optimum reaction yield with immobilized TMAI increased by four percentage points to 43.9% compared with that of free TMAI. The highest productivity of 10 g/L h was achieved with the yield of 23.3%. Continuous production was performed at 70°C; after 168 h, the reaction yield was still above 30%. The resultant syrup was then incubated with Saccharomyces cerevisiae L1 cells. The selective degradation of D-galactose was achieved, obtaining D-tagatose with the purity above 95%. The established production and separation methods further potentiate the industrial production of D-tagatose via bioconversion and biopurification processes.

  2. 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.

  3. 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.

  4. Glucose Oxidase Immobilization on TMAH-Modified Bentonite

    Directory of Open Access Journals (Sweden)

    Ruth Chrisnasari

    2015-03-01

    Full Text Available The influence of bentonite modification by tetramethyl ammonium hydroxide (TMAH on its capability to immobilize glucose oxidase (GOX was studied. Modification of bentonite was conducted by the adding of 0-5% (v/v TMAH. The observed results show that the different concentrations of TMAH affect the percentage of immobilized enzyme. The results of this study show that the best concentration of TMAH is 5% (v/v which can immobilize up to 84.71% of GOX. X-ray diffraction (XRD and Fourier Transforms Infrared Spectroscopy (FTIR studies have been carried out to observe the structural changes in bentonite due to TMAH modification. The obtained immobilized GOX show the optimum catalytic activity on reaction temperature of 40-50 °C and pH of 7. The immobilized GOX kinetics at the optimum conditions determined the Km and Vmax value to be 4.96x10-2 mM and 4.99x10-3 mM.min-1 respectively. In addition, the immobilized GOX on TMAH-modified bentonite is stable enough so it could be re-used six times before its activity decreased by 39.44%.

  5. Glucose oxidase immobilization onto carbon nanotube networking

    International Nuclear Information System (INIS)

    Karachevtsev, V.A.; Glamazda, A.Yu.; Zarudnev, E.S.; Karachevtsev, M.V.; Leontiev, V.S.; Linnik, A.S.; Plokhotnichenko, A.M.; Stepanian, S.G.; Lytvyn, O.S.

    2012-01-01

    The efficient immobilization of GOX onto a carbon nanotube network through the molecular interface formed by PSE is carried out. This conclusion is based on the analysis of AFM images of the network with the adsorbed enzyme, whose globules locate mainly along a nanotube. The band corresponding to the high-frequency component of the G mode in the RR spectrum of the nanotube with adsorbed PSE is downshifted by 0.7 cm -1 relative to this band in the spectrum of pristine nanotubes. The analysis of the intensities of bands assigned to the RBM of nanotubes with adsorbed PSE in comparison with the spectrum of pristine SWNTs revealed the intensity transformation, which can be explained by a change of the resonance condition with variation of the laser energy. Thus, we concluded that PSE molecules create nanohybrids with SWNTs, which ensures the further enzyme immobilization. As the RR spectrum of an SWNT:PSE:GOX film does not essentially differ from SWNT:PSE ones, this indicates that the molecular interface (PSE) isolates the enzyme from nanotubes strongly enough. Our studies on the conductive properties of a single walled carbon nanotube network sprayed onto a quartz substrate from a solution of nanotubes in dichlorobenzene demonstrated that the I(U) dependence has nonlinear character. Most likely, the nonlinearity is related to Schottky barriers, which originate on the contact between nanotubes and the gold electrode, as well as between nanotubes with different conductivities. The deposition of bioorganic compounds (PSE and GOX) on the carbon nanotube network is accompanied by a decrease of their conductivity. Most probably, such a decrease is caused by adsorbed PSE molecules, which induce the appearance of scattering centers for charge carriers on the nanotube surface. The following GOX adsorption has practically no effect on the conductivity of the nanotube network that evidences the reliable isolation of the nanotube surface from the enzyme by means of the molecular

  6. Flow-induced immobilization of glucose oxidase in nonionic micellar nanogels for glucose sensing.

    Science.gov (United States)

    Cardiel, Joshua J; Zhao, Ya; Tonggu, Lige; Wang, Liguo; Chung, Jae-Hyun; Shen, Amy Q

    2014-10-21

    A simple microfluidic platform was utilized to immobilize glucose oxidase (GOx) in a nonionic micellar scaffold. The immobilization of GOx was verified by using a combination of cryogenic electron microscopy (cryo-EM), scanning electron microscopy (SEM), and ultraviolet spectroscopy (UV) techniques. Chronoamperometric measurements were conducted on nanogel-GOx scaffolds under different glucose concentrations, exhibiting linear amperometric responses. Without impacting the lifetime and denaturation of GOx, the nonionic nanogel provides a favorable microenvironment for GOx in biological media. This flow-induced immobilization method in a nonionic nanogel host matrix opens up new pathways for designing a simple, fast, biocompatible, and cost-effective process to immobilize biomolecules that are averse to ionic environments.

  7. 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

  8. 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%.

  9. Study on the preparation of immobilized glucose oxidase membrane and its application in clinic analysis

    International Nuclear Information System (INIS)

    Yu Ye; Cao Jin; Su Zongxian; Chen Zixiong

    1990-01-01

    The paper deals with the preparation of immobilized glucose oxidase membrane by using two steps irradiation (irradiation gratfting, irradiation entrapping). Some properties of membrane were discussed. The immobilized glucose oxidase membrane with oxygen electrode and oxygen analyser can be satisfied with the clinic analysis for the determination of serum glucose

  10. Glucose oxidase immobilization on different modified surfaces of platinum nanowire for application in glucose detection

    International Nuclear Information System (INIS)

    Le, Thi Thanh Tuyen; Tran, Phu Duy; Pham, Xuan Tung; Tong, Duy Hien; Dang, Mau Chien

    2010-01-01

    In this work, the surface of platinum (Pt) nanowires was modified by using several chemicals, including a compound of gelatin gel with SiO 2 , polyvinyl alcohol (PVA) with Prussian blue (PB) mediator and cysteamine self-assembled monolayers (SAM). Then, glucose oxidase (GOD) enzyme was immobilized on the modified surfaces of Pt nanowire electrodes by using techniques of electrochemical adsorption and chemical binding. The GOD immobilized Pt nanowires were used for application in glucose detection by performing a cyclic voltammetry measurement. The detection results showed that GOD was immobilized on all of the tested surfaces and the highest glucose detection sensitivity of 60 μM was obtained when the Pt nanowires were modified by PVA with PB mediator. Moreover, the sensors showed very high current response when the Pt nanowires were modified with the cysteamine SAM. The stability and catalyst activity of GOD are also reported here. For instance, the catalyst activity of GOD retained about 60% of its initial value after it was stored at 4 °C in a 100 mM PBS buffer solution with a pH of 7.2 for a period of 30 days

  11. Glucose oxidase immobilization on different modified surfaces of platinum nanowire for application in glucose detection

    Science.gov (United States)

    Thanh Tuyen Le, Thi; Duy Tran, Phu; Pham, Xuan Tung; Hien Tong, Duy; Chien Dang, Mau

    2010-09-01

    In this work, the surface of platinum (Pt) nanowires was modified by using several chemicals, including a compound of gelatin gel with SiO2, polyvinyl alcohol (PVA) with Prussian blue (PB) mediator and cysteamine self-assembled monolayers (SAM). Then, glucose oxidase (GOD) enzyme was immobilized on the modified surfaces of Pt nanowire electrodes by using techniques of electrochemical adsorption and chemical binding. The GOD immobilized Pt nanowires were used for application in glucose detection by performing a cyclic voltammetry measurement. The detection results showed that GOD was immobilized on all of the tested surfaces and the highest glucose detection sensitivity of 60 μM was obtained when the Pt nanowires were modified by PVA with PB mediator. Moreover, the sensors showed very high current response when the Pt nanowires were modified with the cysteamine SAM. The stability and catalyst activity of GOD are also reported here. For instance, the catalyst activity of GOD retained about 60% of its initial value after it was stored at 4 °C in a 100 mM PBS buffer solution with a pH of 7.2 for a period of 30 days.

  12. Glucose biosensor based on glucose oxidase immobilized on unhybridized titanium dioxide nanotube arrays

    International Nuclear Information System (INIS)

    Wang, Wei; Xie, Yibing; Du, Hongxiu; Xia, Chi; Wang, Yong; Tian, Fang

    2014-01-01

    A glucose biosensor has been fabricated by immobilizing glucose oxidase (GOx) on unhybridized titanium dioxide nanotube arrays using an optimized cross-linking technique. The TiO 2 nanotube arrays were synthesized directly on a titanium substrate by anodic oxidation. The structure and morphology of electrode material were characterized by X-ray diffraction and scanning electron microscopy. The electrochemical performances of the glucose biosensor were conducted by cyclic voltammetry and chronoamperometry measurements. It gives a linear response to glucose in the 0.05 to 0.65 mM concentration range, with a correlation coefficient of 0.9981, a sensitivity of 199.6 μA mM −1 cm −2 , and a detection limit as low as 3.8 µM. This glucose biosensor exhibited high selectivity for glucose determination in the presence of ascorbic acid, sucrose and other common interfering substances. This glucose biosensor also performed good reproducibility and long-time storage stability. This optimized cross-linking technique could open a new avenue for other enzyme biosensors fabrication. (author)

  13. Effect of prior immobilization on muscular glucose clearance in resting and running rats

    International Nuclear Information System (INIS)

    Vissing, J.; Ohkuwa, Tetsuo; Ploug, T.; Galbo, H.

    1988-01-01

    In vitro studies have shown that prior disuse impairs the glucose clearance of red skeletal muscle because of a developed insensitivity to insulin. We studied whether an impaired glucose clearance is present in vivo in 42-h immobilized muscles of resting rats and, furthermore, whether the exercise-induced increase in glucose clearance of red muscles is affected by prior immobilization. The 2-[ 3 H]deoxy-D-glucose (2DG) bolus injection method was used to determine glucose clearance of individual muscles. At rest, glucose clearance was markedly impaired in rats with previously immobilized red muscles compared with nonimmobilized control rats. During running, glucose clearance did not differ between muscles in previously immobilized and control rats. Insulin levels were always similar in the two groups and decreased during exercise. Intracellular nonphosphorylated 2DG was present in tissues with high glucose clearances. In conclusion, 42 h of immobilization markedly impairs glucose clearance of resting red muscle fibers in vivo. Apparently, physical inactivity in particular affects steps involved in insulin-mediated action that are not part of contraction-induced glucose uptake and metabolism. Presence of intracellular 2DG shows that separate determination of phosphorylated 2DG is necessary for accurate estimates of glucose metabolism and that accumulation of phosphorylated 2DG does not accurately reflect glucose transport

  14. A glucose biosensor based on glucose oxidase immobilized on three-dimensional porous carbon electrodes.

    Science.gov (United States)

    Chen, Jingyi; Zhu, Rong; Huang, Jia; Zhang, Man; Liu, Hongyu; Sun, Min; Wang, Li; Song, Yonghai

    2015-08-21

    A novel glucose biosensor was developed by immobilizing glucose oxidase (GOD) on a three-dimensional (3D) porous kenaf stem-derived carbon (3D-KSC) which was firstly proposed as a novel supporting material to load biomolecules for electrochemical biosensing. Here, an integrated 3D-KSC electrode was prepared by using a whole piece of 3D-KSC to load the GOD molecules for glucose biosensing. The morphologies of integrated 3D-KSC and 3D-KSC/GOD electrodes were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The SEM results revealed a 3D honeycomb macroporous structure of the integrated 3D-KSC electrode. The TEM results showed some microporosities and defects in the 3D-KSC electrode. The electrochemical behaviors and electrocatalytic performance of the integrated 3D-KSC/GOD electrode were evaluated by cyclic voltammetry and electrochemical impedance spectroscopy. The effects of pH and scan rates on the electrochemical response of the biosensor have been studied in detail. The glucose biosensor showed a wide linear range from 0.1 mM to 14.0 mM with a high sensitivity of 1.73 μA mM(-1) and a low detection limit of 50.75 μM. Furthermore, the glucose biosensor exhibited high selectivity, good repeatability and reproducibility, and good stability.

  15. A fine pointed glucose oxidase immobilized electrode for low-invasive amperometric glucose monitoring.

    Science.gov (United States)

    Li, Jiang; Koinkar, Pankaj; Fuchiwaki, Yusuke; Yasuzawa, Mikito

    2016-12-15

    A low invasive type glucose sensor, which has a sensing region at the tip of a fine pointed electrode, was developed for continuous glucose monitoring. Platinum-iridium alloy electrode with a surface area of 0.045mm(2) was settled at the middle of pointed PEEK (Polyetheretherketone) tubing and was employed as sensing electrode. Electrodeposition of glucose oxidase in the presence of surfactant, Triton X-100, was performed for high-density enzyme immobilization followed by the electropolymerization of o-phenylenediamine for the formation of functional entrapping and permselective polymer membrane. Ag/AgCl film was coated on the surface of PEEK tubing as reference electrode. Amperometric responses of the prepared sensors to glucose were measured at a potential of 0.60V (vs. Ag/AgCl). The prepared electrode showed the sensitivity of 2.55μA/cm(2) mM with high linearity of 0.9986, within the glucose concentration range up to 21mM. The detection limit (S/N=3) was determined to be 0.11mM. The glucose sensor properties were evaluated in phosphate buffer solution and in vivo monitoring by the implantation of the sensors in rabbit, while conventional needle type sensors as a reference were used. The results showed that change in output current of the proposed sensor fluctuated similar with one in output current of the conventional needle type sensors, which was also in similar accordance with actual blood sugar level measured by commercially glucose meter. One-point calibration method was used to calibrate the sensor output current. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. 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).

  17. Glucose biosensor based on glucose oxidase immobilized at gold nanoparticles decorated graphene-carbon nanotubes.

    Science.gov (United States)

    Devasenathipathy, Rajkumar; Mani, Veerappan; Chen, Shen-Ming; Huang, Sheng-Tung; Huang, Tsung-Tao; Lin, Chun-Mao; Hwa, Kuo-Yuan; Chen, Ting-Yo; Chen, Bo-Jun

    2015-10-01

    Biopolymer pectin stabilized gold nanoparticles were prepared at graphene and multiwalled carbon nanotubes (GR-MWNTs/AuNPs) and employed for the determination of glucose. The formation of GR-MWNTs/AuNPs was confirmed by scanning electron microscopy, X-ray diffraction, UV-vis and FTIR spectroscopy methods. Glucose oxidase (GOx) was successfully immobilized on GR-MWNTs/AuNPs film and direct electron transfer of GOx was investigated. GOx exhibits highly enhanced redox peaks with formal potential of -0.40 V (vs. Ag/AgCl). The amount of electroactive GOx and electron transfer rate constant were found to be 10.5 × 10(-10) mol cm(-2) and 3.36 s(-1), respectively, which were significantly larger than the previous reports. The fabricated amperometric glucose biosensor sensitively detects glucose and showed two linear ranges: (1) 10 μM - 2 mM with LOD of 4.1 μM, (2) 2 mM - 5.2 mM with LOD of 0.95 mM. The comparison of the biosensor performance with reported sensors reveals the significant improvement in overall sensor performance. Moreover, the biosensor exhibited appreciable stability, repeatability, reproducibility and practicality. The other advantages of the fabricated biosensor are simple and green fabrication approach, roughed and stable electrode surface, fast in sensing and highly reproducible. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. 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

  19. 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

  20. Colorimetric Glucose Assay Based on Magnetic Particles Having Pseudo-peroxidase Activity and Immobilized Glucose Oxidase.

    Science.gov (United States)

    Martinkova, Pavla; Opatrilova, Radka; Kruzliak, Peter; Styriak, Igor; Pohanka, Miroslav

    2016-05-01

    Magnetic particles (MPs) are currently used as a suitable alternative for peroxidase in the construction of novel biosensors, analytic and diagnostic methods. Their better chemical and thermal stabilities predestine them as appropriate pseudo-enzymatic catalysts. In this point of view, our research was focused on preparation of simply and fast method for immobilization of glucose oxidase onto surface of MPs with peroxidase-like activity. Spectrophotometric method (wavelength 450 nm) optimized for glucose determination using modified MPs has been successfully developed. Concentration curve for optimization of method was assayed, and Michaelis-Menten constant (K m) calculated, maximum reaction rate (V max), limit of detection, and correlation coefficient were determined to be 0.13 mmol/l (2.34 mg/dl), 1.79 pkat, 3.74 µmol/l (0.067 mg/dl), and 0.996, respectively. Interferences of other sugars such as sucrose, sorbitol, deoxyribose, maltose, and fructose were determined as well as effect of substances presenting in plasma (ascorbic acid, reduced glutathione, trolox, and urea). Results in comparison with positive and negative controls showed no interferences of the other sugars and no influence of plasma substances to measuring of glucose. The constructed method showed corresponding results with linear dependence and a correlation coefficient of 0.997. Possibility of repeated use of modified MPs was successfully proved.

  1. Effect of sulfonylureas administered centrally on the blood glucose level in immobilization stress model.

    Science.gov (United States)

    Sharma, Naveen; Sim, Yun-Beom; Park, Soo-Hyun; Lim, Su-Min; Kim, Sung-Su; Jung, Jun-Sub; Hong, Jae-Seung; Suh, Hong-Won

    2015-05-01

    Sulfonylureas are widely used as an antidiabetic drug. In the present study, the effects of sulfonylurea administered supraspinally on immobilization stress-induced blood glucose level were studied in ICR mice. Mice were once enforced into immobilization stress for 30 min and returned to the cage. The blood glucose level was measured 30, 60, and 120 min after immobilization stress initiation. We found that intracerebroventricular (i.c.v.) injection with 30 µg of glyburide, glipizide, glimepiride or tolazamide attenuated the increased blood glucose level induced by immobilization stress. Immobilization stress causes an elevation of the blood corticosterone and insulin levels. Sulfonylureas pretreated i.c.v. caused a further elevation of the blood corticosterone level when mice were forced into the stress. In addition, sulfonylureas pretreated i.c.v. alone caused an elevation of the plasma insulin level. Furthermore, immobilization stress-induced insulin level was reduced by i.c.v. pretreated sulfonylureas. Our results suggest that lowering effect of sulfonylureas administered supraspinally against immobilization stress-induced increase of the blood glucose level appears to be primarily mediated via elevation of the plasma insulin level.

  2. Co-immobilization of gold nanoparticles with glucose oxidase to improve bioelectrocatalytic glucose oxidation

    Science.gov (United States)

    Aquino Neto, Sidney; Milton, Ross D.; Crepaldi, Laís B.; Hickey, David P.; de Andrade, Adalgisa R.; Minteer, Shelley D.

    2015-07-01

    Recently, there has been much effort in developing metal nanoparticle catalysts for fuel oxidation, as well as the development of enzymatic bioelectrocatalysts for fuel oxidation. However, there has been little study of the synergy of hybrid electrocatalytic systems. We report the preparation of hybrid bioanodes based on Au nanoparticles supported on multi-walled carbon nanotubes (MWCNTs) co-immobilized with glucose oxidase (GOx). Mediated electron transfer was achieved by two strategies: ferrocene entrapped within polypyrrole and a ferrocene-modified linear poly(ethylenimine) (Fc-LPEI) redox polymer. Electrochemical characterization of the Au nanoparticles supported on MWCNTs indicate that this catalyst exhibits an electrocatalytic response for glucose even in acidic conditions. Using the redox polymer Fc-LPEI as the mediator, voltammetric and amperometric data demonstrated that these bioanodes can efficiently achieve mediated electron transfer and also indicated higher catalytic currents with the hybrid bioelectrode. From the amperometry, the maximum current density (Jmax) achieved with the hybrid bioelectrode was 615 ± 39 μA cm-2, whereas the bioanode employing GOx only achieved a Jmax of 409 ± 26 μA cm-2. Biofuel cell tests are consistent with the electrochemical characterization, thus confirming that the addition of the metallic species into the bioanode structure can improve fuel oxidation and consequently, improve the power generated by the system.

  3. Effect of prior immobilization on muscular glucose clearance in resting and running rats

    DEFF Research Database (Denmark)

    Vissing, J; Ohkuwa, T; Ploug, Thorkil

    1988-01-01

    with nonimmobilized control rats (red gastrocnemius 0.46 +/- 0.02 vs. 0.99 +/- 0.08 and soleus 1.10 +/- 0.30 vs. 3.97 +/- 0.54 ml.min-1.100 g-1, P less than 0.005). During running (18 m/min), glucose clearance did not differ between muscles in previously immobilized and control rats. Insulin levels were always......In vitro studies have shown that prior disuse impairs the glucose clearance of red skeletal muscle because of a developed insensitivity to insulin. We studied whether an impaired glucose clearance is present in vivo in 42-h immobilized muscles of resting rats and, furthermore, whether the exercise......-induced increase in glucose clearance of red muscles is affected by prior immobilization. The 2-[3H]deoxy-D-glucose (2DG) bolus injection method was used to determine glucose clearance of individual muscles. At rest, glucose clearance was markedly impaired in rats with previously immobilized red muscles compared...

  4. An amperometric biosensor for glucose detection from glucose oxidase immobilized in polyaniline-polyvinylsulfonate-potassium ferricyanide film.

    Science.gov (United States)

    Arslan, Fatma; Beskan, Umut

    2014-08-01

    In this study, a novel amperometric glucose biosensor with immobilization of glucose oxidase on electrochemically polymerized polyaniline-polyvinylsulphonate-potassium ferricyanide (Pani-Pvs-Fc) films has been accomplished via the entrapment technique. Potassium ferricyanide was used as the mediator. Determination of glucose was carried out by the oxidation of potassium ferrocyanide at 0.3 V vs. Ag/AgCl. The effects of pH and temperature were investigated, and the optimum pH value was found to be 7.5. The storage stability and the operational stability of the enzyme electrode were also studied.

  5. Amperometric Determination of Glucose at Parts per Million Levels with Immobilized Glucose Oxidase.

    Science.gov (United States)

    Sittampalam, G.; Wilson, G. S.

    1982-01-01

    An experiment on the operation and utility of an amperometric immobilized enzyme electrode (or probe) is described, including advantages of the experiment, equipment, reagents, preparation of phosphate buffer, enzyme immobilization techniques, laboratory procedures, precautions, and discussion of experimental results. (SK)

  6. A Glucose Sensor Based on Glucose Oxidase Immobilized by Electrospinning Nanofibrous Polymer Membranes Modified with Carbon Nanotubes

    Directory of Open Access Journals (Sweden)

    You Wang

    2013-05-01

    Full Text Available A glucose biosensor based on glucose oxidase immobilized by electrospinning nanofibrous membranes has been developed. Nanofibrous membranes were electrospun from the solution of poly(acrylonitrile-co-acrylic acid containing carbon nanotubes suspension and directly deposited on Pt electrodes for immobilizing glucose oxidase. The morphologies and structure of the nanofibrous membranes with or without carbon nanotubes were characterized by scanning electron microscopy. The fabrication parameters of nanofibers were optimized such as thickness of the nanofibrous membranes and mass ration of carbon nanotubes. The biosensor showed the relationship with a concentration range of 0.1–10 mM and response time was 60 s. The sensitivity of carbon nanotubes modified biosensors was two times larger than which of no carbon nanotubes modified ones. The pH effect, interference and lifetime of biosensors were discussed.

  7. Direct electrochemistry of glucose oxidase immobilized on nanostructured gold thin films and its application to bioelectrochemical glucose sensor

    International Nuclear Information System (INIS)

    Qiu Cuicui; Wang Xia; Liu Xueying; Hou Shifeng; Ma Houyi

    2012-01-01

    Highlights: ► Au thin films are formed by electrodeposition and galvanic replacement technology. ► Glucose oxidase is stably immobilized via a simple physical adsorption method. ► The direct electrochemical behavior is obtained on the immobilized glucose oxidase. ► An amperometric sensor of glucose with an excellent sensing capability is achieved. - Abstract: Glucose oxidase (GOx) was stably immobilized via a simple physical adsorption method onto the nanostructured Au thin films fabricated by using electrodeposition and galvanic replacement technology, which provides a facile method to prepare morphology-controllable Au films and also facilitates the preparation and application of enzyme modified electrodes. An obvious advantage of the as-prepared enzyme electrode (denoted as GOx/Au/GCE) is that the nano-Au films provide a favorable microenvironment for GOx and facilitate the electron transfer between the active center of GOx and electrodes. Cyclic voltammetry (CV) results indicate that the immobilized GOx displayed a direct, reversible and surface-confined redox reaction in the phosphate buffer solution. Furthermore, the enzyme modified electrode was used as a glucose bioelectrochemical sensor, exhibiting a linear relationship in the concentration ranges of 2.5–32.5 μmol L −1 and 60–130 μmol L −1 with a detection limit of 0.32 μmol L −1 (S/N = 3) at an applied potential of −0.55 V. Due to the excellent stability, sensitivity and anti-interference ability, the Au thin films are hopeful in the construction of glucose biosensors.

  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. 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.

  10. 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.

  11. Improvement of the stability and activity of immobilized glucose oxidase on modified iron oxide magnetic nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Abbasi, Mahboube, E-mail: mahbubeabbasi@yahoo.com; Amiri, Razieh, E-mail: razieh.amiri@gmail.com; Bordbar, Abdol-Kalegh, E-mail: bordbar@chem.ui.ac.ir; Ranjbakhsh, Elnaz, E-mail: e.ranjbakhsh@yahoo.com; Khosropour, Ahmad-Reza, E-mail: khosropour@chem.ui.ac.ir

    2016-02-28

    Graphical abstract: - Highlights: • Modified iron oxide magnetic nanoparticles were synthesized by co-precipitation method and characterized by TEM and XRD. • Covalent attachment of GOX to MIMNs was confirmed by FT-IR technique. • Optimization of the reaction time and initial amount of the GOX were carried out. • Improvement of activity and stability of immobilized GOX have been increased in comparison of free GOX. - Abstract: Immobilized proteins and enzymes are widely investigated in the medical field as well as the food and environmental fields. In this study, glucose oxidase (GOX) was covalently immobilized on the surface of modified iron oxide magnetic nanoparticles (MIMNs) to produce a bioconjugate complex. Transmission electron microscopy (TEM) and X-ray diffraction (XRD) were used to the size, shape and structure characterization of the MIMNs. Binding of GOX to these MIMNs was confirmed by using FT-IR spectroscopy. The stability of the immobilized and free enzyme at different temperature and pH values was investigated by measuring the enzymatic activity. These studies reveal that the enzyme's stability is enhanced by immobilization. Further experiments showed that the storage stability of the enzyme is improved upon binding to the MIMNs. The results of kinetic measurements suggest that the effect of the immobilization process on substrate and product diffusion is small. Such bioconjugates can be considered as a catalytic nanodevice for accelerating the glucose oxidation reaction for biotechnological purposes.

  12. Pilot plant production of glucose from starch with soluble. cap alpha. -amylase and immobilized glucoamylase

    Energy Technology Data Exchange (ETDEWEB)

    Lee, D D; Reilly, P J; Collins, Jr, E V

    1975-01-01

    Pilot plant studies were conducted on cooking and thinning of corn starch with free ..cap alpha..-amylase and the conversion of the resulting dextrin with immobilized glucoamylase adsorbed on porous SiO/sub 2/. Feeds of intermediate DE values gave maximum yields unless the flow rate of low DE feeds was decreased. Final DE values and glucose concentrations after dextrin treated with Thermamyl 60 ..cap alpha..-amylase had been further hydrolyzed in an immobilized glucoamylase column, were slightly lower than they were when free glucoamylase was used. Similar results were obtained when dextrin, thinned with HT-1000 ..cap alpha..-amylase, was hydrolyzed at 38/sup 0/ and pH 4.4 in the immobilized glucoamylase column. Free glucoamylase yielded values of DE and glucose almost identical with dextrin thinned with Thermamyl 60 ..cap alpha..-amylase. Yields with the free glucoamylase were also slightly higher than they were with SiO/sub 2/-bound enzyme.

  13. Immobilization of the Enzyme Glucose Oxidase on Both Bulk and Porous SiO2 Surfaces

    Directory of Open Access Journals (Sweden)

    Fulvia Sinatra

    2008-09-01

    Full Text Available Silicon dioxide surfaces, both bulk and porous, were used to anchor the enzyme glucose oxidase. The immobilization protocol was optimized and the samples characterized using X-ray Photoelectron Spectroscopy, Energy Dispersive X-rays coupled to scanning electron microscopy and enzymatic activity measurements. We show that a uniform layer was obtained by activating the oxide before immobilization. X-ray Photoelectron Spectroscopy measurements carried out on bulk oxide showed that the silicon substrate signal was fully screened after the enzyme deposition showing the absence of uncovered surface regions. The enzyme presence was detected monitoring both the C 1s and N 1s signals. Finally, enzymatic activity measurements confirmed that the glucose oxidase activity was preserved after immobilization and maintained after three months of shelf life if the sample was properly stored. The importance of using porous silicon oxide to maximize the surface area was also evidenced.

  14. 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)

  15. 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.

  16. 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

  17. Significance of membrane bioreactor design on the biocatalytic performance of glucose oxidase and catalase: Free vs. immobilized enzyme systems

    DEFF Research Database (Denmark)

    Morthensen, Sofie Thage; Meyer, Anne S.; Jørgensen, Henning

    2017-01-01

    Membrane separation of xylose and glucose can be accomplished via oxidation of glucose to gluconic acid by enzymatic glucose oxidase catalysis. Oxygen for this reaction can be supplied via decomposition of hydrogen peroxide by enzymatic catalase catalysis. In order to maximize the biocatalytic...... productivity of glucose oxidase and catalase (gluconic acid yield per total amount of enzyme) the following system set-ups were compared: immobilization of glucose oxidase alone; co-immobilization of glucose oxidase and catalase; glucose oxidase and catalase free in the membrane bioreactor. Fouling......-induced enzyme immobilization in the porous support of an ultrafiltration membrane was used as strategy for entrapment of glucose oxidase and catalase. The biocatalytic productivity of the membrane reactor was found to be highly related to the oxygen availability, which in turn depended on the reactor...

  18. Effective immobilization of glucose oxidase on chitosan submicron particles from gladius of Todarodes pacificus for glucose sensing.

    Science.gov (United States)

    Anusha, J R; Fleming, Albin T; Kim, Hee-Je; Kim, Byung Chul; Yu, Kook-Hyun; Raj, C Justin

    2015-08-01

    An effective enzymatic glucose biosensor was developed by immobilizing glucose oxidase on chitosan submicron particles synthesized from the gladius of Todarodes pacificus (GCSP). The chemically synthesized chitosan from gladius was pulverized to submicron particles by ball milling technique, which was further characterized and compared with the standard chitosan (SCS). The degree of deacetylation of GCSP was determined using FTIR spectroscopy which was comparable to the value of standard chitosan. The glucose oxidase (GOx) was immobilized over GCSP on porous zinc oxide/platinum nanoparticle (ZnO/Pt) based electrode. The morphological and structural properties of the electrodes were analyzed using scanning electron microscopy and X-ray diffraction analysis. The glucose sensing behavior of electrode was estimated using electrochemical analysis and showed an excellent analytical performance. The electrode ZnO/Pt/GCSP conjugated with GOx displayed high sensitivity (88.76 μA mM(-1) cm(-2)) with low detection limit in short response time. In addition, the very low value of Michaelis-Menten constant for GCSP based electrode contributes a better affinity of the electrode surface towards glucose oxidase. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Improvement of the stability and activity of immobilized glucose oxidase on modified iron oxide magnetic nanoparticles

    Science.gov (United States)

    Abbasi, Mahboube; Amiri, Razieh; Bordbar, Abdol-Kalegh; Ranjbakhsh, Elnaz; Khosropour, Ahmad-Reza

    2016-02-01

    Immobilized proteins and enzymes are widely investigated in the medical field as well as the food and environmental fields. In this study, glucose oxidase (GOX) was covalently immobilized on the surface of modified iron oxide magnetic nanoparticles (MIMNs) to produce a bioconjugate complex. Transmission electron microscopy (TEM) and X-ray diffraction (XRD) were used to the size, shape and structure characterization of the MIMNs. Binding of GOX to these MIMNs was confirmed by using FT-IR spectroscopy. The stability of the immobilized and free enzyme at different temperature and pH values was investigated by measuring the enzymatic activity. These studies reveal that the enzyme's stability is enhanced by immobilization. Further experiments showed that the storage stability of the enzyme is improved upon binding to the MIMNs. The results of kinetic measurements suggest that the effect of the immobilization process on substrate and product diffusion is small. Such bioconjugates can be considered as a catalytic nanodevice for accelerating the glucose oxidation reaction for biotechnological purposes.

  20. Immobilization of glucose oxidase on sepharose by UV-initiated graft copolymerization

    International Nuclear Information System (INIS)

    D'Angiuro, L.; Cremonesi, P.

    1982-01-01

    The performance of a new method of enzyme immobilization based on photochemically initiated direct graft copolymerization was recently investigated. The immobilization reaction can be carried out in a simple way and by carefully selecting the reaction conditions, the enzyme-graft copolymer can be obtained as the main reaction product. Coupling efficiency of glucose oxidase has been found to depend only on the amount of photocatalyst (FeCl 3 ) fixed on Sepharose used as polysaccharide support. Small quantities of glycidylmethacrylate (GMA) (0.25 g/g dry Sepharose) are sufficient but necessary to achieve the best enzyme coupling efficiency (20-40%). Enzyme immobilization occurs very rapidly and the entire reaction occurs within 60 min. Reaction patterns and physicochemical characteristics of the obtained enzyme-graft copolymers exclude the glucose oxidase entrapment: therefore a covalent attachment mechanism may be proposed. The kinetic parameters of immobilized glucose oxidase (K/sub m/' = 2.0 x 10 -2 M) are quite similar to those of free enzyme (K/sub m/ = 1.93 x 10 -2 M), and no diffusion limitation phenomena are evidenced in samples having different enzyme or polymer content. Lyophilization, thermostability, and long-term continuous operation also have been investigated. The advantages of this method over that using vinylenzyme copolymerization are discussed

  1. Carbon fiber microelectrodes modified with carbon nanotubes as a new support for immobilization of glucose oxidase

    International Nuclear Information System (INIS)

    Wen, H.; Nallathambi, V.; Chakraborty, D.; Barton, S.C.

    2011-01-01

    Carboxylated carbon nanotubes were coated onto carbon microfiber electrodes to create a micron-scale bioelectrode. This material has a high surface area and can serve as a support for immobilization of enzymes such as glucose oxidase. A typical carbon nanotube loading of 13 μg cm -1 yields a coating thickness of 17 μm and a 2000-fold increase in surface capacitance. The modified electrode was further coated with a biocatalytic hydrogel composed of a conductive redox polymer, glucose oxidase, and a crosslinker to create a glucose bioelectrode. The current density on oxidation of glucose is 16.6 mA cm-2 at 0.5 V (vs. Ag/AgCl) in oxygen-free glucose solution. We consider this approach to be useful for designing and characterizing surface treatments for carbon mats and papers by mimicking their local microenvironment. (author)

  2. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Chitosan coated on the layers' glucose oxidase immobilized on cysteamine/Au electrode for use as glucose biosensor.

    Science.gov (United States)

    Zhang, Yawen; Li, Yunqiu; Wu, Wenjian; Jiang, Yuren; Hu, Biru

    2014-10-15

    A glucose biosensor was developed via direct immobilization of glucose oxidase (GOD) by self-assembled cysteamine monolayer on Au electrode surface followed by coating chitosan on the surface of electrode. In this work, chitosan film was coated on the surface of GOD as a protection film to ensure the stability and biocompatibility of the constructed glucose biosensor. The different application ranges of sensors were fabricated by immobilizing varied layers of GOD. The modified surface film was characterized by a scanning electron microscope (SEM) and the fabrication process of the biosensor was confirmed through electrochemical impedance spectroscopy (EIS) of ferrocyanide. The performance of cyclic voltammetry (CV) in the absence and presence of 25 mM glucose and ferrocenemethanol showed a diffusion-controlled electrode process and reflected the different maximum currents between the different GOD layers. With the developed glucose biosensor, the detection limits of the two linear responses are 49.96 μM and 316.8 μM with the sensitivities of 8.91 μA mM(-1)cm(-2) and 2.93 μA mM(-1)cm(-2), respectively. In addition, good stability (up to 30 days) of the developed biosensor was observed. The advantages of this new method for sensors construction was convenient and different width ranges of detection can be obtained by modified varied layers of GOD. The sensor with two layers of enzyme displayed two current linear responses of glucose. The present work provided a simplicity and novelty method for producing biosensors, which may help design enzyme reactors and biosensors in the future. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. 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.

  6. Involvement of α(2)-adrenergic receptor in the regulation of the blood glucose level induced by immobilization stress.

    Science.gov (United States)

    Kang, Yu-Jung; Sim, Yun-Beom; Park, Soo-Hyun; Sharma, Naveen; Suh, Hong-Won

    2015-01-01

    The blood glucose profiles were characterized after mice were forced into immobilization stress with various exposure durations. The blood glucose level was significantly enhanced by immobilization stress for 30 min or 1 h, respectively. On the other hand, the blood glucose level was not affected in the groups which were forced into immobilization stress for 2 or 4 h. We further examined the effect of yohimbine (an α2-adrenergic receptor antagonist) administered systemically or centrally in the immobilization stress model. Mice were pretreated intraperitoneally (i.p.; from 0.5 to 5 mg/kg), intracerebroventricularly (i.c.v.; from 1 to 10 µg/5 µl), or intrathecally (i.t.; from 1 to 10 µg/5 µl) with yohimbine for 10 min and then, forced into immobilization stress for 30 min. The blood glucose level was measured right after immobilization stress. We found that up-regulation of the blood glucose level induced by immobilization stress was abolished by i.p. pretreatment with yohimbine. And the immobilization stress-induced blood glucose level was not inhibited by i.c.v. or i.t. pretreatment with yohimbine at a lower dose (1 µg/5 µl). However, immobilization stress-induced blood glucose level was significantly inhibited by i.c.v. or i.t. pretreatment with yohimbine at higher doses (5 and 10 µg/5 µl). In addition, the i.p. (5 mg/kg), i.c.v. (10 µg/5 µl), or i.t. (10 µg/5 µl) pretreatment with yohimbine reduced hypothalamic glucose transporter 4 expression. The involvement of α2-adrenergic receptor in regulation of immobilization stress- induced blood glucose level was further confirmed by the i.p, i.c.v, or i.t pretreatment with idazoxan, another specific α2-adrenergic receptor antagonist. Finally, i.p., i.c.v., or i.t. pretreatment with yohimbine attenuated the blood glucose level in D-glucose-fed model. We suggest that α2-adrenergic receptors located at the peripheral, the brain and the spinal cord play important roles in the up

  7. Electrochemistry of glucose oxidase immobilized on the carbon nanotube wrapped by polyelectrolyte

    International Nuclear Information System (INIS)

    Wen, Dan; Liu, Ying; Yang, Guocheng; Dong, Shaojun

    2007-01-01

    A more stably dispersing of multi-wall carbon nanotube composite (noted as PDDA-MWNT), which was obtained by wrapping the MWNT with poly(diallydimethylammonium) chloride (PDDA), was used for the immobilization of glucose oxidase (GOD) and its bioelectrochemical studies. The morphologies and structures of the PDDA-MWNT composite were characterized by environment scanning electron microscopy (ESEM) and X-ray photoelectron spectroscopy (XPS). Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry were used to feature the GOD adsorbed onto the electrode modified by PDDA-MWNT composite. The immobilized GOD at the PDDA-MWNT films exhibited a pair of well-defined nearly reversible redox peaks and a fast heterogeneous electron transfer rate with the rate constant (k s ) of 2.76 s -1 . In addition, GOD immobilized in this way retained its bioelectrocatalytic activity for the oxidation of glucose. The method of immobilizing GOD without any additional cross-linking agents presented here is easy and facile, which provides a model for other redox enzymes and proteins

  8. Immobilization of glucose oxidase to nanostructured films of polystyrene-block-poly(2-vinylpyridine).

    Science.gov (United States)

    Bhakta, Samir A; Benavidez, Tomas E; Garcia, Carlos D

    2014-09-15

    A critical step for the development of biosensors is the immobilization of the biorecognition element to the surface of a substrate. Among other materials that can be used as substrates, block copolymers have the untapped potential to provide significant advantages for the immobilization of proteins. To explore such possibility, this manuscript describes the fabrication and characterization of thin-films of polystyrene-block-poly(2-vinylpyridine) (PS-b-P2VP). These films were then used to investigate the immobilization of glucose oxidase, a model enzyme for the development of biosensors. According to the results presented, the nanoporous films can provide significant increases in surface area of the substrate and the immobilization of larger amounts of active enzyme. The characterization of the substrate-enzyme interface discussed in the manuscript aims to provide critical information about relationship between the surface (material, geometry, and density of pores), the protein structure, and the immobilization conditions (pH, and protein concentration) required to improve the catalytic activity and stability of the enzymes. A maximum normalized activity of 3300±700 U m(-2) was achieved for the nanoporous film of PS-b-P2VP. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. 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.

  10. Development of glucose biosensor based on ZnO nanoparticles film and glucose oxidase-immobilized eggshell membrane

    Directory of Open Access Journals (Sweden)

    Bohari Noor Aini

    2015-06-01

    Full Text Available A novel electrochemical glucose biosensor was developed by depositing an ionic liquid (IL (e.g., 1-ethyl-3-methylimidazolium trifluoromethanesulfonate; [EMIM][Otf], ZnO nanoparticles (ZnONPs and eggshell membrane (ESM on a modified glassy carbon electrode (GCE for determination of glucose. Glucose oxidase (GOx was covalently immobilized on eggshell membrane with glutaraldehyde as a cross-linker. Methylene blue was used as a redox indicator to enhance the electron transfer capacity and to ensure stability of both the oxidized and reduced forms in the reaction of enzyme and substrate. The morphological characteristics of microstructures eggshell membranes, chitosan, GOx/ESM, GOx/ZnONPs/IL/ESM and GOx/ZnONPs-IL/CHIT were observed using scanning electron microscopy (SEM. The effects of scan rate, time and pH on the response of glucose biosensors were studied in detail. Under optimal conditions (pH 6.5, 50 s, cyclic voltammetry showed different glucose concentrations on the range of 1 × 10−12 to 0.6 M, with a detection limit of 1 × 10−13 M. The GOx/ZnONPs/IL/ESM was found to be more sensitive as compared to GOx/ZnONPs-IL/CHIT. This developed glucose biosensor detection approach has several advantages such as fast, simple and convenient method, sensitivity, low cost, eco-friendly, low concentrations and remarkable catalytic activities of current signals during glucose reaction.

  11. Immobilized glucose oxidase--catalase and their deactivation in a differential-bed loop reactor.

    Science.gov (United States)

    Prenosil, J E

    1979-01-01

    Glucose oxidase containing catalase was immobilized with a copolymer of phenylenediamine and glutaraldehyde on pumice and titania carrier to study the enzymatic oxidation of glucose in a differential-bed loop reactor. The reaction rate was found to be first order with respect to the concentration of limiting oxygen substrate, suggesting a strong external mass-transfer resistance for all the flow rates used. The partial pressure of oxygen was varied from 21.3 up to 202.6 kPa. The use of a differential-bed loop reactor for the determination of the active enzyme concentration in the catalyst with negligible internal pore diffusion resistance is shown. Catalyst deactivation was studied, especially with respect to the presence of catalase. It is believed that the hydrogen peroxide formed in the oxidation reaction deactivates catalase first; if an excess of catalase is present, the deactivation of glucose oxidase remains small. The mathematical model subsequently developed adequately describes the experimental results.

  12. Immobilization of Glucose Oxidase on a Carbon Nanotubes/Dendrimer-Ferrocene Modified Electrode for Reagentless Glucose Biosensing.

    Science.gov (United States)

    Zhou, Juan; Li, Huan; Yang, Huasong; Cheng, Hui; Lai, Guosong

    2017-01-01

    Ferrocene-grafted dendrimer was covalently linked to the surface of a carbon nanotubes (CNTs)-chitosan (CS) nanocomposite modified electrode for immobilizing high-content glucose oxidase (GOx), which resulted in the successful development a novel reagentless glucose biosensor. Electrochemical impedance spectroscopy, cyclic voltammetry, and amperometry were used to characterize the preparation process and the enzymatically catalytic response of this biosensor. Due to the excellent electron transfer acceleration of the CNTs and the high-content loading of the GOx biomolecule and ferrocene mediator on the electrode matrix, this biosensor showed excellent analytical performance such as fast response time less than 10 s, wide linear range from 0.02 to 2.91 mM and low detection limit down to 7.5 μM as well as satisfactory stability and reproducibility toward the amperometric glucose determination. In addition, satisfactory result was obtained when it was used for the glucose measurements in human blood samples. Thus this biosensor provides great potentials for practical applications.

  13. Immobilization of glucose oxidase on graphene and cobalt phthalocyanine composite and its application for the determination of glucose.

    Science.gov (United States)

    Mani, Veerappan; Devasenathipathy, Rajkumar; Chen, Shen-Ming; Huang, Sheng-Tung; Vasantha, V S

    2014-11-01

    We described a simple and facile chemical reduction strategy for the preparation of graphene (GR)-cobalt phthalocyanine (CoPc) composite and explored it for the enzymatic determination of glucose. CoPc is an active mediator and electrocatalysts for the immobilization of GOx and determination of glucose. However, it is not stable on the electrode surface and also suffers from lack of conductivity. Here, we have employed GR as the suitable support to stabilize CoPc through simple chemical reduction method and the resulting composite has been used for the glucose biosensor application. Scanning electron microscopy, X-ray diffraction and Energy-dispersive X-ray spectroscopy studies confirmed the successful formation of composite. Direct electron transfer of glucose oxidase (GOx) was observed with well defined redox peaks at the formal potential of -0.44 V. The amount of electroactive GOx (Г) and electron transfer rate constant (ks) were calculated to be 3.77×10(-10) mol cm(-2) and 3.57 s(-1), respectively. The fabricated amperometric biosensor detects glucose in wide linear concentration range from 10 μM to 14.8 mM with high sensitivity of 5.0 9μA mM(-1) cm(-2). The sensor offered very low detection limit (LOD) of 1.6 μM. In addition, practical feasibility of the sensor has been explored in screen printing carbon electrode with accurate determination of glucose present in human blood serum and urine samples. Furthermore, the sensor exhibited appreciable stability, repeatability and reproducibility results. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Effect of immobilization technique on performance ZnO nanorods based enzymatic electrochemical glucose biosensor

    Science.gov (United States)

    Shukla, Mayoorika; Pramila; Palani, I. A.; Singh, Vipul

    2017-11-01

    In this paper, ZnO Nanorods (ZNR) have been synthesized over Platinum (Pt) coated glass substrate with in-situ addition KMnO4 during hydrothermal growth process. Significant variation in ZnO nanostructures was observed by KMnO4 addition during the growth. Glucose oxidase was later immobilized over ZNRs. The as-prepared ZNRs were further utilized for glucose detection by employing amperometric electrochemical transduction method. In order to optimize the performance of the prepared biosensor two different immobilization techniques i.e. physical adsorption and cross linking have been employed and compared. Further investigations suggest that immobilization via cross linking method resulted in the improvement of the biosensor performance, thereby significantly affecting the sensitivity and linear range of the fabricated biosensor. Among the two types of biosensors fabricated using ZNR, the best performance was shown by cross linked electrodes. The sensitivity for the same was found to be 17.7 mA-cm-2-M-1, along with a wide linear range of 0.5-8.5 mM.

  15. Immobilized glucose oxidase by radiation induced polymerization of HEMA at low temperature

    International Nuclear Information System (INIS)

    Cao Jin; Su Zongxian

    1988-01-01

    The immobilized glucose oxidase (GOD) by 60 Co-γ induced polymerization of hydroxyethyl methacrylate (HEMA) at -78 deg C was studied. From the experiment results, it was found that the irradation dose until 1 x 10 4 Gy had not a significant effect on the native GOD activity. When the carrier (HEMA) concentration was 50% and the entrapped amount was 1.0 ml GOD/10 ml phosphoric acid buffer solution, the immobilized GOD had not only elastic, but also had high remaining activity. The native GOD was less sensitive to pH value than the immobilized GOD, but both the proper pH values didn't change. The kinetic reaction results showed, Michaelis constant k'm=1.42 x 10 -2 mol (native GOD km=1.0 x 10 -2 mol). This value indicated that diffuse velocity of substitue was restricted. The activation energies of the immobilized GOD were found to be 13.7kJ/mol

  16. 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

  17. Immobilization of Glucose Oxidase on Modified-Carbon-Paste-Electrodes for Microfuel Cell

    Directory of Open Access Journals (Sweden)

    Laksmi Ambarsari

    2016-03-01

    Full Text Available Glucose oxidase (GOx is being developed for many applications such as an implantable fuel cell, due to its attractive property of operating under physiological conditions. This study reports the functional immobilization of glucose oxidase onto polyaniline-nanofiber-modified-carbon-paste-electrodes (GOx/MCPE as bioanodes in fuel cell applications. In particular, GOx is immobilized onto the electrode surface via a linker molecule (glutaraldehyde. Polyaniline, synthesized by the interfacial polymerization method, produces a morphological form of nanofibers (100-120 nm which have good conductivity. The performance of the polyaniline-modified-carbon-paste-electrode (MCPE was better than the carbon- paste-electrode (CPE alone. The optimal pH and temperature of the GOx/MCPE were 4.5 (in 100 mM acetate buffer and 65 °C, respectively. The GOx/MCPE exhibit high catalytic performances (activation energy 16.4 kJ mol-1, have a high affinity for glucose (Km value 37.79 µM and can have a maximum current (Imax of 3.95 mA. The sensitivity of the bioelectrode also was high at 57.79 mA mM-1 cm-2.

  18. 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.

  19. Oxidative polymerization of 5-hydroxytryptamine to physically and chemically immobilize glucose oxidase for electrochemical biosensing.

    Science.gov (United States)

    Huang, Ting; Liu, Zaichun; Li, Yunlong; Li, Yanqiu; Chao, Long; Chen, Chao; Tan, Yueming; Xie, Qingji; Yao, Shouzhuo; Wu, Yuping

    2018-07-12

    Poly(5-hydroxytryptamine) (poly(5-HT)) is exploited as a new and efficient enzyme-immobilization matrix for amperometric and biofuel cell (BFC)-based biosensing. A GOx-poly(5-HT)-Pd nanoparticles (PdNPs) bionanocomposite is prepared by Na 2 PdCl 4 -initiated oxidized polymerization of 5-hydroxytryptamine (5-HT) in a neutral aqueous solution containing glucose oxidase (GOx), and this bionanocomposite and then chitosan (CS) are cast-coated on a Pd-plated Au electrode to yield a CS/GOx-poly(5-HT)-PdNPs/Pd plate /Au enzyme electrode. Scanning/transmission electron microscopy, UV-vis spectrophotometry and electrochemical quartz crystal microbalance are employed for material characterization and/or process monitoring. Under optimized conditions, the amperometric response of the enzyme electrode is linear with glucose concentration from 2.0 μM to 6.66 mM with a sensitivity of 110 μA mM -1  cm -2 , a limit of detection of 0.2 μM, and excellent operation/storage stability in the first-generation biosensing mode. The sensitivity is larger than those of some conventional electrodes under identical conditions. The enzyme electrode also works well in the second-generation biosensing mode. By using the enzyme electrode as the anode for glucose oxidation and a Pd plate /Au electrode as the cathode for KMnO 4 reduction, a monopolar BFC is constructed as a self-powered biosensor, the current response of which is linear with glucose concentration from 50 μM to 34.5 mM. Experiments also show that poly(5-HT) is a physical and chemical dual-immobilization matrix of enzyme, since the abundant amino groups in poly(5-HT) can be used for chemical bonding of GOx. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. Direct electrochemical sensing of glucose using glucose oxidase immobilized on functionalized carbon nanotubes via a novel metal chelate-based affinity method

    International Nuclear Information System (INIS)

    Tu, X.; Zhao, Y.; Luo, S.; Luo, X.; Feng, L.

    2012-01-01

    We report on a novel amperometric glassy carbon biosensing electrode for glucose. It is based on the immobilization of a highly sensitive glucose oxidase (GOx) by affinity interaction on carbon nanotubes (CNTs) functionalized with iminodiacetic acid and metal chelates. The new technique for immobilization is exploiting the affinity of Co(II) ions to the histidine and cysteine moieties on the surface of GOx. The direct electrochemistry of immobilized GOx revealed that the functionalized CNTs greatly improve the direct electron transfer between GOx and the surface of the electrode to give a pair of well-defined and almost reversible redox peaks and undergoes fast heterogeneous electron transfer with a rate constant (k s) of 0. 59 s -1 . The GOx immobilized in this way fully retained its activity for the oxidation of glucose. The resulting biosensor is capable of detecting glucose at levels as low as 0.01 mM, and has excellent operational stability (with no decrease in the activity of enzyme over a 10 days period). The method of immobilizing GOx is easy and also provides a model technique for potential use with other redox enzymes and proteins. (author)

  1. 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.

  2. Surface modification of polyvinyl alcohol/malonic acid nanofibers by gaseous dielectric barrier discharge plasma for glucose oxidase immobilization

    International Nuclear Information System (INIS)

    Afshari, Esmail; Mazinani, Saeedeh; Ranaei-Siadat, Seyed-Omid; Ghomi, Hamid

    2016-01-01

    Highlights: • We fabricated polyvinyl alcohol/malonic acid nanofibers using electrospinning. • The surface nanofibers were modified by gaseous (air, nitrogen, CO_2 and argon) dielectric barrier discharge. • Among them, air plasma had the most significant effect on glucose oxidase immobilization. • Chemical analysis showed that after modification of nanofibers by air plasma, the carboxyl group increased. • After air plasma treatment, reusability and storage stability of glucose oxidase immobilized on nanofibers improved. - Abstract: Polymeric nanofiber prepares a suitable situation for enzyme immobilization for variety of applications. In this research, we have fabricated polyvinyl alcohol (PVA)/malonic acid nanofibers using electrospinning. After fabrication of nanofibers, the effect of air, nitrogen, CO_2, and argon DBD (dielectric barrier discharge) plasmas on PVA/malonic acid nanofibers were analysed. Among them, air plasma had the most significant effect on glucose oxidase (GOx) immobilization. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectrum analysis and X-ray photoelectron spectroscopy (XPS) results revealed that in case of air plasma modified nanofibers, the carboxyl groups on the surface are increased. The scanning electron microscopy (SEM) images showed that, after GOx immobilization, the modified nanofibers with plasma has retained its nanofiber structure. Finally, we analysed reusability and storage stability of GOx immobilized on plasma modified and unmodified nanofibers. The results were more satisfactory for modified nanofibers with respect to unmodified ones.

  3. Surface modification of polyvinyl alcohol/malonic acid nanofibers by gaseous dielectric barrier discharge plasma for glucose oxidase immobilization

    Energy Technology Data Exchange (ETDEWEB)

    Afshari, Esmail, E-mail: e.afshari@mail.sbu.ac.ir [Laser and Plasma Research Institute, Shahid Beheshti University, Evin, 1983963113 Tehran (Iran, Islamic Republic of); Mazinani, Saeedeh [Amirkabir Nanotechnology Research Institute (ANTRI), Amirkabir University of Technology, 15875-4413, Tehran (Iran, Islamic Republic of); Ranaei-Siadat, Seyed-Omid [Protein Research Center, Shahid Beheshti University, Evin, 1983963113 Tehran (Iran, Islamic Republic of); Ghomi, Hamid [Laser and Plasma Research Institute, Shahid Beheshti University, Evin, 1983963113 Tehran (Iran, Islamic Republic of)

    2016-11-01

    Highlights: • We fabricated polyvinyl alcohol/malonic acid nanofibers using electrospinning. • The surface nanofibers were modified by gaseous (air, nitrogen, CO{sub 2} and argon) dielectric barrier discharge. • Among them, air plasma had the most significant effect on glucose oxidase immobilization. • Chemical analysis showed that after modification of nanofibers by air plasma, the carboxyl group increased. • After air plasma treatment, reusability and storage stability of glucose oxidase immobilized on nanofibers improved. - Abstract: Polymeric nanofiber prepares a suitable situation for enzyme immobilization for variety of applications. In this research, we have fabricated polyvinyl alcohol (PVA)/malonic acid nanofibers using electrospinning. After fabrication of nanofibers, the effect of air, nitrogen, CO{sub 2}, and argon DBD (dielectric barrier discharge) plasmas on PVA/malonic acid nanofibers were analysed. Among them, air plasma had the most significant effect on glucose oxidase (GOx) immobilization. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectrum analysis and X-ray photoelectron spectroscopy (XPS) results revealed that in case of air plasma modified nanofibers, the carboxyl groups on the surface are increased. The scanning electron microscopy (SEM) images showed that, after GOx immobilization, the modified nanofibers with plasma has retained its nanofiber structure. Finally, we analysed reusability and storage stability of GOx immobilized on plasma modified and unmodified nanofibers. The results were more satisfactory for modified nanofibers with respect to unmodified ones.

  4. Immobilization of glucose oxidase using CoFe2O4/SiO2 nanoparticles as carrier

    Science.gov (United States)

    Wang, Hai; Huang, Jun; Wang, Chao; Li, Dapeng; Ding, Liyun; Han, Yun

    2011-04-01

    Aminated-CoFe2O4/SiO2 magnetic nanoparticles (NPs) were prepared from primary silica particles using modified StÖber method. Glucose oxidase (GOD) was immobilized on CoFe2O4/SiO2 NPs via cross-linking with glutaraldehyde (GA). The optimal immobilization condition was achieved with 1% (v/v) GA, cross-linking time of 3 h, solution pH of 7.0 and 0.4 mg GOD (in 3.0 mg carrier). The immobilized GOD showed maximal catalytic activity at pH 6.5 and 40 °C. After immobilization, the GOD exhibited improved thermal, storage and operation stability. The immobilized GOD still maintained 80% of its initial activity after the incubation at 50 °C for 25 min, whereas free enzyme had only 20% of initial activity after the same incubation. After kept at 4 °C for 28 days, the immobilized and free enzyme retained 87% and 40% of initial activity, respectively. The immobilized GOD maintained approximately 57% of initial activity after reused 7 times. The KM (Michaelis-Menten constant) values for immobilized GOD and free GOD were 14.6 mM and 27.1 mM, respectively.

  5. Direct electron transfer from glucose oxidase immobilized on a nano-porous glassy carbon electrode

    International Nuclear Information System (INIS)

    Haghighi, Behzad; Tabrizi, Mahmoud Amouzadeh

    2011-01-01

    Highlights: → A direct electron transfer reaction of glucose oxidase was observed on the surface of a nano-porous glassy carbon electrode. → A pair of well-defined and reversible redox peaks was observed at the formal potential of approximately -0.439 V. → The apparent electron transfer rate constant was measured to be 5.27 s -1 . → A mechanism for the observed direct electron transfer reaction was proposed, which consists of a two-electron and a two-proton transfer. - Abstract: A pair of well-defined and reversible redox peaks was observed for the direct electron transfer (DET) reaction of an immobilized glucose oxidase (GOx) on the surface of a nano-porous glassy carbon electrode at the formal potential (E o ') of -0.439 V versus Ag/AgCl/saturated KCl. The electron transfer rate constant (k s ) was calculated to be 5.27 s -1 . The dependence of E o ' on pH indicated that the direct electron transfer of the GOx was a two-electron transfer process, coupled with two-proton transfer. The results clearly demonstrate that the nano-porous glassy carbon electrode is a cost-effective and ready-to-use scaffold for the fabrication of a glucose biosensor.

  6. Direct electron transfer from glucose oxidase immobilized on a nano-porous glassy carbon electrode

    Energy Technology Data Exchange (ETDEWEB)

    Haghighi, Behzad, E-mail: haghighi@iasbs.ac.ir [Department of Chemistry, Institute for Advanced Studies in Basic Sciences, P.O. Box 45195-1159, Gava Zang, Zanjan (Iran, Islamic Republic of); Tabrizi, Mahmoud Amouzadeh [Department of Chemistry, Institute for Advanced Studies in Basic Sciences, P.O. Box 45195-1159, Gava Zang, Zanjan (Iran, Islamic Republic of)

    2011-11-30

    Highlights: > A direct electron transfer reaction of glucose oxidase was observed on the surface of a nano-porous glassy carbon electrode. > A pair of well-defined and reversible redox peaks was observed at the formal potential of approximately -0.439 V. > The apparent electron transfer rate constant was measured to be 5.27 s{sup -1}. > A mechanism for the observed direct electron transfer reaction was proposed, which consists of a two-electron and a two-proton transfer. - Abstract: A pair of well-defined and reversible redox peaks was observed for the direct electron transfer (DET) reaction of an immobilized glucose oxidase (GOx) on the surface of a nano-porous glassy carbon electrode at the formal potential (E{sup o}') of -0.439 V versus Ag/AgCl/saturated KCl. The electron transfer rate constant (k{sub s}) was calculated to be 5.27 s{sup -1}. The dependence of E{sup o}' on pH indicated that the direct electron transfer of the GOx was a two-electron transfer process, coupled with two-proton transfer. The results clearly demonstrate that the nano-porous glassy carbon electrode is a cost-effective and ready-to-use scaffold for the fabrication of a glucose biosensor.

  7. A multisyringe flow injection system with immobilized glucose oxidase based on homogeneous chemiluminescence detection

    International Nuclear Information System (INIS)

    Manera, Matias; Miro, Manuel; Estela, Jose Manuel; Cerda, Victor

    2004-01-01

    In this paper, enzyme containing reactors are for the first time implemented in the multisyringe flow injection analysis (MSFIA) technique interfaced with chemiluminescence detection for biochemical assays. The automated methodology is based on the on-line substrate conversion in an oxidase packed-bed reactor and the post-column chemiluminogenic catalysed-reaction of the generated oxidising species with an organic molecule (namely, 3-aminophthalhydrazide) in front of the photosensor module. Various catalysts in homogeneous phase are compared taking advantage of the benefits of the MSFIA concept. On one hand, mineral catalysts (namely, Co(II)) are assessed, on the other hand, minute and accurate volumes of soluble organic species (viz., horseradish peroxidase (HRP)) are readily handled without requiring further immobilization protocols. The potentials of the MSFIA-CL concept with immobilisation of the proper oxidase protein are demonstrated using glucose as a model of substrate. Despite the different pH and kinetic requirements for both the substrate conversion in the enzyme-reactor and the Co(II)/HRP-mediated luminol oxidation integrated in the flow system, the MSFIA approach warrants maximum yields owing to the independent optimisation of the physical and chemical parameters of the various reactions involved. Under the optimised configurations and experimental variables, dynamic working ranges from 2.5x10 -6 to 1.0x10 -3 mol l -1 glucose may be obtained for both detection schemes by proper photomultiplier gain selection. The detection and determination limits calculated at the 3σ and 10σ level were 8.6x10 -7 and 2.0x10 -6 mol l -1 glucose, respectively, for the Co(II)-luminol system, and 1.3x10 -6 and 2.3x10 -6 mol l -1 glucose, respectively, for the HRP-luminol procedure. The repeatability (n=10) at the 1.0x10 -5 mol l -1 level was slightly better for the Co(II)-catalysed reaction (2.5% versus 4.0%). The developed MSFIA-CL methodology was used for kinetic

  8. Preparation of immobilized glucose oxidase wafer enzyme on calcium-bentonite modified by surfactant

    Science.gov (United States)

    Widi, R. K.; Trisulo, D. C.; Budhyantoro, A.; Chrisnasari, R.

    2017-07-01

    Wafer glucose oxidase (GOx) enzymes was produced by addition of PAH (Poly-Allyamine Hydrochloride) polymer into immobilized GOx enzyme on modified-Tetramethylammonium Hydroxide (TMAH) 5%-calsium-bentonite. The use of surfactant molecul (TMAH) is to modify the surface properties and pore size distribution of the Ca-bentonite. These properties are very important to ensure GOx molecules can be bound on the Ca-bentonit surface to be immobilized. The addition of the polymer (PAH) is expected to lead the substrates to be adsorbed onto the enzyme. In this study, wafer enzymes were made in various concentration ratio (Ca-bentonite : PAH) which are 1:0, 1:1, 1:2 and 1:3. The effect of PAH (Poly-Allyamine Hydrochloride) polymer added with various ratios of concentrations can be shown from the capacitance value on LCR meter and enzyme activity using DNS method. The addition of the polymer (PAH) showed effect on the activity of GOx, it can be shown from the decreasing of capacitance value by increasing of PAH concentration.

  9. Radiation target analyses of free and immobilized glucose 6-phosphate dehydrogenase

    International Nuclear Information System (INIS)

    Kempner, E.S.; Miller, J.H.

    2010-01-01

    The sensitivity of the enzyme glucose 6-phosphate dehydrogenase to ionizing radiation was examined under several conditions, including the presence of several free-radical scavengers. The enzyme was also irradiated when covalently bound to polyacrylamide beads whose structure is very similar to the polypeptide backbone of proteins. All the enzyme forms were irradiated in the frozen state with high-energy electrons from a linear accelerator. Surviving enzyme activity and surviving monomers were determined; the data were analyzed by target theory. Free-radical scavengers reduced the radiation target size of both the activity and monomers of the free enzyme, but not that of the immobilized enzyme activity. The target size of the activity of the free enzyme was that of a dimer mass, but in the case of the immobilized enzyme it was equal to the smaller mass of the monomer. Free-radical scavengers reduce the target size by modifying radiation energy transfer. The target size of the polyacrylamide-bound enzyme activity was expected to be very large since the connection between polyacrylamide and protein is a peptide bond which permits transfer of radiation-deposited energy. Several explanations concerning energy transfer are suggested for this result.

  10. A glucose biosensor based on direct electron transfer of glucose oxidase immobilized onto glassy carbon electrode modified with nitrophenyl diazonium salt

    International Nuclear Information System (INIS)

    Nasri, Zahra; Shams, Esmaeil

    2013-01-01

    Graphical abstract: - Abstract: This study reports a novel, simple and fast approach for construction of a highly stable glucose biosensor based on the immobilization of glucose oxidase (GOx) onto a glassy carbon electrode (GCE) electrografted with 4-aminophenyl (AP) by diazonium chemistry. Aminophenyl was used as cross-linker for covalent attachment of glucose oxidase to the electrode surface. Cyclic voltammograms of the GOx-modified GCE in phosphate buffer solution exhibited a pair of well-defined redox peaks, attesting the direct electron transfer (DET) of GOx with the underlying electrode. The proposed biosensor could be used to detect glucose based on the consumption of O 2 with the oxidation of glucose catalyzed by GOx and exhibited a wide linear range of glucose from 0.05 mM to 4.5 mM and low detection limit of 10 μM. The surface coverage of active GOx, heterogeneous electron transfer rate constant (k s ) and Michaelis–Menten constant (K M ) of immobilized GOx were 1.23 × 10 −12 mol cm −2 , 4.25 s −1 and 2.95 mM, respectively. The great stability of this biosensor, technically simple and possibility of preparation at short period of time make this method suitable for fabrication of low-cost glucose biosensors

  11. 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.

  12. 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.

  13. Surface modification of polyvinyl alcohol/malonic acid nanofibers by gaseous dielectric barrier discharge plasma for glucose oxidase immobilization

    Science.gov (United States)

    Afshari, Esmail; Mazinani, Saeedeh; Ranaei-Siadat, Seyed-Omid; Ghomi, Hamid

    2016-11-01

    Polymeric nanofiber prepares a suitable situation for enzyme immobilization for variety of applications. In this research, we have fabricated polyvinyl alcohol (PVA)/malonic acid nanofibers using electrospinning. After fabrication of nanofibers, the effect of air, nitrogen, CO2, and argon DBD (dielectric barrier discharge) plasmas on PVA/malonic acid nanofibers were analysed. Among them, air plasma had the most significant effect on glucose oxidase (GOx) immobilization. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectrum analysis and X-ray photoelectron spectroscopy (XPS) results revealed that in case of air plasma modified nanofibers, the carboxyl groups on the surface are increased. The scanning electron microscopy (SEM) images showed that, after GOx immobilization, the modified nanofibers with plasma has retained its nanofiber structure. Finally, we analysed reusability and storage stability of GOx immobilized on plasma modified and unmodified nanofibers. The results were more satisfactory for modified nanofibers with respect to unmodified ones.

  14. A comparative study of enzyme immobilization strategies for multi-walled carbon nanotube glucose biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Jin; Jaroch, David; Rickus, Jenna L; Marshall Porterfield, D [Weldon School of Biomedical Engineering, Purdue University (United States); Claussen, Jonathan C; Ul Haque, Aeraj; Diggs, Alfred R [Physiological Sensing Facility, Bindley Bioscience Center and Birck Nanotechnology Center, Purdue University (United States); McLamore, Eric S [Department of Agricultural and Biological Engineering, University of Florida (United States); Calvo-Marzal, Percy, E-mail: porterf@purdue.edu [Department of Chemistry, Purdue University (United States)

    2011-09-02

    This work addresses the comparison of different strategies for improving biosensor performance using nanomaterials. Glucose biosensors based on commonly applied enzyme immobilization approaches, including sol-gel encapsulation approaches and glutaraldehyde cross-linking strategies, were studied in the presence and absence of multi-walled carbon nanotubes (MWNTs). Although direct comparison of design parameters such as linear range and sensitivity is intuitive, this comparison alone is not an accurate indicator of biosensor efficacy, due to the wide range of electrodes and nanomaterials available for use in current biosensor designs. We proposed a comparative protocol which considers both the active area available for transduction following nanomaterial deposition and the sensitivity. Based on the protocol, when no nanomaterials were involved, TEOS/GOx biosensors exhibited the highest efficacy, followed by BSA/GA/GOx and TMOS/GOx biosensors. A novel biosensor containing carboxylated MWNTs modified with glucose oxidase and an overlying TMOS layer demonstrated optimum efficacy in terms of enhanced current density (18.3 {+-} 0.5 {mu}A mM{sup -1} cm{sup -2}), linear range (0.0037-12 mM), detection limit (3.7 {mu}M), coefficient of variation (2%), response time (less than 8 s), and stability/selectivity/reproducibility. H{sub 2}O{sub 2} response tests demonstrated that the most possible reason for the performance enhancement was an increased enzyme loading. This design is an excellent platform for versatile biosensing applications.

  15. A comparative study of enzyme immobilization strategies for multi-walled carbon nanotube glucose biosensors

    International Nuclear Information System (INIS)

    Shi, Jin; Jaroch, David; Rickus, Jenna L; Marshall Porterfield, D; Claussen, Jonathan C; Ul Haque, Aeraj; Diggs, Alfred R; McLamore, Eric S; Calvo-Marzal, Percy

    2011-01-01

    This work addresses the comparison of different strategies for improving biosensor performance using nanomaterials. Glucose biosensors based on commonly applied enzyme immobilization approaches, including sol-gel encapsulation approaches and glutaraldehyde cross-linking strategies, were studied in the presence and absence of multi-walled carbon nanotubes (MWNTs). Although direct comparison of design parameters such as linear range and sensitivity is intuitive, this comparison alone is not an accurate indicator of biosensor efficacy, due to the wide range of electrodes and nanomaterials available for use in current biosensor designs. We proposed a comparative protocol which considers both the active area available for transduction following nanomaterial deposition and the sensitivity. Based on the protocol, when no nanomaterials were involved, TEOS/GOx biosensors exhibited the highest efficacy, followed by BSA/GA/GOx and TMOS/GOx biosensors. A novel biosensor containing carboxylated MWNTs modified with glucose oxidase and an overlying TMOS layer demonstrated optimum efficacy in terms of enhanced current density (18.3 ± 0.5 μA mM -1 cm -2 ), linear range (0.0037-12 mM), detection limit (3.7 μM), coefficient of variation (2%), response time (less than 8 s), and stability/selectivity/reproducibility. H 2 O 2 response tests demonstrated that the most possible reason for the performance enhancement was an increased enzyme loading. This design is an excellent platform for versatile biosensing applications.

  16. A comparative study of enzyme immobilization strategies for multi-walled carbon nanotube glucose biosensors

    Science.gov (United States)

    Shi, Jin; Claussen, Jonathan C.; McLamore, Eric S.; Haque, Aeraj ul; Jaroch, David; Diggs, Alfred R.; Calvo-Marzal, Percy; Rickus, Jenna L.; Porterfield, D. Marshall

    2011-09-01

    This work addresses the comparison of different strategies for improving biosensor performance using nanomaterials. Glucose biosensors based on commonly applied enzyme immobilization approaches, including sol-gel encapsulation approaches and glutaraldehyde cross-linking strategies, were studied in the presence and absence of multi-walled carbon nanotubes (MWNTs). Although direct comparison of design parameters such as linear range and sensitivity is intuitive, this comparison alone is not an accurate indicator of biosensor efficacy, due to the wide range of electrodes and nanomaterials available for use in current biosensor designs. We proposed a comparative protocol which considers both the active area available for transduction following nanomaterial deposition and the sensitivity. Based on the protocol, when no nanomaterials were involved, TEOS/GOx biosensors exhibited the highest efficacy, followed by BSA/GA/GOx and TMOS/GOx biosensors. A novel biosensor containing carboxylated MWNTs modified with glucose oxidase and an overlying TMOS layer demonstrated optimum efficacy in terms of enhanced current density (18.3 ± 0.5 µA mM - 1 cm - 2), linear range (0.0037-12 mM), detection limit (3.7 µM), coefficient of variation (2%), response time (less than 8 s), and stability/selectivity/reproducibility. H2O2 response tests demonstrated that the most possible reason for the performance enhancement was an increased enzyme loading. This design is an excellent platform for versatile biosensing applications.

  17. A preliminary investigation on the interaction between sol-gel immobilized glucose oxidase and freely diffusing glucose by means of two-photon microscopy

    Science.gov (United States)

    Delfino, I.; Portaccio, M.; De Rosa, M.; Lepore, M.

    2013-02-01

    To study immobilized protein interactions with dissolved substrates is a very important topic both from a fundamental and technological standpoint. In the present report we illustrate the preliminary results obtained on sol-gel immobilized glucose oxidase (GOD) using a standard de-scanned two-photon microscope based on a modified confocal scanhead with internal detectors and a Ti:sapphire laser as a source. Data acquisition conditions were preliminary defined using functionalized beads of different dimensions. Various sol-gel supports were then investigated by monitoring endogeneous fluorescence due to the flavoadenine (FAD) molecules, present in GOD. Linear absorption and fluorescence spectroscopy along with Fourier Transform Infrared microscopy were employed for a full-optical characterization of the samples. The results show that GOD immobilization processes can be successfully monitored in some cases and also the interaction with glucose could be studied by this approach. This assessment holds potentials to better understand the characteristic of immobilized enzymes biocatalysis and to develop new biosensing schemes.

  18. Controlled fabrication of gold nanoparticles biomediated by glucose oxidase immobilized on chitosan layer-by-layer films

    International Nuclear Information System (INIS)

    Caseli, Luciano; Santos, David S. dos; Aroca, Ricardo F.; Oliveira, Osvaldo N.

    2009-01-01

    The control of size and shape of metallic nanoparticles is a fundamental goal in nanochemistry, and crucial for applications exploiting nanoscale properties of materials. We present here an approach to the synthesis of gold nanoparticles mediated by glucose oxidase (GOD) immobilized on solid substrates using the Layer-by-Layer (LbL) technique. The LbL films contained four alternated layers of chitosan and poly(styrene sulfonate) (PSS), with GOD in the uppermost bilayer adsorbed on a fifth chitosan layer: (chitosan/PSS) 4 /(chitosan/GOD). The films were inserted into a solution containing gold salt and glucose, at various pHs. Optimum conditions were achieved at pH 9, producing gold nanoparticles of ca. 30 nm according to transmission electron microscopy. A comparative study with the enzyme in solution demonstrated that the synthesis of gold nanoparticles is more efficient using immobilized GOD.

  19. High-performance glucose biosensor based on chitosan-glucose oxidase immobilized polypyrrole/Nafion/functionalized multi-walled carbon nanotubes bio-nanohybrid film.

    Science.gov (United States)

    Shrestha, Bishnu Kumar; Ahmad, Rafiq; Mousa, Hamouda M; Kim, In-Gi; Kim, Jeong In; Neupane, Madhav Prasad; Park, Chan Hee; Kim, Cheol Sang

    2016-11-15

    A highly electroactive bio-nanohybrid film of polypyrrole (PPy)-Nafion (Nf)-functionalized multi-walled carbon nanotubes (fMWCNTs) nanocomposite was prepared on the glassy carbon electrode (GCE) by a facile one-step electrochemical polymerization technique followed by chitosan-glucose oxidase (CH-GOx) immobilization on its surface to achieve a high-performance glucose biosensor. The as-fabricated nanohybrid composite provides high surface area for GOx immobilization and thus enhances the enzyme-loading efficiency. The structural characterization revealed that the PPy-Nf-fMWCNTs nanocomposite films were uniformly formed on GCE and after GOx immobilization, the surface porosities of the film were decreased due to enzyme encapsulation inside the bio-nanohybrid composite materials. The electrochemical behavior of the fabricated biosensor was investigated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and amperometry measurements. The results indicated an excellent catalytic property of bio-nanohybrid film for glucose detection with improved sensitivity of 2860.3μAmM(-1)cm(-2), the linear range up to 4.7mM (R(2)=0.9992), and a low detection limit of 5μM under a signal/noise (S/N) ratio of 3. Furthermore, the resulting biosensor presented reliable selectivity, better long-term stability, good repeatability, reproducibility, and acceptable measurement of glucose concentration in real serum samples. Thus, this fabricated biosensor provides an efficient and highly sensitive platform for glucose sensing and can open up new avenues for clinical applications. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Surface modification of Fe_2O_3/Fe_3O_4 nanocomposites for use in immobilization of glucose oxidase

    International Nuclear Information System (INIS)

    Albuquerque, I.L.T.; Santos, P.T.A.; Costa, A.C.F.M.; Oliveira, L.S.C.

    2017-01-01

    The increase in the number of people with diabetes in recent years and the high cost-benefit ratio of the existing biosensor technology have increased the interest for the development of glucose detection biosensor based on immobilization of glucose-oxidase (GOD) mainly using magnetic nanoparticles. In this context, nanocomposites of Fe_2O_3/Fe_3O_4 were prepared by combustion reaction and their surface was functionalized with 3-aminopropyltriethoxysilane via silanization reaction and with chitosan via functionalization to obtain a hybrid material that was evaluated as possible GOD immobilizer. The samples were characterized by powder X-ray diffraction, Fourier transform infrared spectroscopy, thermogravimetry, scanning electron microscopy, transmission electron microscopy, magnetic properties and in vitro cytotoxicity. The results revealed that it was possible to obtain the ferrimagnetic composite, the surface modification reduced the saturation magnetization, but maintained the ferrimagnetic characteristics, and all samples were considered non-toxic. For preliminary testing of the GOD immobilization it was revealed that the nanocomposite modified with silane and chitosan showed the better result, about 2.7 mg of immobilized GOD for 100 mg of nanocomposite, which makes this material a potential alternative to manufacture GOD biosensors. (author)

  1. Flow-injection amperometric determination of glucose using a biosensor based on immobilization of glucose oxidase onto Au seeds decorated on core Fe₃O₄ nanoparticles.

    Science.gov (United States)

    Samphao, Anchalee; Butmee, Preeyanut; Jitcharoen, Juthamas; Švorc, Ľubomír; Raber, Georg; Kalcher, Kurt

    2015-09-01

    An amperometric biosensor based on chemisorption of glucose oxidase (GOx) on Au seeds decorated on magnetic core Fe3O4 nanoparticles (Fe3O4@Au) and their immobilization on screen-printed carbon electrode bulk-modified with manganese oxide (SPCE{MnO2}) was designed for the determination of glucose. The Fe3O4@Au/GOx modified SPCE{MnO2} was used in a flow-injection analysis (FIA) arrangement. The experimental conditions were investigated in amperometric mode with the following optimized parameters: flow rate 1.7 mL min(-1), applied potential +0.38 V, phosphate buffer solution (PBS; 0.1 mol L(-1), pH 7.0) as carrier and 3.89 unit mm(-2) enzyme glucose oxidase loading on the active surface of the SPCE. The designed biosensor in FIA arrangement yielded a linear dynamic range for glucose from 0.2 to 9.0 mmol L(-1) with a sensitivity of 2.52 µA mM(-1) cm(-2), a detection limit of 0.1 mmol L(-1) and a quantification limit of 0.3 mmol L(-1). Moreover, a good repeatability of 2.8% (number of measurements n=10) and a sufficient reproducibility of 4.0% (number of sensors n=3) were achieved. It was found that the studied system Fe3O4@Au facilitated not only a simpler enzyme immobilization but also provided wider linear range. The practical application of the proposed biosensor for FIA quantification of glucose was tested in glucose sirup samples, honeys and energy drinks with the results in good accordance with those obtained by an optical glucose meter and with the contents declared by the producers. Copyright © 2015. Published by Elsevier B.V.

  2. Development of an Amperometric Glucose Biosensor Based on the Immobilization of Glucose Oxidase on the Se-MCM-41 Mesoporous Composite

    Directory of Open Access Journals (Sweden)

    Sabriye Yusan

    2018-01-01

    Full Text Available A new bioenzymatic glucose biosensor for selective and sensitive detection of glucose was developed by the immobilization of glucose oxidase (GOD onto selenium nanoparticle-mesoporous silica composite (MCM-41 matrix and then prepared as a carbon paste electrode (CPE. Cyclic voltammetry was employed to probe the catalytic behavior of the biosensor. A linear calibration plot is obtained over a wide concentration range of glucose from 1 × 10−5 to 2 × 10−3 M. Under optimal conditions, the biosensor exhibits high sensitivity (0.34 µA·mM−1, low detection limit (1 × 10−4 M, high affinity to glucose (Km = 0.02 mM, and also good reproducibility (R.S.D. 2.8%, n=10 and a stability of about ten days when stored dry at +4°C. Besides, the effects of pH value, scan rate, mediator effects on the glucose current, and electroactive interference of the biosensor were also discussed. As a result, the biosensor exhibited an excellent electrocatalytic response to glucose as well as unique stability and reproducibility.

  3. A Novel Microdialysis Glucose Sensor System Based on Co-immobilizing on AU Micro-Electrode by SOL-GEL Technique

    National Research Council Canada - National Science Library

    Yu, Ping

    2001-01-01

    .... The sensor is based on co_immobilizing glucose oxidase (COD) with the catalase by sol-gel technique on the surface of the silicon bases with two pattern of An microelectrodes. A graduated ("sandwich...

  4. Influence of gamma irradiation on polymerization of pyrrole and glucose oxidase immobilization onto poly (pyrrole)/poly (vinyl alcohol) matrix

    Energy Technology Data Exchange (ETDEWEB)

    Idris, Sarada, E-mail: sarada@nuclearmalaysia.gov.my [Department of Radiation Technology, Malaysian Nuclear Agency, 43000, Bangi, Selangor (Malaysia); Department of Electrical, Electronic and Systems Engineering, Faculty of Engineering & Built Environment, Universiti Kebangsaan Malaysia, Bangi, 43600 (Malaysia); Bakar, Ahmad Ashrif A., E-mail: ashrif@ukm.edu.my [Department of Electrical, Electronic and Systems Engineering, Faculty of Engineering & Built Environment, Universiti Kebangsaan Malaysia, Bangi, 43600 (Malaysia); Thevy Ratnam, Chantara [Department of Radiation Technology, Malaysian Nuclear Agency, 43000, Bangi, Selangor (Malaysia); Kamaruddin, Nur Hasiba [Department of Electrical, Electronic and Systems Engineering, Faculty of Engineering & Built Environment, Universiti Kebangsaan Malaysia, Bangi, 43600 (Malaysia); Shaari, Sahbudin [Institute of Microengineering and Nanoelectronics, Universiti Kebangsaan Malaysia, Bangi, 43600 (Malaysia)

    2017-04-01

    Graphical abstract: The illustration of pyrrole polymerization, PVA crosslinking and immobilization of GOx onto polymer matrix. - Highlights: • Immobilization of glucose oxidase onto polymer matrices by gamma irradiation is proposed. • Crosslinking and grafting of polymers implies the immobilization reaction. • The mechanisms relies on gamma irradiation doses. • A simple single step process of polymerization, cross linking and immobilization by mean of gamma irradiation as was shown in Graphical abstract. - Abstract: This paper describes the immobilization of glucose oxidase, GOx onto polymer matrix comprising of poly(pyrrole), PPy and poly(vinyl alcohol), PVA using gamma irradiation technique. Py/PVA-GOx film was prepared by spreading PVA:GOx, 1:1 solution onto dried pyrrole film and exposed to gamma irradiation from cobalt 60 source at doses ranging from 0 to 60 kGy. The films were subjected to structural and morphological analyses by using Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), Scanning electron microscope (SEM), Field emission scanning electron microscope (FESEM) and Atomic-force microscopy (AFM) techniques. Similar studies were also made on pristine pyrrole film which served as control. The SEM and FTIR spectra of Py/PVA-GOx film revealed that pyrrole has been successfully polymerized through irradiation-induced reactions. The results on the morphological properties of the samples characterize using FESEM, SEM and AFM further confirmed the occurrence of radiation-induced modification of Py/PVA-GOx film. The FTIR spectra showed the existence of intermolecular interaction between polymer matrix and GOx indicating that GOx had been successfully immobilized onto Ppy/PVA matrix by radiation-induced reactions. Results revealed that radiation induced reactions such as polymerization of pyrrole, crosslinking of PVA, grafting between the adjacent PVA and pyrrole molecules as well as immobilization of GOx onto Ppy

  5. Influence of gamma irradiation on polymerization of pyrrole and glucose oxidase immobilization onto poly (pyrrole)/poly (vinyl alcohol) matrix

    International Nuclear Information System (INIS)

    Idris, Sarada; Bakar, Ahmad Ashrif A.; Thevy Ratnam, Chantara; Kamaruddin, Nur Hasiba; Shaari, Sahbudin

    2017-01-01

    Graphical abstract: The illustration of pyrrole polymerization, PVA crosslinking and immobilization of GOx onto polymer matrix. - Highlights: • Immobilization of glucose oxidase onto polymer matrices by gamma irradiation is proposed. • Crosslinking and grafting of polymers implies the immobilization reaction. • The mechanisms relies on gamma irradiation doses. • A simple single step process of polymerization, cross linking and immobilization by mean of gamma irradiation as was shown in Graphical abstract. - Abstract: This paper describes the immobilization of glucose oxidase, GOx onto polymer matrix comprising of poly(pyrrole), PPy and poly(vinyl alcohol), PVA using gamma irradiation technique. Py/PVA-GOx film was prepared by spreading PVA:GOx, 1:1 solution onto dried pyrrole film and exposed to gamma irradiation from cobalt 60 source at doses ranging from 0 to 60 kGy. The films were subjected to structural and morphological analyses by using Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), Scanning electron microscope (SEM), Field emission scanning electron microscope (FESEM) and Atomic-force microscopy (AFM) techniques. Similar studies were also made on pristine pyrrole film which served as control. The SEM and FTIR spectra of Py/PVA-GOx film revealed that pyrrole has been successfully polymerized through irradiation-induced reactions. The results on the morphological properties of the samples characterize using FESEM, SEM and AFM further confirmed the occurrence of radiation-induced modification of Py/PVA-GOx film. The FTIR spectra showed the existence of intermolecular interaction between polymer matrix and GOx indicating that GOx had been successfully immobilized onto Ppy/PVA matrix by radiation-induced reactions. Results revealed that radiation induced reactions such as polymerization of pyrrole, crosslinking of PVA, grafting between the adjacent PVA and pyrrole molecules as well as immobilization of GOx onto Ppy

  6. Electrochemical quartz crystal impedance study on immobilization of glucose oxidase in a polymer grown from dopamine oxidation at an Au electrode for glucose sensing

    International Nuclear Information System (INIS)

    Li Mingrui; Deng Chunyan; Xie Qingji; Yang Yang; Yao Shouzhuo

    2006-01-01

    Glucose oxidase (GOD) was codeposited into a polymer grown from oxidation of dopamine (DA) at an Au electrode in a neutral phosphate aqueous solution for the first time. The electrochemical quartz crystal impedance analysis (EQCIA) method was used to monitor the GOD-immobilization process. Effects of concentrations of phosphate buffer, DA and GOD were investigated, and the optimal concentrations were found to be 20.0mM phosphate buffer (pH 7.0), 30.0mM DA and 5.00mgml -1 GOD. A glucose biosensor was thus constructed, and effects of various experimental parameters on the sensor performance, including applied potential, solution pH and electroactive interferents, were examined. At an optimal potential of 0.6V versus the KCl-saturated calomel electrode (SCE), the current response of the biosensor in the selected phosphate buffer (pH 7.0) was linear with the concentration of glucose from 0.05 to 9mM, with a lower detection limit of 3μM (S/N=3), short response time (within 15s) and good anti-interferent ability. The Michaelis constant (K m app ) was estimated to be 9.6mM. The biosensor exhibited good storage stability, i.e. 96% of its initial response was retained after 7-day storage in the selected phosphate buffer at 4deg. C, and even after another 3 weeks the biosensor retained 86% of its initial response. In addition, the enzymatic specific activity and enzymatic relative activity of the GOD immobilized in the polymer from dopamine oxidation (PFDO) were estimated from the EQCIA method to be 1.43kUg -1 and 3.7%, respectively, which were larger than the relevant values obtained experimentally using poly(o-aminophenol) and poly(N-methylpyrrole) matrices, suggesting that the PFDO is a better matrix to immobilize GOD

  7. Radiation pretreatment of cellulosic wastes and immobilization of cells producing cellulase for their conversion to glucose

    International Nuclear Information System (INIS)

    Kumakura, Minoru; Kaetsu, Isao

    1988-01-01

    Radiation pretreatment of cellulosic wastes such as saw dust and chaff was studied by using electron beam accelerator, in which irradiation effect was increased by increasing irradiation dose and dose rate, by after heating irradiated materials at 100∼140deg C, and by irradiation in the addition of alkaline solution. Trichoderma reesei cells producing cellulase were immobilized by using fibrous porous carrier obtained from radiation polymerization. The filter paper, cellobiose, and CMC activities in the immobilized growing cells were higher than those in free cells. The activity in the immobilized cells obtained with hydrophobic carrier was higher than that obtained with hydrophilic one. Durability of the immobilized cells was examined by repeated batch culture. It was found that the enzyme solution produced in the culture of the immobilized cells can hydrolyze effectively saw dust pretreated by radiation. (author)

  8. Design of Amperometric Biosensors for the Detection of Glucose Prepared by Immobilization of Glucose Oxidase on Conducting (PolyThiophene Films

    Directory of Open Access Journals (Sweden)

    Maria Pilo

    2018-01-01

    Full Text Available Enzyme-based sensors have emerged as important analytical tools with application in diverse fields, and biosensors for the detection of glucose using the enzyme glucose oxidase have been widely investigated. In this work, the preparation of biosensors by electrochemical polymerization of (polythiophenes, namely 2,2′-bithiophene (2,2′-BT and 4,4′-bis(2-methyl-3-butyn-2-ol-2,2′-bithiophene (4,4′-bBT, followed by immobilization of glucose oxidase on the films, is described. N-cyclohexyl-N′-(2-morpholinoethylcarbodiimide metho-p-toluenesulfonate (CMC was used as a condensing agent, and p-benzoquinone (BQ was used as a redox mediator in solution. The glucose oxidase electrodes with films of 2,2′-BT and 4,4′-bBT were then tested for their ability in detecting glucose from synthetic and real samples (pear, apricot, and peach fruit juices.

  9. Optical determination of glucose and hydrogen peroxide using a nanocomposite prepared from glucose oxidase and magnetite nanoparticles immobilized on graphene oxide

    International Nuclear Information System (INIS)

    Chang, Qing; Tang, Heqing

    2014-01-01

    Fe 3 O 4 nanoparticles were deposited on sheets of graphene oxide (GO) by a precipitation method, and glucose oxidase (GOx) was then immobilized on this material to produce a GOx/Fe 3 O 4 /GO magnetic nanocomposite containing crosslinked enzyme clusters. The 3-component composite functions as a binary enzyme that was employed in a photometric method for the determination of glucose and hydrogen peroxide where the GOx/Fe 3 O 4 /GO nanoparticles cause the generation of H 2 O 2 which, in turn, oxidize the substrate N,N-diethyl-p-phenylenediamine to form a purple product with an absorption maximum at 550 nm. The absorbance at 550 nm can be correlated to the concentration of glucose and/or hydrogen peroxide. Under optimized conditions, the calibration plot is linear in the 0.5 to 600 μM glucose concentration range, and the detection limit is 0.2 μM. The respective plot for H 2 O 2 ranges from 0.1 to 10 μM, and the detection limit is 0.04 μM. The method was successfully applied to the determination of glucose in human serum samples. The GOx/Fe 3 O 4 /GO nanoparticles are reusable. (author)

  10. Direct electrochemistry and electrocatalysis of glucose oxidase immobilized on reduced graphene oxide and silver nanoparticles nanocomposite modified electrode.

    Science.gov (United States)

    Palanisamy, Selvakumar; Karuppiah, Chelladurai; Chen, Shen-Ming

    2014-02-01

    The direct electrochemistry of glucose oxidase (GOx) was successfully realized on electrochemically reduced graphene oxide and silver nanoparticles (RGO/Ag) nanocomposite modified electrode. The fabricated nanocomposite was characterized by field emission scanning electron microscope and energy dispersive spectroscopy. The GOx immobilized nanocomposite modified electrode showed a pair of well-defined redox peaks with a formal potential (E°) of -0.422 V, indicating that the bioactivity of GOx was retained. The heterogeneous electron transfer rate constant (Ks) of GOx at the nanocomposite was calculated to be 5.27 s(-1), revealing a fast direct electron transfer of GOx. The GOx immobilized RGO/Ag nanocomposite electrode exhibited a good electrocatalytic activity toward glucose over a linear concentration range from 0.5 to 12.5 mM with a detection limit of 0.16 mM. Besides, the fabricated biosensor showed an acceptable sensitivity and selectivity for glucose. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Direct electron transfer and biosensing of glucose oxidase immobilized at multiwalled carbon nanotube-alumina-coated silica modified electrode

    International Nuclear Information System (INIS)

    Wu, Wei-Che; Huang, Jian-Lung; Tsai, Yu-Chen

    2012-01-01

    Investigations are reported regarding the direct electrochemical performance of glucose oxidase (GOD) immobilized on a film of multiwalled carbon nanotube-alumina-coated silica (MWCNT-ACS). The surface morphology of the GOD/MWCNT-ACS nanobiocomposite is characterized by scanning electron microscopy. In cyclic voltammetric response, the immobilized GOD displays a pair of well-defined redox peaks, with a formal potential (E°′) of − 0.466 V versus Ag/AgCl in a 0.1 M phosphate buffer solution (pH 7.5) at a scan rate of 0.05 V s −1 ; also the electrochemical response indicates a surface-controlled electrode process. The dependence of formal potential on solution pH indicates that the direct electron transfer reaction of GOD is a reversible two-electron coupled with a two-proton electrochemical reaction process. The glucose biosensor based on the GOD/MWCNT-ACS nanobiocomposite shows a sensitivity of 0.127 A M −1 cm −2 and an apparent Michaelis–Menten constant of 0.5 mM. Furthermore, the prepared biosensor exhibits excellent anti-interference ability to the commonly co-existed uric acid and ascorbic acid. - Highlights: ► A film composed of MWCNT-ACS was used for biosensor application. ► High sensitivity and good selectivity were obtained for the detection of glucose. ► This approach is potential for fabrication of mediator-free biosensor.

  12. Evaluation Of Enzyme Immobilization Methods For Paper-based Devices-a Glucose Oxidase Study

    OpenAIRE

    Nery; Emilia Witkowska; Kubota; Lauro T.

    2016-01-01

    Paper-based sensors gained almost explosive attention during the last few years. A large number of systems, often destined to resource limited settings is based on enzymatic reactions. Choice of an adequate immobilization method could significantly prolong the shelf-life of such sensors, especially in applications, where exposure to high temperatures during storage and transport is more than a threat. We are seeking to compare a variety of immobilization methods based on different phenomena (...

  13. Immobilization of Glucose Oxidase to Nanostructured Films of Polystyrene-block-poly(2-vinylpyridine)

    OpenAIRE

    Bhakta, Samir A; Benavidez, Tomas E; Garcia, Carlos D

    2014-01-01

    A critical step for the development of biosensors is the immobilization of the biorecognition element to the surface of a substrate. Among other materials that can be used as substrates, block copolymers have the untapped potential to provide significant advantages for the immobilization of proteins. To explore such possibility, this manuscript describes the fabrication and characterization of thin-films of polystyrene-block-poly(2-vinylpyridine) (PS-b-P2VP). These films were then used to inv...

  14. Glucose biosensor based on glucose oxidase immobilized on a nanofilm composed of mesoporous hydroxyapatite, titanium dioxide, and modified with multi-walled carbon nanotubes

    International Nuclear Information System (INIS)

    Li, J.; Kuang, D.; Feng, Y.; Zhang, F.; Liu, M.

    2012-01-01

    We report on a highly sensitive glucose biosensor that was fabricated from a composite made from mesoporous hydroxyapatite and mesoporous titanium dioxide which then were ultrasonically mixed with multi-walled carbon nanotubes to form a rough nanocomposite film. This film served as a platform to immobilize glucose oxidase onto a glassy carbon electrode. The morphological and electrochemical properties of the film were examined by scanning electron microscopy and electrochemical impedance spectroscopy. Cyclic voltammetry and chronoamperometry were used to characterize the electrochemical performances of the biosensor which exhibited excellent electrocatalytic activity to the oxidation of glucose. At an operating potential of 0. 3 V and pH 6. 8, the sensor displays a sensitivity of 57. 0 μA mM -1 cm -2 , a response time of <5 s, a linear dynamic range from 0. 01 to 15. 2 mM, a correlation coefficient of 0. 9985, and a detection limit of 2 μM at an SNR of 3. No interferences are found for uric acid, ascorbic acid, dopamine and most carbohydrates. The sensor is stable and was successfully applied to the determination of glucose in real samples. (author)

  15. Glucose biosensor based on immobilization of glucose oxidase on a carbon paste electrode modified with microsphere-attached l-glycine.

    Science.gov (United States)

    Donmez, Soner; Arslan, Fatma; Sarı, Nurşen; Hasanoğlu Özkan, Elvan; Arslan, Halit

    2017-09-01

    In the present study, a novel biosensor that is sensitive to glucose was prepared using the microspheres modified with (4-formyl-3-methoxyphenoxymethyl)polystyrene (FMPS) with l-glycine. Polymeric microspheres having Schiff bases were prepared from FMPS using the glycine condensation method. Glucose oxidase enzyme was immobilized onto modified carbon paste electrode by cross-linking with glutaraldehyde. Oxidation of enzymatically produced H 2 O 2 (+0.5 V vs. Ag/AgCl) was used for determination of glucose. Optimal temperature and pH were found as 50 °C and 8.0, respectively. The glucose biosensor showed a linear working range from 5.0 × 10 -4 to 1.0 × 10 -2 M, R 2 = 0.999. Storage and operational stability of the biosensor were also investigated. The biosensor gave perfect reproducible results after 20 measurements with 3.3% relative standard deviation. It also had good storage stability. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  16. Influence of gamma irradiation on polymerization of pyrrole and glucose oxidase immobilization onto poly (pyrrole)/poly (vinyl alcohol) matrix

    Science.gov (United States)

    Idris, Sarada; A. Bakar, Ahmad Ashrif; Thevy Ratnam, Chantara; Kamaruddin, Nur Hasiba; Shaari, Sahbudin

    2017-04-01

    This paper describes the immobilization of glucose oxidase, GOx onto polymer matrix comprising of poly(pyrrole), PPy and poly(vinyl alcohol), PVA using gamma irradiation technique. Py/PVA-GOx film was prepared by spreading PVA:GOx, 1:1 solution onto dried pyrrole film and exposed to gamma irradiation from cobalt 60 source at doses ranging from 0 to 60 kGy. The films were subjected to structural and morphological analyses by using Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), Scanning electron microscope (SEM), Field emission scanning electron microscope (FESEM) and Atomic-force microscopy (AFM) techniques. Similar studies were also made on pristine pyrrole film which served as control. The SEM and FTIR spectra of Py/PVA-GOx film revealed that pyrrole has been successfully polymerized through irradiation-induced reactions. The results on the morphological properties of the samples characterize using FESEM, SEM and AFM further confirmed the occurrence of radiation-induced modification of Py/PVA-GOx film. The FTIR spectra showed the existence of intermolecular interaction between polymer matrix and GOx indicating that GOx had been successfully immobilized onto Ppy/PVA matrix by radiation-induced reactions. Results revealed that radiation induced reactions such as polymerization of pyrrole, crosslinking of PVA, grafting between the adjacent PVA and pyrrole molecules as well as immobilization of GOx onto Ppy/PVA matrix occurred simultaneously upon gamma irradiation. The optimum dose for GOx immobilization in the polymer matrix found to be 40 kGy. Therefore it is clear that this irradiation technique offered a simple single process to produce Py/PVA-GOx film without additional crosslinking and polymerization agents.

  17. Glucose biosensor based on a platinum electrode modified with rhodium nanoparticles and with glucose oxidase immobilized on gold nanoparticles

    International Nuclear Information System (INIS)

    Guo, Xishan; Jian, Jinming; Liang, Bo; Ye, Xuesong; Zhang, Yelei

    2014-01-01

    We have developed an enzymatic glucose biosensor that is based on a flat platinum electrode which was covered with electrophoretically deposited rhodium (Rh) nanoparticles and then sintered to form a large surface area. The biosensor was obtained by depositing glucose oxidase (GOx), Nafion, and gold nanoparticles (AuNPs) on the Rh electrode. The electrical potential and the fractions of Nafion and GOx were optimized. The resulting biosensor has a very high sensitivity (68.1 μA mM −1 cm −2 ) and good linearity in the range from 0.05 to 15 mM (r = 0.989). The limit of detection is as low as 0.03 mM (at an SNR of 3). The glucose biosensor also is quite selective and is not interfered by electroactive substances including ascorbic acid, uric acid and acetaminophen. The lifespan is up to 90 days. It was applied to the determination of glucose in blood serum, and the results compare very well with those obtained with a clinical analyzer. (author)

  18. Direct electron transfer and biosensing of glucose oxidase immobilized at multiwalled carbon nanotube-alumina-coated silica modified electrode

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Wei-Che; Huang, Jian-Lung; Tsai, Yu-Chen, E-mail: yctsai@dragon.nchu.edu.tw

    2012-05-01

    Investigations are reported regarding the direct electrochemical performance of glucose oxidase (GOD) immobilized on a film of multiwalled carbon nanotube-alumina-coated silica (MWCNT-ACS). The surface morphology of the GOD/MWCNT-ACS nanobiocomposite is characterized by scanning electron microscopy. In cyclic voltammetric response, the immobilized GOD displays a pair of well-defined redox peaks, with a formal potential (E Degree-Sign Prime ) of - 0.466 V versus Ag/AgCl in a 0.1 M phosphate buffer solution (pH 7.5) at a scan rate of 0.05 V s{sup -1}; also the electrochemical response indicates a surface-controlled electrode process. The dependence of formal potential on solution pH indicates that the direct electron transfer reaction of GOD is a reversible two-electron coupled with a two-proton electrochemical reaction process. The glucose biosensor based on the GOD/MWCNT-ACS nanobiocomposite shows a sensitivity of 0.127 A M{sup -1} cm{sup -2} and an apparent Michaelis-Menten constant of 0.5 mM. Furthermore, the prepared biosensor exhibits excellent anti-interference ability to the commonly co-existed uric acid and ascorbic acid. - Highlights: Black-Right-Pointing-Pointer A film composed of MWCNT-ACS was used for biosensor application. Black-Right-Pointing-Pointer High sensitivity and good selectivity were obtained for the detection of glucose. Black-Right-Pointing-Pointer This approach is potential for fabrication of mediator-free biosensor.

  19. Photochemical immobilization of protein on the inner wall of a microchannel and Its application in a glucose sensor

    International Nuclear Information System (INIS)

    Nakajima, Hizuru; Ishino, Satomi; Masuda, Hironori; Nakagama, Tatsuro; Shimosaka, Takuya; Uchiyama, Katsumi

    2006-01-01

    A new protein immobilization technique has been developed for patterning enzymes in a specific position inside a microchannel. First, bovine serum albumin (BSA) was adsorbed onto the internal surface of a polydimethylsiloxane microchannel. The microchannel was then filled with the conjugate solution of a photoreactive cross-linker, 4-azido-2,3,5,6-tetrafluorobenzoic acid succinimidyl ester (ATFB-SE), and an enzyme, horseradish peroxidase (HRP). An irradiation by a He-Cd laser activated the azido group of the conjugates and these conjugates became covalently attached to the adsorbed BSA on the microchannel. The enzyme turnover was observed from only the HRP zone. This technique was successfully applied to the enzymatic glucose sensor. Glucose oxidase (GOD) and HRP were sequentially patterned in a single microchannel, i.e., the HRP zone was located downstream from the GOD zone. The calibration curve of a glucose standard solution was linear over the range of 0-128 μM with a correlation coefficient of 0.993. Compared to the traditional method using a 96-well microtiter plate, the present technique on the microchip shortened the reaction time from 30 min to 4.8 s, i.e., to 1/375

  20. 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

  1. 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.

  2. Direct electron transfer and electrocatalysis of glucose oxidase immobilized on glassy carbon electrode modified with Nafion and mesoporous carbon FDU-15

    International Nuclear Information System (INIS)

    Wang Kunqi; Yang Hua; Zhu Lin; Ma Zhongsu; Xing Shenyang; Lv Qiang; Liao Jianhui; Liu Changpeng; Xing Wei

    2009-01-01

    In this paper, it was found that glucose oxidase (GOD) has been stably immobilized on glassy carbon electrode modified with mesoporous carbon FDU-15 (MC-FDU-15) and Nafion by simple technique. The sorption behavior of GOD immobilized on MC-FDU-15 matrix was characterized by transmission electron microscopy (TEM), ultraviolet-visible (UV-vis), FTIR, respectively, which demonstrated that MC-FDU-15 could facilitate the electron exchange between the active center of GOD and electrode. The direct electrochemistry and electrocatalysis behavior of GOD on the modified electrode were characterized by cyclic voltammogram (CV) which indicated that GOD immobilized on Nafion and MC-FDU-15 matrices display direct, reversible and surface-controlled redox reaction with an enhanced electron transfer rate constant of 4.095 s -1 in 0.1 M phosphate buffer solution (PBS) (pH 7.12). Furthermore, it was also discovered that, in the presence of O 2 , GOD immobilized on Nafion and MC-FDU-15 matrices could produce a linear response to glucose. Thus, Nafion/GOD-MC-FDU-15/GC electrode is hopeful to be used in glucose biosensor. In addition, GOD immobilized on MC-FDU-15 and Nafion matrices possesses an excellent bioelectrocatalytic activity for the reduction of O 2 . So, the Nafion/GOD-MC-FDU-15/GC electrode can be utilized as the cathode in biofuel cell.

  3. Glucose Oxidase Directly Immobilized onto Highly Porous Gold Electrodes for Sensing and Fuel Cell applications

    International Nuclear Information System (INIS)

    Toit, Hendrik du; Di Lorenzo, Mirella

    2014-01-01

    Highlights: • Electrochemical adsorption of glucose oxidase (GOx) on highly porous gold (hPG); • Rapid one-step immobilisation protocol with no use of expensive and/or harsh reagents; • Linear response to glucose in the range 50 μM -10 mM; • Lower detection limit, stable over 5 days: 25 μM. • The use of the GOx-hPG in a fuel cell lead to the peak power density of 6 μW cm −2 . - Abstract: The successful implementation of redox-enzyme electrodes in biosensors and enzymatic biofuel cells has been the subject of extensive research. For high sensitivity and high energy-conversion efficiency, the effective electron transfer at the protein-electrode interface has a key role. This is difficult to achieve in the case of glucose oxidase, due to the fact that for this enzyme the redox centre is buried inside the structure, far from any feasible electrode binding sites. This study reports, a simple and rapid methodology for the direct immobilisation of glucose oxidase into highly porous gold electrodes. When the resulting electrode was tested as glucose sensor, a Michaelis-Menten kinetic trend was observed, with a detection limit of 25 μM. The bioelectrode sensitivity, calculated against the superficial surface area of the bioelectrode, was of 22.7 ± 0.1 μA mM −1 cm −2 . This glucose oxidase electrode was also tested as an anode in a glucose/O 2 enzymatic biofuel cell, leading to a peak power density of 6 μW cm −2 at a potential of 0.2 V

  4. Effect of oleic acid on the production of ethanol and fructose from glucose/fructose mixtures in an immobilized cell reactor

    Energy Technology Data Exchange (ETDEWEB)

    Guenette, M E [Ottawa Univ., ON (Canada). Dept. of Chemical Engineering; [IOGEN Corp., Ottawa, ON (Canada); Duvnjak, Z [Ottawa Univ., ON (Canada). Dept. of Chemical Engineering; [IOGEN Corp., Ottawa, ON (Canada)

    1996-12-31

    Saccharomyces cerevisiae ATCC 39859 was immobilized onto small cubes of wood to produce ethanol and very enriched fructose syrup from glucose/fructose mixtures through the selective fermentation of glucose. A maximum ethanol productivity of 21.9 g/l.h was attained from a feed containing 9.7% (w/v) glucose and 9.9% (w/v) fructose. An ethanol concentration, glucose conversion and fructose yield of 29.6 g/l, 62% and 99% were obtained, respectively. This resulted in a final fructose/glucose ratio of 2.7. At lower ethanol productivity levels the fructose/glucose ratio increases, as does the ethanol concentration in the effluent. The addition of 30 mg/l oleic acid to the medium increased the ethanol productivity and its concentration by 13% at a dilution rate of 0.74 h{sup -1}. (orig.)

  5. Covalent immobilization of glucose oxidase on amino MOFs via post-synthetic modification

    NARCIS (Netherlands)

    Tudisco, C.G.; Zolubas, G.; Seoane de la Cuesta, B.; Zafarani, H.; Kazemzad Asiabi, M.; Gascon Sabate, J.; Hagedoorn, P.L.; Rassaei, L.

    2016-01-01

    The post-synthetic modification (PSM) of two amino-MOFs with glucose oxidase is reported in this study. The multi-step approach preserved the MOFs' structure and allowed the production of enzyme-functionalized MOFs (MOFs@GOx), which retained the enzymatic activity and showed selective properties

  6. Co-immobilization of cyclohexanone monooxygenase and glucose-6-phosphate dehydrogenase onto polyethylenimine-porous agarose polymeric composite using γ irradiation to use in biotechnological processes

    International Nuclear Information System (INIS)

    Atia, K.S.

    2005-01-01

    The co-immobilization of cyclohexanone monooxygenase (CHMO) and glucose-6-phosphate dehydrogenase (G6PDH) was optimized by completely coating, via covalent immobilization, the surface aldehyde groups of porous agarose (glyoxyl-agarose) with amine groups of polyethylenimine (PEI). The highest immobilization efficiency (∼87%) (activity of enzyme per amount of immobilized enzyme) was obtained with a CHMO/G6PDH ratio 2:1. The effects of different ratios of the support to the amount of enzymes (CHMO:G6PDH=2:1), the optimum incubation pH and the incubation time on the enzymatic activity of the enzymes were determined and found to be 5:1, 8.5 and 30 min, respectively. Subjecting the co-immobilized enzymes to doses of γ-radiation (5-100 kGy) resulted in complete loss in the activity of the free enzymes at a dose of 40 kGy, while the co-immobilized ones showed relatively high resistance to γ-radiation up to a dose of 50 kGy

  7. Development of high-performance functional materials for enzyme immobilization by the use of ionizing radiation

    International Nuclear Information System (INIS)

    SALIM, R.D.M.

    2013-01-01

    Isomerization of glucose to fructose was carried out using Glucose isomerase (GI) that immobilized by entrapment into Poly (acrylic acid) P (AA) and Poly (acrylic acid-co- 2-Acrylamido 2- methyl Propane sulfonic acid) P (AA-co-AMPS) polymer networks, the enzyme carriers were prepared by radiation induced co-polymerization in presence of (Methylene- bis acrylamide) (MBAA) as a crosslinking agent. Effects of immobilization conditions such as irradiation dose, methylene bis acrylamide concentration, comonomer composition, and amount of GI were investigated. The influence of reaction conditions on the activity of immobilized GI were studied, the optimum ph value of reaction solution is 7.5 and reaction temperature is 65 degree C. The immobilized GI into P (AA-co-AMPS) and P (AA) polymer networks retained 81% and 69%,respectively, of its initial activity after recycled for 15 times while it retained 87% and 71% ,respectively ,of its initial activity after stored at 4 degree C for 48 days , The Km values of free and immobilized GI onto P(AA-co-AMPS) and onto P(AA) matrices were found to be 34, 29.2 , 14.5 mg/ml respectively while the Vmax Values calculated to be 3.87 ,1.6,0.79 mg/ml.min, respectively, Therefore , the bio conversion of glucose to fructose can be successfully performed by GI entrapped into P (AA-co-AMPS) hydrogel .

  8. Co-immobilization of multiple enzymes by metal coordinated nucleotide hydrogel nanofibers: improved stability and an enzyme cascade for glucose detection.

    Science.gov (United States)

    Liang, Hao; Jiang, Shuhui; Yuan, Qipeng; Li, Guofeng; Wang, Feng; Zhang, Zijie; Liu, Juewen

    2016-03-21

    Preserving enzyme activity and promoting synergistic activity via co-localization of multiple enzymes are key topics in bionanotechnology, materials science, and analytical chemistry. This study reports a facile method for co-immobilizing multiple enzymes in metal coordinated hydrogel nanofibers. Specifically, four types of protein enzymes, including glucose oxidase, Candida rugosa lipase, α-amylase, and horseradish peroxidase, were respectively encapsulated in a gel nanofiber made of Zn(2+) and adenosine monophosphate (AMP) with a simple mixing step. Most enzymes achieved quantitative loading and retained full activity. At the same time, the entrapped enzymes were more stable against temperature variation (by 7.5 °C), protease attack, extreme pH (by 2-fold), and organic solvents. After storing for 15 days, the entrapped enzyme still retained 70% activity while the free enzyme nearly completely lost its activity. Compared to nanoparticles formed with AMP and lanthanide ions, the nanofiber gels allowed much higher enzyme activity. Finally, a highly sensitive and selective biosensor for glucose was prepared using the gel nanofiber to co-immobilize glucose oxidase and horseradish peroxidase for an enzyme cascade system. A detection limit of 0.3 μM glucose with excellent selectivity was achieved. This work indicates that metal coordinated materials using nucleotides are highly useful for interfacing with biomolecules.

  9. Immobilization of glucose oxidase onto a novel platform based on modified TiO{sub 2} and graphene oxide, direct electrochemistry, catalytic and photocatalytic activity

    Energy Technology Data Exchange (ETDEWEB)

    Haghighi, Nasibeh, E-mail: Haghighi.nasibeh@yahoo.com [Department of Chemistry, University of Kurdistan, P.O. Box 416, Sanandaj (Iran, Islamic Republic of); Hallaj, Rahman, E-mail: Rhallaj@uok.ac.ir [Department of Chemistry, University of Kurdistan, P.O. Box 416, Sanandaj (Iran, Islamic Republic of); Nanotechnology Research Center, University of Kurdistan, P.O. Box 416, Sanandaj (Iran, Islamic Republic of); Salimi, Abdollah [Department of Chemistry, University of Kurdistan, P.O. Box 416, Sanandaj (Iran, Islamic Republic of); Nanotechnology Research Center, University of Kurdistan, P.O. Box 416, Sanandaj (Iran, Islamic Republic of)

    2017-04-01

    In this work a new organic–inorganic nanocomposite has been introduced for enzyme immobilization. The composite consisting of graphene oxide (GO) and titanium oxide nanoparticles (TiO{sub 2}) modified with 2, 2′-dithioxo-3, 3′-bis (3-(triethoxysilyl) propyl)-2H, 2′H-[5, 5′-bithiazolylidene]-4, 4′(3H, 3′H)-dione as Organic-Inorganic Supporting Ligand (OISL). The OISL was covalently attached to TiO{sub 2} nanoparticles and employed for obtaining a suitable solid surface to enzyme attachment. The glucose oxidase (GOD) was irreversibly loaded on the GC/GO/TiO{sub 2}-OISL using consecutive cyclic voltammetry. The enzyme immobilization and the enzymatic activity were determined by electrochemical methods. The cyclic voltammogram displayed a pair of well-defined and nearly symmetric redox peaks with a formal potential of − 0.465 V and an apparent electron transfer rate constant of 1.74 s{sup −1}. The GO/TiO{sub 2}-OISL can catalyze the electroreduction and electrooxidation of hydrogen peroxide. The GC/GO/TiO{sub 2}-OISL/GOD electrode was used in the hydrogen peroxide determination. The fabricated nanobiocomposite shows dramatic photoelectrocatalytic activity which evaluated by studying the electrocatalytic activity of the fabricated electrode toward hydrogen peroxide in darkness and in the presences of light. - Highlights: • In this work a novel platform used to successful immobilization of glucose oxidase. Due to its large functional group this modified nanoparticles load enzyme (GOD) and remain enzyme whit out denaturation for a long time. • The loaded enzyme shows direct electron transfer and excellent charge transfer kinetic. Also the fabricated nano-bio-composite has good catalytic activity toward hydrogen peroxide during electrooxidation and electro reduction process. • The nano-bio-composite shows excellent photocatalytic activity.

  10. Immobilization of glucose oxidase onto a novel platform based on modified TiO2 and graphene oxide, direct electrochemistry, catalytic and photocatalytic activity

    International Nuclear Information System (INIS)

    Haghighi, Nasibeh; Hallaj, Rahman; Salimi, Abdollah

    2017-01-01

    In this work a new organic–inorganic nanocomposite has been introduced for enzyme immobilization. The composite consisting of graphene oxide (GO) and titanium oxide nanoparticles (TiO 2 ) modified with 2, 2′-dithioxo-3, 3′-bis (3-(triethoxysilyl) propyl)-2H, 2′H-[5, 5′-bithiazolylidene]-4, 4′(3H, 3′H)-dione as Organic-Inorganic Supporting Ligand (OISL). The OISL was covalently attached to TiO 2 nanoparticles and employed for obtaining a suitable solid surface to enzyme attachment. The glucose oxidase (GOD) was irreversibly loaded on the GC/GO/TiO 2 -OISL using consecutive cyclic voltammetry. The enzyme immobilization and the enzymatic activity were determined by electrochemical methods. The cyclic voltammogram displayed a pair of well-defined and nearly symmetric redox peaks with a formal potential of − 0.465 V and an apparent electron transfer rate constant of 1.74 s −1 . The GO/TiO 2 -OISL can catalyze the electroreduction and electrooxidation of hydrogen peroxide. The GC/GO/TiO 2 -OISL/GOD electrode was used in the hydrogen peroxide determination. The fabricated nanobiocomposite shows dramatic photoelectrocatalytic activity which evaluated by studying the electrocatalytic activity of the fabricated electrode toward hydrogen peroxide in darkness and in the presences of light. - Highlights: • In this work a novel platform used to successful immobilization of glucose oxidase. Due to its large functional group this modified nanoparticles load enzyme (GOD) and remain enzyme whit out denaturation for a long time. • The loaded enzyme shows direct electron transfer and excellent charge transfer kinetic. Also the fabricated nano-bio-composite has good catalytic activity toward hydrogen peroxide during electrooxidation and electro reduction process. • The nano-bio-composite shows excellent photocatalytic activity.

  11. High performance glucose/O2 compartment-less biofuel cell using DNA/CNTs as platform for immobilizing bilirubin oxidase as novel biocathode and integrated NH2-CNTs/dendrimer/glucose dehydrogenase/nile blue as bioanode

    International Nuclear Information System (INIS)

    Korani, Aazam; Salimi, Abdollah

    2015-01-01

    Highlights: • A biocathode based on immobilization of bilirubin oxidase onto MWCNTs/DNA is designed. • The performance of MWCNTs/DNA/BOD biocathode for O 2 reduction reaction is improved. • Compared to MWCNTs/BOD,at present biocathode current density to ORR increased 3 folds. • The onset potential for ORR is 0.57 V and its current density increased to 270 μA cm −2 . • A glucose/O 2 BFC with voltage of 0.66 V, J = 172 μAcm −2 and power of 45 μW cm −2 fabricated. - Abstract: Herein, deoxyribonucleic acid (DNA)/multi-walled carbon nanotube (MWCNTs) with enhanced negative charged density was used as a novel electrochemical platform for oriented immobilization of bilirubin oxidase. The proposed support improved the direct electron transfer kinetics of BOD and its catalytic activity toward oxygen reduction reaction (ORR). In comparison to BOD enzyme which immobilized directly onto MWCNTs the current density increased three folds and reached to 270 μA cm −2 at 0.405 V with an onset potential of 0.57 V (vs. Ag/AgCl). The ability of this modified electrode as a biocathode is investigated after assembling with bioanode. The bioanode prepared with covalent attachment of glucose dehydrogenase enzyme (GDH) and nile blue (NB) as an efficient mediator for coenzyme regeneration onto glassy carbon electrode modified with amino-carbon nanotubes(MWCNTs-NH 2 ) and carboxyl terminated polyamidoamin dendrimer (PAMAM-Den) as a multifunctional linker. Finally, the performance of one-compartment glucose/O 2 biofuel cell without separators is also investigated. The open circuit voltage of the cell and maximum current density are obtained 660 mV and 172 μA cm −2 , respectively, while the maximum power density of 45 μW cm −2 is achieved at 428 mV of the cell voltage in buffer solution saturated with O 2 and containing 50 mM of glucose. The stability of the constructed EBFC is investigated under continuous operation at maximum power. It is observed that the biofuel

  12. Immobilization of Styrene-acrylamide Co-polymer on Either Silica Particles or Inner Surface of Silica Capillary for the Separation of D-Glucose Anomers

    International Nuclear Information System (INIS)

    Ali, Faiz; Kim, Yune Sung; Cheong, Won Jo

    2014-01-01

    Styrene-acrylamide co-polymer was immobilized on porous partially sub-2 μm silica monolith particles and inner surface of fused silica capillary (50 μm ID and 28 cm length) to result in μLC and CEC stationary phases, respectively, for separation of anomeric D-glucose derivatives. Reversed addition-fragmentation transfer (RAFT) polymerization was incorporated to induce surface polymerization. Acrylamide was employed to incorporate amide-functionality in the stationary phase. The resultant μLC and CEC stationary phases were able to separate isomers of D-glucose derivatives with high selectivity and efficiency. The mobile phase of 75/ 25 (v/v) acetonitrile (ACN)/water with 0.1% TFA, was used for HPLC with a packed column (1 mm ID, 300 mm length). The effects of pH and ACN composition on anomeric separation of D-glucose in CEC have been examined. A mobile phase of 85/15 (v/v) ACN/30 mM sodium acetate pH 6.7 was found the optimized mobile phase for CEC. The CEC stationary phase also gave good separation of other saccharides such as maltotriose and Dextran 1500 (MW∼1500) with good separation efficiency (number of theoretical plates ∼300,000/m)

  13. Immobilization of Styrene-acrylamide Co-polymer on Either Silica Particles or Inner Surface of Silica Capillary for the Separation of D-Glucose Anomers

    Energy Technology Data Exchange (ETDEWEB)

    Ali, Faiz; Kim, Yune Sung; Cheong, Won Jo [Inha Univ., Incheon (Korea, Republic of)

    2014-02-15

    Styrene-acrylamide co-polymer was immobilized on porous partially sub-2 μm silica monolith particles and inner surface of fused silica capillary (50 μm ID and 28 cm length) to result in μLC and CEC stationary phases, respectively, for separation of anomeric D-glucose derivatives. Reversed addition-fragmentation transfer (RAFT) polymerization was incorporated to induce surface polymerization. Acrylamide was employed to incorporate amide-functionality in the stationary phase. The resultant μLC and CEC stationary phases were able to separate isomers of D-glucose derivatives with high selectivity and efficiency. The mobile phase of 75/ 25 (v/v) acetonitrile (ACN)/water with 0.1% TFA, was used for HPLC with a packed column (1 mm ID, 300 mm length). The effects of pH and ACN composition on anomeric separation of D-glucose in CEC have been examined. A mobile phase of 85/15 (v/v) ACN/30 mM sodium acetate pH 6.7 was found the optimized mobile phase for CEC. The CEC stationary phase also gave good separation of other saccharides such as maltotriose and Dextran 1500 (MW∼1500) with good separation efficiency (number of theoretical plates ∼300,000/m)

  14. Non-enzymatic glucose sensing platform using self assembled cobalt oxide/graphene nanocomposites immobilized graphite modified electrode

    DEFF Research Database (Denmark)

    Vivekananth, R.; Babu, R. Suresh; Prasanna, K.

    2018-01-01

    A new strategy to prepare the densely packed cobalt oxide (Co3O4)/graphene nanocomposites by a self-assembly method were adopted in this work. A new non-enzymatic glucose determination has been fabricated by using Co3O4/graphene nanocomposites modified electrode as a sensing material. The nanocom...... of the modified electrode for glucose determination has been evaluated in urine samples....

  15. Enhanced performance of a glucose/O(2) biofuel cell assembled with laccase-covalently immobilized three-dimensional macroporous gold film-based biocathode and bacterial surface displayed glucose dehydrogenase-based bioanode.

    Science.gov (United States)

    Hou, Chuantao; Yang, Dapeng; Liang, Bo; Liu, Aihua

    2014-06-17

    The power output and stability of enzyme-based biofuel cells (BFCs) is greatly dependent on the properties of both the biocathode and bioanode, which may be adapted for portable power production. In this paper, a novel highly uniform three-dimensional (3D) macroporous gold (MP-Au) film was prepared by heating the gold "supraspheres", which were synthesized by a bottom-up protein templating approach, and followed by modification of laccase on the MP-Au film by covalent immobilization. The as-prepared laccase/MP-Au biocathode exihibited an onset potential of 0.62 V versus saturated calomel electrode (SCE, or 0.86 V vs NHE, normal hydrogen electrode) toward O2 reduction and a high catalytic current of 0.61 mAcm(-2). On the other hand, mutated glucose dehydrogenase (GDH) surface displayed bacteria (GDH-bacteria) were used to improve the stability of the glucose oxidation at the bioanode. The as-assembled membraneless glucose/O2 fuel cell showed a high power output of 55.8 ± 2.0 μW cm(-2) and open circuit potential of 0.80 V, contributing to the improved electrocatalysis toward O2 reduction at the laccase/MP-Au biocathode. Moreover, the BFC retained 84% of its maximal power density even after continuous operation for 55 h because of the high stability of the bacterial surface displayed GDH mutant toward glucose oxidation. Our findings may be promising for the development of more efficient glucose BFC for portable battery or self-powered device applications.

  16. A resettable and reprogrammable keypad lock based on electrochromic Prussian blue films and biocatalysis of immobilized glucose oxidase in a bipolar electrode system.

    Science.gov (United States)

    Yu, Xue; Liang, Jiying; Yang, Tiangang; Gong, Mengjie; Xi, Dongman; Liu, Hongyun

    2018-01-15

    Herein, a resettable and reprogrammable biomolecular keypad lock on the basis of a closed bipolar electrode (BPE) system was established. In this system, one ITO electrode with immobilized chitosan (CS) and glucose oxidase (GOD), designated as CS-GOD, acted as one pole of BPE in the sensing cell; another ITO with electrodeposited Prussian blue (PB) films as the other pole in the reporting cell. The addition of ascorbic acid (AA) in the sensing cell with driving voltage (V tot ) at +2.5V would make the PB films become Prussian white (PW) in the reporting cell, accompanied by the color change from blue to nearly transparent. On the other hand, with the help of oxygen, the addition of glucose in the sensing cell with V tot at -1.5V would induce PW back to PB. The change of color and the corresponding UV-vis absorbance at 700nm for the PB/PW films in the reporting cell could be reversibly switched by changing the solute in the sensing cell between AA and glucose and then switching V tot between +2.5 and -1.5V. Based on these, a keypad lock was developed with AA, glucose and V tot as 3 inputs, and the color change of the PB/PW films as the output. This keypad lock system combined enzymatic catalysis with bipolar electrochemistry, demonstrating some unique advantages such as good reprogrammability, easy resettability and visual readout by naked eye. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. 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

  18. Immobilization of enzymes on radiation-modified gelatine gel by using a chemical cross-linking agent

    International Nuclear Information System (INIS)

    Bachmann, S.; Gebicka, L.; Galant, S.

    1981-01-01

    Investigations into the effect of ionizing radiation on the gelatine gels have shown that water-insoluble gel can be formed under suitable irradiation conditions. To establish the optimal conditions for the processing of the insoluble gel, the yield of cross-linking has been determined for gelatine solutions and its gels irradiated with various doses in the absence and in the presence of oxygen. Glucose isomerase (GI) was used as a test enzyme for immobilization on the gelatine gel. This enzyme which catalyses the isomerization of glucose to fructose has been used on the commercial-scale production of high fructose syrups. The support matrix chosen for the enzyme immobilization has been obtained by irradiating 4% wt/vol. de-aerated gelatine gel at a dose of 1.5 x 10 4 kGy at 15 0 C. Actinoplanes missouriensis cells containing GI were mixed with gelatine gel particles and cross-linked with glutaraldehyde. It was found that the immobilized GI can be successfully applied in the continuous isomerization of glucose to fructose. (author)

  19. 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

  20. Biofabrication Using Pyrrole Electropolymerization for the Immobilization of Glucose Oxidase and Lactate Oxidase on Implanted Microfabricated Biotransducers

    Directory of Open Access Journals (Sweden)

    Christian N. Kotanen

    2014-03-01

    Full Text Available The dual responsive Electrochemical Cell-on-a-Chip Microdisc Electrode Array (ECC MDEA 5037 is a recently developed electrochemical transducer for use in a wireless, implantable biosensor system for the continuous measurement of interstitial glucose and lactate. Fabrication of the biorecognition membrane via pyrrole electropolymerization and both in vitro and in vivo characterization of the resulting biotransducer is described. The influence of EDC-NHS covalent conjugation of glucose oxidase with 4-(3-pyrrolyl butyric acid (monomerization and with 4-sulfobenzoic acid (sulfonization on biosensor performance was examined. As the extent of enzyme conjugation was increased sensitivity decreased for monomerized enzymes but increased for sulfonized enzymes. Implanted biotransducers were examined in a Sprague-Dawley rat hemorrhage model. Resection after 4 h and subsequent in vitro re-characterization showed a decreased sensitivity from 0.68 (±0.40 to 0.22 (±0.17 µA·cm−2·mM−1, an increase in the limit of detection from 0.05 (±0.03 to 0.27 (±0.27 mM and a six-fold increase in the response time from 41 (±18 to 244 (±193 s. This evidence reconfirms the importance of biofouling at the bio-abio interface and the need for mitigation strategies to address the foreign body response.

  1. Adaptation of the hypothalamic-pituitary-adrenal axis and glucose to repeated immobilization or restraint stress is not influenced by associative signals.

    Science.gov (United States)

    Rabasa, Cristina; Delgado-Morales, Raúl; Muñoz-Abellán, Cristina; Nadal, Roser; Armario, Antonio

    2011-02-02

    Repeated exposure to the same stressor very often results in a reduction of some prototypical stress responses, namely those related to the hypothalamic-pituitary-adrenal (HPA) and sympatho-medullo-adrenal (SMA) axes. This reduced response to repeated exposure to the same (homotypic) stressor (adaptation) is usually considered as a habituation-like process, and therefore, a non-associative type of learning. However, there is some evidence that contextual cues and therefore associative processes could contribute to adaptation. In the present study we demonstrated in two experiments using adult male rats that repeated daily exposure to restraint (REST) or immobilization on boards (IMO) reduced the HPA (plasma levels of ACTH and corticosterone) and glucose responses to the homotypic stressor and such reduced responses remained intact when all putative cues associated to the procedure (experimenter, way of transporting to the stress room, stress boxes, stress room and colour of the restrainer in the case of REST) were modified on the next day. Therefore, the present results do not favour the view that adaptation after repeated exposure to a stressor may involve associative processes related to signals predicting the imminence of the stressors, but more studies are needed on this issue. Copyright © 2010 Elsevier B.V. All rights reserved.

  2. 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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  4. Ethanol production from glucose and xylose by immobilized Thermoanaerobacter pentosaceus at 70 °C in an up-flow anaerobic sludge blanket (UASB) reactor

    DEFF Research Database (Denmark)

    Sittijunda, Sureewan; Tomás, Ana Faria; Reungsang, Alissara

    2013-01-01

    The newly isolated extreme thermophilic ethanologen Thermoanaerobacter pentosaceus was immobilized in different support materials in order to improve its ethanol production ability. In batch fermentation, a maximum ethanol yield of 1.36 mol mol-1 consumed sugars was obtained by T. pentosaceus...... occurred at an HRT of 6 h. The maximum ethanol yield and concentration, 1.50 mol mol-1 consumed sugars and 12.4 g l-1, were obtained with an HRT of 12 h. The latter represented an improvement of 60 % in relation to previously obtained results. This indicates that immobilization of T. pentosaceus...... immobilized on rapeseed straw. Additionally, immobilized T. pentosaceus’ ethanol production was improved by 11 % in comparison to free cells. In continuous mode, it was shown that hydraulic retention time (HRT) affected ethanol yield, and a dramatic shift from ethanol to acetate and lactate production...

  5. 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...

  6. Direct measurement of glucose profiles in immobilized yeast gels with a pH-insensitive micro-electrode under anaerobic conditions

    NARCIS (Netherlands)

    Cronenberg, C.C.H.; Heuvel, van den J.C.; Ottengraf, S.P.P.

    1993-01-01

    A 10 µm glucose sensor was developed based on a glucose oxidase coated Pt-electrode inserted in a capillary shaft. The internal buffer medium effected in a glucose response that was insensitive for the external pH. The sensor was successfully utilized at pH 4 under anaerobic conditions in gel

  7. Analytical systems as a basis for immobilized enzymes. 3. Use of a glucose enzyme electrode to determine carbohydrates in biological solutions

    Energy Technology Data Exchange (ETDEWEB)

    Kulys, J; Pesliakiene, M

    1981-01-01

    A method is described for determination of glucose, sucrose, and lactose in biological solutions using a glucose enzyme electrode characterized by high sensitivity and selectivity. The enzyme membrane (15 nm thick) is prepared from glucose oxidase isolated from Penicillium vitale. Glucose is determined in one minute (using static currents) or in 12 s (using registered current in a kinetic regime). Phosphate buffer (5-10 mM) is the only reagent required for analysis. Determination of sucrose and lactose require prior hydrolysis with 17.8% HCl at 70 degrees Celcius for O.5 and lO.7 minutes, respectively.

  8. 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.

  9. 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.

  10. Microorganism immobilization

    Science.gov (United States)

    Compere, Alicia L.; Griffith, William L.

    1981-01-01

    Live metabolically active microorganisms are immobilized on a solid support by contacting particles of aggregate material with a water dispersible polyelectrolyte such as gelatin, crosslinking the polyelectrolyte by reacting it with a crosslinking agent such as glutaraldehyde to provide a crosslinked coating on the particles of aggregate material, contacting the coated particles with live microorganisms and incubating the microorganisms in contact with the crosslinked coating to provide a coating of metabolically active microorganisms. The immobilized microorganisms have continued growth and reproduction functions.

  11. Chitosan-guar gum-silver nanoparticles hybrid matrix with immobilized enzymes for fabrication of beta-glucan and glucose sensing photometric flow injection system.

    Science.gov (United States)

    Bagal-Kestwal, Dipali R; Kestwal, Rakesh Mohan; Hsieh, Wen-Ting; Chiang, Been-Huang

    2014-01-01

    Simple and fast photometric flow injection analysis system was developed for sensing of β-1,3-glucan from medicinal mushroom Ganoderma lucidum during fermentation. For this purpose, the chitosan-guar gum-silver nanoparticle-beta glucanase (Ch-GG-AgNPs-βG) beads and Ch-GG-AgNPs-GOD (glucose oxidase) beads were prepared. The bead packed mini-columns were then used to assemble a flow injection analysis (FIA) system for the detection of β-(1→3)-d-glucan biomarker or glucose. This colorimetric flow system can detect glucose and glucan with detection limits as low as 50ngmL(-1) and 100ngmL(-1) (S/N=3), respectively. The analysis time of this FIA was approximately 40s, which is faster than the previously reported glucan sensors. The glucose and glucan calibration curves were obtained in the range of 0.25-1.25μgmL(-1) (R(2)=0.988) and 0.2-1.0μgmL(-1)(R(2)=0.979), respectively. The applicability of the nano-bio-composite FIA sensor system for spiked and real β-(1→3)-d-glucan samples were tested, and the accuracy of the results were greater than 95%. Thus, the designed FIA provides a simple, interference free and rapid tool for monitoring glucose and β-glucan content, which can be used for various food samples with a little modification. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Immobilization of glucose oxidase into a nanoporous TiO₂ film layered on metallophthalocyanine modified vertically-aligned carbon nanotubes for efficient direct electron transfer.

    Science.gov (United States)

    Cui, Hui-Fang; Zhang, Kuan; Zhang, Yong-Fang; Sun, Yu-Long; Wang, Jia; Zhang, Wei-De; Luong, John H T

    2013-08-15

    Glucose oxidase (GOD) was adsorbed into a nanoporous TiO₂ film layered on the surface of an iron phthalocyanine (FePc) vertically-aligned carbon nanotube (CNT) modified electrode. A Nafion film was then dropcast on the electrode's surface to improve operational and storage stabilities of the GOD-based electrode. Scanning electron microscopy (SEM) micrographs revealed the formation of FePc and nanoporous TiO₂ nanoparticles along the sidewall and the tip of CNTs. Cyclic voltammograms of the GOD electrode in neutral PBS exhibited a pair of well-defined redox peaks, attesting the direct electron transfer of GOD (FAD/FADH₂) with the underlying electrode. The potential of glucose electro-oxidation under nitrogen was ∼+0.12 V with an oxidation current density of 65.3 μA cm(-2) at +0.77 V. Voltammetric and amperometric responses were virtually unaffected by oxygen, illustrating an efficient and fast direct electron transfer. The modification of the CNT surface with FePc resulted in a biosensor with remarkable detection sensitivity with an oxygen-independent bioelectrocatalysis. In deaerated PBS, the biosensor displayed average response time of 12 s, linearity from 50 μM to 4 mM, and a detection limit of 30 μM (S/N=3) for glucose. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. 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.

  14. 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

  15. Multi-input and -output logic circuits based on bioelectrocatalysis with horseradish peroxidase and glucose oxidase immobilized in multi-responsive copolymer films on electrodes.

    Science.gov (United States)

    Yu, Xue; Lian, Wenjing; Zhang, Jiannan; Liu, Hongyun

    2016-06-15

    Herein, poly(N-isopropylacrylamide-co-N,N'-dimethylaminoethylmethacrylate) copolymer films were polymerized on electrode surface with a simple one-step method, and the enzyme horseradish peroxidase (HRP) was embedded in the films simultaneously, which were designated as P(NiPAAm-co-DMEM)-HRP. The films exhibited a reversible structure change with the external stimuli, such as pH, CO2, temperature and SO4(2-), causing the cyclic voltammetric (CV) response of electroactive K3Fe(CN)6 at the film electrodes to display the corresponding multi-stimuli sensitive ON-OFF behavior. Based on the switchable CV property of the system and the electrochemical reduction of H2O2 catalyzed by HRP in the films and mediated by Fe(CN)6(3-) in solution, a 5-input/3-output logic gate was established. To further increase the complexity of the logic system, another enzyme glucose oxidase (GOD) was added into the films, designated as P(NiPAAm-co-DMEM)-HRP-GOD. In the presence of oxygen, the oxidation of glucose in the solution was catalyzed by GOD in the films, and the produced H2O2 in situ was recognized and electrocatalytically reduced by HRP and mediated by Fe(CN)6(3-). Based on the bienzyme films, a cascaded or concatenated 4-input/3-output logic gate system was proposed. The present work combined the multi-responsive interface with bioelectrocatalysis to construct cascaded logic circuits, which might open a new avenue to develop biocomputing elements with more sophisticated functions and design novel glucose biosensors. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. 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...

  17. 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.

  18. Surface modification of Fe{sub 2}O{sub 3}/Fe{sub 3}O{sub 4} nanocomposites for use in immobilization of glucose oxidase; Modificacao da superficie de nanocompositos de Fe{sub 2}O{sub 3}/Fe{sub 3}O{sub 4} visando seu uso para imobilizacao da glicose oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Albuquerque, I.L.T.; Santos, P.T.A.; Costa, A.C.F.M., E-mail: izabelleliz@hotmail.com, E-mail: patytaraujo@gmail.com, E-mail: ana.costa@ufcg.edu.br [Universidade Federal de Campina Grande (UFCG), PB (Brazil). Dept. de Engenharia de Materiais; Cornejo, D.R., E-mail: daniel.r.cornejo@gmail.com [Universidade de Sao Paulo (USP), SP (Brazil). Inst. de Fisica; Bicalho, S.M.C.M., E-mail: dbrandao@jhs.med.br [JHS Lab. Quimico, Sabara, MG (Brazil); Oliveira, L.S.C., E-mail: libiaconrado@yahoo.com.br [Universidade Federal de Campina Grande (UFCG), PB (Brazil). Dept. de Engenharia Quimica

    2017-04-15

    The increase in the number of people with diabetes in recent years and the high cost-benefit ratio of the existing biosensor technology have increased the interest for the development of glucose detection biosensor based on immobilization of glucose-oxidase (GOD) mainly using magnetic nanoparticles. In this context, nanocomposites of Fe{sub 2}O{sub 3}/Fe{sub 3}O{sub 4} were prepared by combustion reaction and their surface was functionalized with 3-aminopropyltriethoxysilane via silanization reaction and with chitosan via functionalization to obtain a hybrid material that was evaluated as possible GOD immobilizer. The samples were characterized by powder X-ray diffraction, Fourier transform infrared spectroscopy, thermogravimetry, scanning electron microscopy, transmission electron microscopy, magnetic properties and in vitro cytotoxicity. The results revealed that it was possible to obtain the ferrimagnetic composite, the surface modification reduced the saturation magnetization, but maintained the ferrimagnetic characteristics, and all samples were considered non-toxic. For preliminary testing of the GOD immobilization it was revealed that the nanocomposite modified with silane and chitosan showed the better result, about 2.7 mg of immobilized GOD for 100 mg of nanocomposite, which makes this material a potential alternative to manufacture GOD biosensors. (author)

  19. 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.

  20. 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.

  1. 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. ...

  2. 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...

  3. Enzyme-immobilized SiO2-Si electrode: Fast interfacial electron transfer with preserved enzymatic activity

    Science.gov (United States)

    Wang, Gang; Yau, Siu-Tung

    2005-12-01

    The enzyme, glucose oxidase (GOx), is immobilized using electrostatic interaction on the native oxide of heavily doped n-type silicon. Voltammetric measurement shows that the immobilized GOx gives rise to a very fast enzyme-silicon interfacial electron transfer rate constant of 7.9s-1. The measurement also suggests that the enzyme retains its native conformation when immobilized on the silicon surface. The preserved native conformation of GOx is further confirmed by testing the enzymatic activity of the immobilized GOx using glucose. The GOx-immobilized silicon is shown to behave as a glucose sensor that detects glucose with concentrations as low as 50μM.

  4. Immobilization of oxidases and their analytical applications

    International Nuclear Information System (INIS)

    Yasinzai, M.

    2007-01-01

    Immobilized enzymes are replacing their soluble counter-parts in nearly every field of application. These enzyme modifications have evolved from a research curiosity into an entire branch of Biotechnology. An immobilization method for flavin containing oxidases and their use in flow injection system is described. An electrochemical detector for H/sub 2/O/sub 2/ is assembled which is used effectively for the determination of glucose using more common glucose oxidase and the simultaneous determination of sugars. The combination of oxidases with hydrolases have been used for the determination of maltose and starch. (author)

  5. Glucose production for cellulose

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, S; Karube, I

    1977-04-16

    Glucose was produced from cellulose by passing a cellulose solution through a column of an immobilized cellulase which was prepared by coating an inorganic carrier such as macadam or stainless steel beads with collagen containing the cellulase. Thus, 4 mL of 5% cellulase T-AP (60,000 units/g) solution was dissolved in 100 g of 0.9% collagen solution and the solution mixed with 60 g of macadam (diam. = 0.5 to 1.5 mm) and stirred for 10 min. The treated beads were dried in air at 10/sup 0/ to yield an immobilized enzyme retaining 64% of its activity. Through a column (0.8 x 20 cm) packed with 3 g of the immobilized enzyme, 100 mL of 0.33% Avicel SF solution was circulated at 26.4 mL/min at 30/sup 0/ for 60 h. The Avicel SF conversion to glucose was 23%.

  6. 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, ...

  7. 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 ...

  8. [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.

  9. 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.

  10. Preparation of immobilized growing cells and enzymatic hydrolysis of sawdust

    International Nuclear Information System (INIS)

    Kumakura, M.; Kaetsu, I.

    1984-01-01

    Trichoderma reesei cells were immobilized by radiation polymerization using porous materials such as non-woven material and sawdust, and the enzymatic hydrolysis of sawdust with the enzyme solution from the immobilized growing cells was studied. The filter paper activity, which shows the magnitude of cellulase production in the immobilized cells, was comparable with that in the intact cells. The filter paper activity was affected by addition concentration of monomer and porous materials. The cells in the immobilized cells grew to be adhered on the surface of the fibrous polymers. Sawdust, which was pretreated by irradiation technique, was effectively hydrolyzed with the enzyme solution resulting from the culture of the immobilized cells, in which the glucose yield increased increasing the culture time of the immobilized cells. (author)

  11. Insulin action in human thighs after one-legged immobilization

    DEFF Research Database (Denmark)

    Richter, Erik; Kiens, Bente; Mizuno, M.

    1989-01-01

    Insulin action was assessed in thighs of five healthy young males who had one knee immobilized for 7 days by a splint. The splint was not worn in bed. Subjects also used crutches to prevent weight bearing of the immobilized leg. Immobilization decreased the activity of citrate synthase and 3-OH......-acyl-CoA-dehydrogenase in the vastus lateralis muscle by 9 and 14%, respectively, and thigh volume by 5%. After 7 days of immobilization, a two-step euglycemic hyperinsulinemic clamp procedure combined with arterial and bilateral femoral venous catheterization was performed. Insulin action on glucose uptake and tyrosine release...... of the thighs at mean plasma insulin concentrations of 67 (clamp step I) and 447 microU/ml (clamp step II) was decreased by immobilization, whereas immobilization did not affect insulin action on thigh exchange of free fatty acids, glycerol, O2, or potassium. Before and during the clamp step I, lactate release...

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

    Science.gov (United States)

    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.

  13. 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

  14. 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

  15. 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.

  16. Halloysite Clay Nanotubes for Enzyme Immobilization.

    Science.gov (United States)

    Tully, Joshua; Yendluri, Raghuvara; Lvov, Yuri

    2016-02-08

    Halloysite clay is an aluminosilicate nanotube formed by rolling flat sheets of kaolinite clay. They have a 15 nm lumen, 50-70 nm external diameter, length of 0.5-1 μm, and different inside/outside chemistry. Due to these nanoscale properties, they are used for loading, storage, and controlled release of active chemical agents, including anticorrosions, biocides, and drugs. We studied the immobilization in halloysite of laccase, glucose oxidase, and lipase. Overall, negatively charged proteins taken above their isoelectric points were mostly loaded into the positively charged tube's lumen. Typical tube loading with proteins was 6-7 wt % from which one-third was released in 5-10 h and the other two-thirds remained, providing enhanced biocatalysis in nanoconfined conditions. Immobilized lipase showed enhanced stability at acidic pH, and the optimum pH shifted to more alkaline pH. Immobilized laccase was more stable with respect to time, and immobilized glucose oxidase showed retention of enzymatic activity up to 70 °C, whereas the native sample was inactive.

  17. 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.

  18. Studies on the preparation of immobilized enzymes by radiopolymerization, (9)

    International Nuclear Information System (INIS)

    Kawashima, Koji; Fujino, Satomi; Hayashi, Toru; Kim, Sung-K.

    1982-01-01

    Glucose Oxidase (GOD, EC 1, 1, 3, 4) was immobilized in the form of the beads by the radiation polymerization method under low temperature and the enzymatic characteristics were investigated. 1) Polyethyleneglycol dimethacrylate and acrylamide were favorable compounds for the immobilization of GOD. 2) Neither optimum pH nor pH stability was changed after immobilization treatment. 3) Optimum reaction temperature was shifted by 5 0 C to the higher side and heat stability was improved. 4) Immobilized GOD showed activity up to 60U per gram of dried polymer. 5) The small beads had retained high activities (10 - 80%) 6) The immobilized GOD was not leached out from the polymer matrix. (author)

  19. 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 ...

  20. Studies of the immobilization of enzymes and microorganism pt.1

    International Nuclear Information System (INIS)

    Kim, S.K.

    1979-01-01

    A new method of immobilization of glucose oxidase by the aerobic gamma radiation of synthetic monomers was developed. The radiocopolymerization was conducted aerobically at -70 to-80 degC with the mixture of several polyfunctional esters, acrylates and native enzyme. The retained activity of immobilized glucoseoxidase was about 50 to 55% when a NK 23G ester, acrylamide-bis and water mixture (1:1:2) in cold toluene treated with 450 Krad of gamma radiation. The radiation dose did not influence significantly to the enzyme activity. The solvents used to prepare the beads of glucose oxidase and monomers were toluene, n-hexane, petoleum ether and chloroform. 0.05M tris-gycerol(pH 7.0) was a more suitable buffer solution for immobilizing the enzyme than was 0.02M phosphate. Immobilization of glucose oxidase shifted the optimum pH for its reaction from 6.0 to 6.5. The pH profile for the immobilized enzyme showed a broad range of optimum activity while the native enzyme gave a sharp pick for its optimum pH value. The immobilized enzyme reaction temperature was at the range of 30-40 degreesC. (Author)

  1. Immobilized waste leaching

    International Nuclear Information System (INIS)

    Suarez, A.A.

    1989-01-01

    The main mechanism by which the immobilized radioactive materials can return to biosphere is the leaching due to the intrusion of water into the repositories. Some mathematical models and experiments utilized to evaluate the leaching rates in different immobilization matrices are described. (author) [pt

  2. Preparation and activity of bubbling-immobilized cellobiase within chitosan-alginate composite.

    Science.gov (United States)

    Wang, Fang; Su, Rong-Xin; Qi, Wei; Zhang, Ming-Jia; He, Zhi-Min

    2010-01-01

    Cellobiase can hydrolyze cellobiose into glucose; it plays a key role in the process of cellulose hydrolysis by reducing the product inhibition. To reuse the enzyme and improve the economic value of cellulosic ethanol, cellobiase was immobilized using sodium alginate and chitosan as carriers by the bubbling method. The immobilization conditions were optimized as follows: enzyme loading of 100 U cellobiase/g carrier, 30 min immobilization, 3.5 wt% sodium alginate, 0.25 wt% chitosan, and 2 wt% calcium chloride. Compared to free enzyme, the immobilized cellobiase had a decreased apparent K(m) and the maximum activity at a lower pH, indicating its higher acidic and thermal stability. The immobilized cellobiase was further tested in the hydrolysis of cellobiose and various cellulosic substrates (microcrystalline cellulose, filter paper, and ammonia-pretreated corn cobs). Together with cellulases, the immobilized cellobiase converted the cellulosic substrates into glucose with the rate and extent similar to the free enzyme.

  3. Immobilizing live Escherichia coli for AFM studies of surface dynamics

    International Nuclear Information System (INIS)

    Lonergan, N.E.; Britt, L.D.; Sullivan, C.J.

    2014-01-01

    Atomic force microscopy (AFM) is a probe-based technique that permits high resolution imaging of live bacterial cells. However, stably immobilizing cells to withstand the probe-based lateral forces remains an obstacle in AFM mediated studies, especially those of live, rod shaped bacteria in nutrient media. Consequently, AFM has been under-utilized in the research of bacterial surface dynamics. The aim of the current study was to immobilize a less adherent Escherichia coli strain in a method that both facilitates AFM imaging in nutrient broth and preserves overall cell viability. Immobilization reagents and buffers were systematically evaluated and the cell membrane integrity was monitored in all sample preparations. As expected, the biocompatible gelatin coated surfaces facilitated stable cell attachment in lower ionic strength buffers, yet poorly immobilized cells in higher ionic strength buffers. In comparison, poly-L-lysine surfaces bound cells in both low and high ionic strength buffers. The benefit of the poly-L-lysine binding capacity was offset by the compromised membrane integrity exhibited by cells on poly-L-lysine surfaces. However, the addition of divalent cations and glucose to the immobilization buffer was found to mitigate this unfavorable effect. Ultimately, immobilization of E. coli cells on poly-L-lysine surfaces in a lower ionic strength buffer supplemented with Mg 2+ and Ca 2+ was determined to provide optimal cell attachment without compromising the overall cell viability. Cells immobilized in this method were stably imaged in media through multiple division cycles. Furthermore, permeability assays indicated that E. coli cells recover from the hypoosmotic stress caused by immobilization in low ionic strength buffers. Taken together, this data suggests that stable immobilization of viable cells on poly-L-lysine surfaces can be accomplished in lower ionic strength buffers that are supplemented with divalent cations for membrane stabilization while

  4. 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...

  5. Kinetic and mass transfer studies on the isomerization of cellulose hydrolyzate using immobilized Streptomyces cells

    Energy Technology Data Exchange (ETDEWEB)

    Ghose, T K; Chand, S

    1978-01-01

    Streptomyces cells possessing glucose isomerase activity, heat-treated and confined within polyester sacs have been used in batch/continuous isomerization of enzymatically hydrolyzed microcrystalline cellulose. Conversion data at different concentrations of substrate closely follow the reactor performance equation based on the reaction kinetics. The effect of external film and pore diffusional resistances were experimentally found to be negligible. The dispersion effects in the packed bed column have been evaluated by pulse input tracer analysis. Continuous operation of the column to isomerize cellulose hydrolyzate (2.0 M glucose) showed an exponential deactivation of enzyme activity with a half-life of 447 h.

  6. 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.

  7. Plutonium Disposition by Immobilization

    International Nuclear Information System (INIS)

    Gould, T.; DiSabatino, A.; Mitchell, M.

    2000-01-01

    The ultimate goal of the Department of Energy (DOE) Immobilization Project is to develop, construct, and operate facilities that will immobilize between 17 to 50 tonnes (MT) of U.S. surplus weapons-usable plutonium materials in waste forms that meet the ''spent fuel'' standard and are acceptable for disposal in a geologic repository. Using the ceramic can-in-canister technology selected for immobilization, surplus plutonium materials will be chemically combined into ceramic forms which will be encapsulated within large canisters of high level waste (HLW) glass. Deployment of the immobilization capability should occur by 2008 and be completed within 10 years. In support of this goal, the DOE Office of Fissile Materials Disposition (MD) is conducting development and testing (D and T) activities at four DOE laboratories under the technical leadership of Lawrence Livermore National Laboratory (LLNL). The Savannah River Site has been selected as the site for the planned Plutonium Immobilization Plant (PIP). The D and T effort, now in its third year, will establish the technical bases for the design, construction, and operation of the U. S. capability to immobilize surplus plutonium in a suitable and cost-effective manner. Based on the D and T effort and on the development of a conceptual design of the PIP, automation is expected to play a key role in the design and operation of the Immobilization Plant. Automation and remote handling are needed to achieve required dose reduction and to enhance operational efficiency

  8. Alcoholic fermentation by immobilized yeast at high sugar concentrations

    Energy Technology Data Exchange (ETDEWEB)

    Holcberg, I.B.; Margalith, P.

    1981-01-01

    Glucose fermentation by Saccharomyces cerevisiae immobilized by entrapment in agar, carrageenan, alginate and polyacrylamide gels, was compared to that of freely suspended cells at concentration of 10-50% (w.w.) sugar. The rate of ethanol production by the entrapped cells was 20-25% higher than that of the free cells. Concentrations of up to 14.5% w/w ethanol (30% glucose initial concentration) could be obtained. A number of hypotheses for the improved alcoholic fermentation are discussed.

  9. 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

  10. 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

  11. Ethanol production by immobilized cells with forced substrate supply

    Energy Technology Data Exchange (ETDEWEB)

    Mitani, Y.; Nishizawa, Y.; Nagai, S.

    1984-01-01

    Ethanol fermentation by a forced substrate supply into an immobilized cell layer was carried out to increase the ethanol production rate and to eliminate the diffusion dependency of substrate supply in an ordinary immobilized cell reaction. Saccharomyces cerevisiae IFO 2347 was immobilized in a mixture of k-carrageenan, locust bean gum, and celite (2: 0.5: 40 wt/vol %). A glucose minimal medium was fed into the immobilized cell layer (5 to 22 mm in thickness) at retention times between 0.6 and 2.8 h under pressure. The stable ethanol fermentation could be maintained for more than 3 weeks with an ethanol yield of 0.48 g ethanol/g glucose and ethanol productivity of 63 g.(l gel)/sup -1/.h/sup -1/ at a retention time of 1.5 h. The yeast cells were well distributed through the gel layer with a vertical gradient, and an average cell density was ca. 8.0 X 10/sup 9/ cells/ml gel, 4-fold higher than that of ordinary immobilized cells. A small filter press reactor was constructed to examine the applicability of ethanol fermentation with this forced substrate supply. The operation could be continued for a month at a retention time of 2 h yielding 96 g/l of ethanol from 200 g/l of glucose. 6 references, 5 figures, 3 tables.

  12. Ethanol fermentation by immobilized cells of Zymomonas mobilis

    Energy Technology Data Exchange (ETDEWEB)

    Grote, W.

    1985-01-01

    Previous studies have shown that immobilized yeast cell cultures have commercial potential for fuel ethanol production. In this study the suitability of strains of Z. mobilis for whole cell immobilization was investigated. Experiments revealed that immobilization in Ca-alginate or K-carrageenan gel or use of flocculating strains was effective for ethanol production at relatively high productivities. Two laboratory size reactors were designed and constructed. These were a compartmented multiple discshaft column and a tower fermentor. Results of this work supported other studies that established that growth and fermentation could be uncoupled. The data indicated that specific metabolic rates were dependent on the nature of the fermentation media. The addition of lactobacilli to Z. mobilis continuous fermentations had only a transient effect, and was unlikely to affect an immobilized Z. mobilis process. With 150 gl/sup -1/ glucose media and a Z. mobilis ZM4 immobilized cell reactor, a maximum volumetric ethanol productivity of 55 gl/sup -1/h/sup -1/ was obtained. The fermentation of sucrose media or sucrose-based raw materials (molasses, cane juice, synthetic mill liquor) by immobilized Z. mobilis ZM4 revealed a pattern of rapid sucrose hydrolysis, preferential glucose utilization and the conversion of fructose to the undesirable by-products levan and sorbitol.

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

    Science.gov (United States)

    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.

  14. 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...

  15. 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

  16. 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.

  17. Glucose Sensing

    CERN Document Server

    Geddes, Chris D

    2006-01-01

    Topics in Fluorescence Spectroscopy, Glucose Sensing is the eleventh volume in the popular series Topics in Fluorescence Spectroscopy, edited by Drs. Chris D. Geddes and Joseph R. Lakowicz. This volume incorporates authoritative analytical fluorescence-based glucose sensing reviews specialized enough to be attractive to professional researchers, yet also appealing to the wider audience of scientists in related disciplines of fluorescence. Glucose Sensing is an essential reference for any lab working in the analytical fluorescence glucose sensing field. All academics, bench scientists, and industry professionals wishing to take advantage of the latest and greatest in the continuously emerging field of glucose sensing, and diabetes care & management, will find this volume an invaluable resource. Topics in Fluorescence Spectroscopy Volume 11, Glucose Sensing Chapters include: Implantable Sensors for Interstitial Fluid Smart Tattoo Glucose Sensors Optical Enzyme-based Glucose Biosensors Plasmonic Glucose Sens...

  18. Mediator-free interaction of glucose oxidase, as model enzyme for immobilization, with Al-doped and undoped ZnO thin films laser-deposited on polycarbonate supports.

    Science.gov (United States)

    V T K P, Fidal; Inguva, Saikumar; Krishnamurthy, Satheesh; Marsili, Enrico; Mosnier, Jean-Paul; T S, Chandra

    2017-01-01

    Al doped and undoped ZnO thin films were deposited by pulsed-laser deposition on polycarbonate sheets. The films were characterized by optical transmission, Hall effect measurement, XRD and SEM. Optical transmission and surface reflectometry studies showed good transparency with thicknesses ∼100nm and surface roughness of 10nm. Hall effect measurements showed that the sheet carrier concentration was -1.44×10 15 cm -2 for AZO and -6×10 14 cm -2 for ZnO. The films were then modified by drop-casting glucose oxidase (GOx) without the use of any mediators. Higher protein concentration was observed on ZnO as compared to AZO with higher specific activity for ZnO (0.042Umg -1 ) compared to AZO (0.032Umg -1 ), and was in agreement with cyclic voltemmetry (CV). X-ray photoelectron spectroscopy (XPS) suggested that the protein was bound by dipole interactions between AZO lattice oxygen and the amino group of the enzyme. Chronoamperometry showed sensitivity of 5.5μAmM -1 cm -2 towards glucose for GOx/AZO and 2.2μAmM -1 cm -2 for GOx/ZnO. The limit of detection (LoD) was 167μM of glucose for GOx/AZO, as compared to 360μM for GOx/ZnO. The linearity was 0.28-28mM for GOx/AZO whereas it was 0.6-28mM for GOx/ZnO with a response time of 10s. Possibly due to higher enzyme loading, the decrease of impedance in presence of glucose was larger for GOx/ZnO as compared to GOx/AZO in electrochemical impedance spectroscopy (EIS). Analyses with clinical blood serum samples showed that the systems had good reproducibility and accuracy. The characteristics of novel ZnO and AZO thin films with GOx as a model enzyme, should prove useful for the future fabrication of inexpensive, highly sensitive, disposable electrochemical biosensors for high throughput diagnostics. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Development of High-Productivity Continuous Ethanol Production using PVA-Immobilized Zymomonas mobilis in an Immobilized-Cells Fermenter

    Directory of Open Access Journals (Sweden)

    Nurhayati Nurhayati

    2015-07-01

    Full Text Available Ethanol as one of renewable energy was being considered an excellent alternative clean-burning fuel to replace gasoline. Continuous ethanol fermentation systems had offered important economic advantages compared to traditional systems. Fermentation rates were significantly improved, especially when continuous fermentation was integrated with cell immobilization techniques to enrich the cells concentration in fermentor. Growing cells of Zymomonas mobilis immobilized in polyvinyl alcohol (PVA gel beads were employed in an immobilized-cells fermentor for continuous ethanol fermentation from glucose. The glucose loading, dilution rate, and cells loading were varied in order to determine which best condition employed in obtaining both high ethanol production and low residual glucose with high dilution rate. In this study, 20 g/L, 100 g/L, 125 g/L and 150 g/L of glucose concentration and 20% (w/v, 40% (w/v and 50% (w/v of cells loading were employed with range of dilution rate at 0.25 to 1 h-1. The most stable production was obtained for 25 days by employing 100 g/L of glucose loading. Meanwhile, the results also exhibited that 125 g/L of glucose loading as well as 40% (w/v of cells loading yielded high ethanol concentration, high ethanol productivity, and acceptable residual glucose at 62.97 g/L, 15.74 g/L/h and 0.16 g/L, respectively. Furthermore, the dilution rate of 4 hour with 100 g/L and 40% (w/v of glucose and cells loading was considered as the optimum condition with ethanol production, ethanol productivity and residual glucose obtained were 49.89 g/L, 12.47 g/L/h, and 2.04 g/L, respectively. This recent study investigated ethanol inhibition as well. The present research had proved that high sugar concentration was successfully converted to ethanol. These achieved results were promising for further study.

  20. Glucose oxidase and graphene bionanocomposite bridged by ionic liquid unit for glucose biosensing application

    DEFF Research Database (Denmark)

    Jiang, Yuanyuan; Zhang, Qixian; Li, Fenghua

    2012-01-01

    activity towards the reduction of O2 and H2O2. Then negatively charged glucose oxidase (GOD) was immobilized onto the composite matrix simply by ionic exchange. The ionic liquid here could improve the dispersibility of graphene and provide a favorable conductive microenvironment for the immobilized GOD......, thus promote its direct electron transfer at the GC electrode. This novel IL-graphene–GOD bionanocomposite could act as a biosensor towards the detection of glucose with a linear response up to 16mM. In this report, the method for immobilizing GOD by ionic interaction is of universality and has...... widespread use, even in other biological systems, which brings a forceful combination between GOD and IL-graphene. Besides, the biosensor is easy to prepare, have good stability, and will have potential application in glucose detection....

  1. Electrochemical Glucose Biosensor Based on Glucose Oxidase Displayed on Yeast Surface.

    Science.gov (United States)

    Wang, Hongwei; Lang, Qiaolin; Liang, Bo; Liu, Aihua

    2015-01-01

    The conventional enzyme-based biosensor requires chemical or physical immobilization of purified enzymes on electrode surface, which often results in loss of enzyme activity and/or fractions immobilized over time. It is also costly. A major advantage of yeast surface display is that it enables the direct utilization of whole cell catalysts with eukaryote-produced proteins being displayed on the cell surface, providing an economic alternative to traditional production of purified enzymes. Herein, we describe the details of the display of glucose oxidase (GOx) on yeast cell surface and its application in the development of electrochemical glucose sensor. In order to achieve a direct electrochemistry of GOx, the entire cell catalyst (yeast-GOx) was immobilized together with multiwalled carbon nanotubes on the electrode, which allowed sensitive and selective glucose detection.

  2. 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.

  3. 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.

  4. Immobilization of microorganisms. Part 1. Preparation of immobilized Lactobacillus bulgaricus

    Energy Technology Data Exchange (ETDEWEB)

    Lee, K H

    1981-01-01

    The immobilization of Lactobacillus bulgaricus on polyacrylamide and on alginate beads was investigated. The most active immobilized cells were obtained by entrapment in Ca alginate beads. These immobilized microbial cells, when introduced into 4.5% lactose solution and whey solution showed maximum relative activity of 28% for lactose and 18% for whey compared to free cells.

  5. Limb immobilization and corticobasal syndrome.

    Science.gov (United States)

    Graff-Radford, Jonathan; Boeve, Bradley F; Drubach, Daniel A; Knopman, David S; Ahlskog, J Eric; Golden, Erin C; Drubach, Dina I; Petersen, Ronald C; Josephs, Keith A

    2012-12-01

    Recently, we evaluated two patients with corticobasal syndrome (CBS) who reported symptom onset after limb immobilization. Our objective was to investigate the association between trauma, immobilization and CBS. The charts of forty-four consecutive CBS patients seen in the Mayo Clinic Alzheimer Disease Research Center were reviewed with attention to trauma and limb immobilization. 10 CBS patients (23%) had immobilization or trauma on the most affected limb preceding the onset or acceleration of symptoms. The median age at onset was 61. Six patients manifested their first symptoms after immobilization from surgery or fracture with one after leg trauma. Four patients had pre-existing symptoms of limb dysfunction but significantly worsened after immobilization or surgery. 23 percent of patients had immobilization or trauma of the affected limb. This might have implications for management of CBS, for avoiding injury, limiting immobilization and increasing movement in the affected limb. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Membranes suited for immobilizing biomolecules

    NARCIS (Netherlands)

    2009-01-01

    The present invention relates to flow-through membranes suitable for the immobilization of biomols., methods for the prepn. of such membranes and the use of such membranes for the immobilization of biomols. and subsequent detection of immobilized biomols. The invention concerns a flow-through

  7. 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.

  8. 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

  9. 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.

  10. 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...

  11. 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...

  12. Glucose allostasis

    DEFF Research Database (Denmark)

    Stumvoll, Michael; Tataranni, P Antonio; Stefan, Norbert

    2003-01-01

    individuals with normal glucose tolerance, normoglycemia can always be maintained by compensatorily increasing AIR in response to decreasing M (and vice versa). This has been mathematically described by the hyperbolic relationship between AIR and M and referred to as glucose homeostasis, with glucose......In many organisms, normoglycemia is achieved by a tight coupling of nutrient-stimulated insulin secretion in the pancreatic beta-cell (acute insulin response [AIR]) and the metabolic action of insulin to stimulate glucose disposal (insulin action [M]). It is widely accepted that in healthy...... concentration assumed to remain constant along the hyperbola. Conceivably, glucose is one of the signals stimulating AIR in response to decreasing M. Hypothetically, as with any normally functioning feed-forward system, AIR should not fully compensate for worsening M, since this would remove the stimulus...

  13. Glucose and fructose from starch-containing plant products

    Energy Technology Data Exchange (ETDEWEB)

    Mueller, H

    1981-03-13

    Enzymic hydrolysis and isomerization of cocoa kernels or potatoes gave fructose (I)-rich glucose (II) syrup. Thus, a mixture of 150 g cocoa powder and 0.3 g a-amylase in 500 ml H/sub 2/O at pH 7.5 was stirred for approx. 0.5 h at 70/sup 0/ treated with 0.3 amidoglucosidase, stirred for 24 h at 55/sup 0/, neutralized, treated with 0.15 g isomerase, and kept for approx. 10 h at 50-60/sup 0/ to give II syrup containing 50% I.

  14. Pseudo-bi-enzyme glucose sensor: ZnS hollow spheres and glucose oxidase concerted catalysis glucose.

    Science.gov (United States)

    Shuai, Ying; Liu, Changhua; Wang, Jia; Cui, Xiaoyan; Nie, Ling

    2013-06-07

    This work creatively uses peroxidase-like ZnS hollow spheres (ZnS HSs) to cooperate with glucose oxidase (GOx) for glucose determinations. This approach is that the ZnS HSs electrocatalytically oxidate the enzymatically generated H2O2 to O2, and then the O2 circularly participates in the previous glucose oxidation by glucose oxidase. Au nanoparticles (AuNPs) and carbon nanotubes (CNTs) are used as electron transfer and enzyme immobilization matrices, respectively. The biosensor of glucose oxidase-carbon nanotubes-Au nanoparticles-ZnS hollow spheres-gold electrode (GOx-CNT-AuNPs-ZnS HSs-GE) exhibits a rapid response, a low detection limit (10 μM), a wide linear range (20 μM to 7 mM) as well as good anti-interference, long-term longevity and reproducibility.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. 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.

  17. 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

  18. Continuous enzyme reactions with immobilized enzyme tubes prepared by radiation cast-polymerization

    International Nuclear Information System (INIS)

    Kumakura, Minoru; Kaetsu, Isao

    1986-01-01

    Immobilized glucose oxidase tubes were prepared by radiation cast-polymerization of 2-hydroxyethyl methacrylate and tetraethyleneglycol diacrylate monomer at low temperatures. The immobilized enzyme tubes which were spirally set in a water bath were used as reactor, in which the enzyme activity varied with tube size and flow rate of the substrate. The conversion yield of the substrate in continuous enzyme reaction was about 80%. (author)

  19. State and Kinetic Parameters Estimation of Bio-Ethanol Production with Immobilized Cells

    OpenAIRE

    Mihaylova, Iva; Popova, Silviya; Kostov, Georgi; Ignatova, Maya; Lubenova, Velislava; Naydenova, Vessela; Pircheva, Desislava; Angelov, Mihail

    2013-01-01

    In this paper, state and kinetic parameters estimation based on extended Kalman filter (EKF) is proposed. Experimental data from alcoholic fermentation process with immobilized cells is used. The measurements of glucose and ethanol concentration are used as on-line measurements for observers design and biomass concentration is used for results verification. Biomass, substrate and product concentrations inside immobilized compounds are estimated using the proposed algorithm. Monitoring of the ...

  20. Immobilization of enzymes by radiation

    International Nuclear Information System (INIS)

    Kaetsu, I.; Kumakura, M.; Yoshida, M.; Asano, M.; Himei, M.; Tamura, M.; Hayashi, K.

    1979-01-01

    Immobilization of various enzymes was performed by radiation-induced polymerization of glass-forming monomers at low temperatures. Alpha-amylase and glucoamylase were effectively immobilized in hydrophilic polymer carrier such as poly(2-hydroxyethyl methacrylate) and also in rather hydrophobic carrier such as poly(tetraethylene-glycol diacrylate). Immobilized human hemoglobin underwent the reversible oxygenation concomitantly with change of oxygen concentration outside of the matrices. (author)

  1. Effects of immobilization on spermiogenesis

    Science.gov (United States)

    Meitner, E. R.

    1980-01-01

    The influence of immobilization stress on spermiogenesis in rats was investigated. After 96 hour immobilization, histological changes began to manifest themselves in the form of practically complete disappearance of cell population of the wall of seminiferous tubule as well as a markedly increased number of cells with pathologic mitoses. Enzymological investigations showed various changes of activity (of acid and alkaline phosphatase and nonspecific esterase) in the 24, 48, and 96 hour immobilization groups.

  2. 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

  3. 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

  4. Facile direct electron transfer in glucose oxidase modified electrodes

    International Nuclear Information System (INIS)

    Wang Dan; Chen Liwei

    2009-01-01

    Glucose oxidase (GOx) is widely used in the glucose biosensor industry. However, mediatorless direct electron transfer (DET) from GOx to electrode surfaces is very slow. Recently, mediatorless DET has been reported via the incorporation of nanomaterials such as carbon nanotubes and nanoparticles in the modification of electrodes. Here we report GOx electrodes showing DET without the need for any nanomaterials. The enzyme after immobilization with poly-L-lysine (PLL) and Nafion retains the biocatalytic activities and oxidizes glucose efficiently. The amperometric response of Nafion-PLL-GOx modified electrode is linearly proportional to the concentration of glucose up to 10 mM with a sensitivity of 0.75 μA/mM at a low detection potential (-0.460 V vs. Ag/AgCl). The methodology developed in this study will have impact on glucose biosensors and biofuel cells and may potentially simplify enzyme immobilization in other biosensing systems.

  5. 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

  6. Immobilization of microbial cell and yeast cell and its application to biomass conversion using radiation techniques

    International Nuclear Information System (INIS)

    Kaetsu, Isao; Kumakura, Minoru; Fujimura, Takashi; Kasai, Noboru; Tamada, Masao

    1987-01-01

    The recent results of immobilization of cellulase-producing cells and ethanol-fermentation yeast by radiation were reported. The enzyme of cellulase produced by immobilized cells was used for saccharification of lignocellulosic wastes and immobilized yeast cells were used for fermentation reaction from glucose to ethanol. The wastes such as chaff and bagasse were treated by γ-ray or electron-beam irradiation in the presence of alkali and subsequent mechanical crushing, to form a fine powder less than 50 μm in diameter. On the other hand, Trichoderma reesei as a cellulase-producing microbial cell was immobilized on a fibrous carrier having a specific porous structure and cultured to produce cellulase. The enzymatic saccharification of the pretreated waste was carried out using the produced cellulase. The enhanced fermentation process to produce ethanol from glucose with the immobilized yeast by radiation was also studied. The ethanol productivity of immobilized growing yeast cells thus obtained was thirteen times that of free yeast cells in a 1:1 volume of liquid medium to immobilized yeast cells. (author)

  7. Immobilization of microbial cell and yeast cell and its application to biomass conversion using radiation techniques

    Science.gov (United States)

    Kaetsu, Isao; Kumakura, Minoru; Fujimura, Takashi; Kasai, Noboru; Tamada, Masao

    The recent results of immobilization of cellulase-producing cells and ethanol-fermentation yeast by radiation were reported. The enzyme of cellulase produced by immobilized cells was used for saccharification of lignocellulosic wastes and immobilized yeast cells were used for fermentation reaction from glucose to ethanol. The wastes such as chaff and bagasse were treated by γ-ray or electron-beam irradiation in the presence of alkali and subsequent mechanical crushing, to form a fine powder less than 50 μm in diameter. On the other hand, Trichoderma reesei as a cellulase-producing microbial cell was immobilized on a fibrous carrier having a specific porous structure and cultured to produce cellulase. The enzymatic saccharification of the pretreated waste was carried out using the produced cellulase. The enhanced fermentation process to produce ethanol from glucose with the immobilized yeast by radiation was also studied. The ethanol productivity of immobilized growing yeast cells thus obtained was thirteen times that of free yeast cells in a 1:1 volume of liquid medium to immobilized yeast cells.

  8. Immobilization of Aspergillus awamori β-glucosidase on commercial gelatin: An inexpensive and efficient process.

    Science.gov (United States)

    Nishida, Verônica S; de Oliveira, Roselene F; Brugnari, Tatiane; Correa, Rúbia Carvalho G; Peralta, Rosely A; Castoldi, Rafael; de Souza, Cristina G M; Bracht, Adelar; Peralta, Rosane M

    2018-05-01

    In this work, a β-glucosidase of Aspergillus awamori with a molecular weight of 180 kDa was produced in solid-state cultures using a mixture of pineapple crown leaves and wheat bran. Maximum production of the enzyme (820 ± 30 U/g substrate) was obtained after 8 days of culture at 28 °C and initial moisture of 80%. The crude enzyme was efficiently immobilized on glutaraldehyde cross-linked commercial gelatin. Immobilization changed the kinetics of the enzyme, whose behavior could no longer be described by a saturation function of the Michaelis-Menten type. Comparative evaluation of the free and immobilized enzyme showed that the immobilized enzyme was more thermostable and less inhibited by glucose than the free form. In consequence of these properties, the immobilized enzyme was able to hydrolyze cellobiose more extensively. In association with Trichoderma reesei cellulase, the free and immobilized β-glucosidase increased the liberation of glucose from cellulose 3- and 5-fold, respectively. Immobilization of the A. awamori β-glucosidase on glutaraldehyde cross-linked commercial gelatin is an efficient and cheap method allowing the reuse of the enzyme by at least 10 times. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Biodegradation of chlorobenzene using immobilized crude extracts ...

    African Journals Online (AJOL)

    SERVER

    2007-10-04

    Oct 4, 2007 ... immobilized crude extracts were reused for all other experiments and found that immobilization .... India which are of analytical reagent grade. .... 9. 60. 3. 1. Figure 3. Degradation of chlorobenzene by immobilized crude.

  10. Supramolecular protein immobilization on lipid bilayers

    NARCIS (Netherlands)

    Bosmans, R.P.G.; Hendriksen, W.E.; Verheijden, Mark Lloyd; Eelkema, R.; Jonkheijm, Pascal; van Esch, J.H.; Brunsveld, Luc

    2015-01-01

    Protein immobilization on surfaces, and on lipid bilayers specifically, has great potential in biomolecular and biotechnological research. Of current special interest is the immobilization of proteins using supramolecular noncovalent interactions. This allows for a reversible immobilization and

  11. Iodine immobilization in apatites

    International Nuclear Information System (INIS)

    Audubert, F.; Lartigue, J.E.

    2000-01-01

    In the context of a scientific program on long-lived radionuclide conditioning, a matrix for iodine 129 immobilization has been studied. A lead vanado-phosphate apatite was prepared from the melt of lead vanado-phosphate Pb 3 (VO 4 ) 1.6 (PO 4 ) 0.4 and lead iodide PbI 2 in stoichiometric proportions by calcination at 700 deg. C during 3 hours. Natural sintering of this apatite is not possible because the product decomposition occurs at 400 deg. C. Reactive sintering is the solution. The principle depends on the coating of lead iodide with lead vanado-phosphate. Lead vanado-phosphate coating is used as iodo-apatite reactant and as dense covering to confine iodine during synthesis. So the best condition to immobilize iodine during iodo-apatite synthesis is a reactive sintering at 700 deg. C under 25 MPa. We obtained an iodo-apatite surrounded with dense lead vanadate. Leaching behaviour of the matrix synthesized by solid-solid reaction is under progress in order to determine chemical durability, basic mechanisms of the iodo-apatite alteration and kinetic rate law. Iodo-apatite dissolution rates were pH and temperature dependent. We obtained a rate of 2.5 10 -3 g.m -2 .d -1 at 90 deg. C in initially de-ionised water. (authors)

  12. A low perfusion rate microreactor for the continous monitoring of enzyme characteristics: applications for glucose oxidase.

    NARCIS (Netherlands)

    Posthuma-Trumpie, G.A.; Venema, K.; Berkel, van W.J.H.; Korf, J.

    2007-01-01

    This report describes a versatile and robust microreactor for bioactive proteins physically immobilized on a polyether sulfone filter. The potential of the reactor is illustrated with glucose oxidase immobilized on a filter with a cut-off value of 30 kDa. A flow-injection system was used to deliver

  13. A low perfusion rate microreactor for continuous monitoring of enzyme characteristics : application to glucose oxidase

    NARCIS (Netherlands)

    Posthuma-Trumpie, G. A.; Venema, K.; van Berkel, W. J. H.; Korf, J.

    This report describes a versatile and robust microreactor for bioactive proteins physically immobilized on a polyether sulfone filter. The potential of the reactor is illustrated with glucose oxidase immobilized on a filter with a cut-off value of 30 kDa. A flow-injection system was used to deliver

  14. 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.

  15. 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.

  16. Assessing attitudes toward spinal immobilization.

    Science.gov (United States)

    Bouland, Andrew J; Jenkins, J Lee; Levy, Matthew J

    2013-10-01

    Prospective studies have improved knowledge of prehospital spinal immobilization. The opinion of Emergency Medical Services (EMS) providers regarding spinal immobilization is unknown, as is their knowledge of recent research advances. To examine the attitudes, knowledge, and comfort of prehospital and Emergency Department (ED) EMS providers regarding spinal immobilization performed under a non-selective protocol. An online survey was conducted from May to July of 2011. Participants were drawn from the Howard County Department of Fire and Rescue Services and the Howard County General Hospital ED. The survey included multiple choice questions and responses on a modified Likert scale. Correlation analysis and descriptive data were used to analyze results. Comfort using the Kendrick Extrication Device was low among ED providers. Experienced providers were more likely to indicate comfort using this device. Respondents often believed that spinal immobilization is appropriate in the management of penetrating trauma to the chest and abdomen. Reported use of padding decreased along with the frequency with which providers practice and encounter immobilized patients. Respondents often indicated that they perform spinal immobilization due solely to mechanism of injury. Providers who feel as if spinal immobilization is often performed unnecessarily were more likely to agree that immobilization causes an unnecessary delay in patient care. The results demonstrate the need for improved EMS education in the use of the Kendrick Extrication Device, backboard padding, and spinal immobilization in the management of penetrating trauma. The attitudes highlighted in this study are relevant to the implementation of a selective spinal immobilization protocol. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. A highly sensitive electrochemical glucose sensor structuring with nickel hydroxide and enzyme glucose oxidase

    International Nuclear Information System (INIS)

    Mathew, Manjusha; Sandhyarani, N.

    2013-01-01

    Graphical abstract: A combination of Ni 2+ /Ni 3+ redox couple and glucose oxidase has successfully been exploited for the realization of a highly sensitive glucose sensor for the first time. -- Highlights: • A multilayered glucose biosensor with enhanced sensitivity was fabricated. • Combination of Ni 2+ /Ni 3+ redox couple and glucose oxidase has been exploited for the first time. • Exhibits a lower detection limit of 100 nM with a high sensitivity of 16,840 μA mM −1 cm −2 . • The surface shows a low Michaelis–Menten constant value of 2.4 μM. • Detailed mechanism of sensing was proposed and justified. -- Abstract: A multilayered glucose biosensor with enhanced electron transport was fabricated via the sequential electrodeposition of chitosan gold nanocomposite (CGNC) and nickel hydroxide (Ni(OH) 2 ) on a bare gold electrode and subsequent immobilization of glucose oxidase. A thin film of Ni(OH) 2 deposited on CGNC modified gold electrode serves as an electrochemical redox probe as well as a matrix for the immobilization of glucose oxidase retaining its activity. Electron transport property of CGNC has been exploited to enhance the electron transport between the analyte and electrode. Electrochemical characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. Under optimal conditions the biosensor exhibits a linear range from 1 μM to 100 μM with a limit of detection (lod) down to 100 nM. The sensor shows a low Michaelis-Menten constant value of 2.4 μM indicates the high affinity of enzyme to the analyte points to the retained activity of enzyme after immobilization. The present glucose sensor with the high selectivity, sensitivity and stability is promising for practical clinical applications

  18. 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...

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

    Science.gov (United States)

    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.

  20. HLW immobilization in glass

    International Nuclear Information System (INIS)

    Leroy, P.; Jacquet-Francillon, N.; Runge, S.

    1992-01-01

    The immobilization of High Level Waste in glass in France is a long history which started as early as in the 1950's. More than 30 years of Research and Development have been invested in that field. Two industrial facilities are operating (AVM and R7) and a third one (T7), under cold testing, is planned to start active operation in the mid-92. While vitrification has been demonstrated to be an industrially mastered process, the question of the quality of the final waste product, i.e. the HLW glass, must be addressed. The scope of the present paper is to focus on the latter point from both standpoints of the R and D and of the industrial reality

  1. 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.

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

    Science.gov (United States)

    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.

  3. 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

  4. 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.

  5. 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...

  6. 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.

  7. 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

  8. 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...

  9. Enhanced accumulation of starch and total carbohydrates in alginate-immobilized Chlorella spp. induced by Azospirillum brasilense: II. Heterotrophic conditions.

    Science.gov (United States)

    Choix, Francisco J; de-Bashan, Luz E; Bashan, Yoav

    2012-10-10

    The effect of the bacterium Azospirillum brasilense jointly immobilized with Chlorella vulgaris or C. sorokiniana in alginate beads on total carbohydrates and starch was studied under dark and heterotrophic conditions for 144 h in synthetic growth medium supplemented with either d-glucose or Na-acetate as carbon sources. In all treatments, enhanced total carbohydrates and starch content per culture and per cell was obtained after 24h; only jointly immobilized C. vulgaris growing on d-glucose significantly increased total carbohydrates and starch content after 96 h. Enhanced accumulation of carbohydrate and starch under jointly immobilized conditions was variable with time of sampling and substrate used. Similar results occurred when the microalgae was immobilized alone. In both microalgae growing on either carbon sources, the bacterium promoted accumulation of carbohydrates and starch; when the microalgae were immobilized alone, they used the carbon sources for cell multiplication. In jointly immobilized conditions with Chlorella spp., affinity to carbon source and volumetric productivity and yield were higher than when Chlorella spp. were immobilized alone; however, the growth rate was higher in microalgae immobilized alone. This study demonstrates that under heterotrophic conditions, A. brasilense promotes the accumulation of carbohydrates in two strains Chlorella spp. under certain time-substrate combinations, producing mainly starch. As such, this bacterium is a biological factor that can change the composition of compounds in microalgae in dark, heterotrophic conditions. Copyright © 2012. Published by Elsevier Inc.

  10. Ethanol fermentation of molasses by Saccharomyces cerevisiae cells immobilized onto sugar beet pulp

    Directory of Open Access Journals (Sweden)

    Vučurović Vesna M.

    2012-01-01

    Full Text Available Natural adhesion of Saccharomyces cerevisiae onto sugar beet pulp (SBP is a very simple and cheap immobilization method for retaining high cells density in the ethanol fermentation system. In the present study, yeast cells were immobilized by adhesion onto SBP suspended in the synthetic culture media under different conditions such as: glucose concentration (100, 120 and 150 g/l, inoculum concentration (5, 10 and 15 g/l dry mass and temperature (25, 30, 35 and 40°C. In order to estimate the optimal immobilization conditions the yeast cells retention (R, after each immobilization experiment was analyzed. The highest R value of 0.486 g dry mass yeast /g dry mass SBP was obtained at 30°C, glucose concentration of 150 g/l, and inoculum concentration of 15 g/l. The yeast immobilized under these conditions was used for ethanol fermentation of sugar beet molasses containing 150.2 g/l of reducing sugar. Efficient ethanol fermentation (ethanol concentration of 70.57 g/l, fermentation efficiency 93.98% of sugar beet molasses was achieved using S. cerevisiae immobilized by natural adhesion on SBP. [Projekat Ministarstva nauke Republike Srbije, br. TR-31002

  11. Immobilization of cellulose producing cells (sporotrichum cellulophilum) using irradiated rice husk as a substrate

    International Nuclear Information System (INIS)

    Lina, M.R.; Tamada, M.; Kumakura, M.

    1991-01-01

    An experiment to study the effect of irradiated rice husk as a substrate on cellulase production of free and immobilized cells of S. cellulophium was carried out. Radiation pretreatment of rice husk was done using electron beam accelerator (Dynamitron IEA 3000-25,2), with doses of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 MGy. The substrate used in cellulase production of free and immobilized cells were cellulose powder as a standard, and 1.0 MGy irradiated rice husk. Concentrations of cellulose powder for free and immobilized cells were 1, 2, 3, 5, and 8% (w/v). Irradiated rice husk concentrations for free cells were 3, 6, 9, 15, and 24% (w/v), whereas for immobilized cells were 3, 6, and 9% (w/v). Results showed that glucose concentration in 1.0 MGy irradiated rice husk was the highest of all irradiated and unirradiated rice husks. The GPA (glucose production activity) values used of free immobilized cells of S. cellulophium in medium containing 1.0 MGy irradiated rice husk were about 50% lower than in cellulose powder medium. Cellulase solution resulted by immobilized cells, either in cellulose powder or in irradiated rice husk media, were clear and did not contain mycelium. (authors). 7 refs, 7 figs

  12. High-level-waste immobilization

    International Nuclear Information System (INIS)

    Crandall, J.L.

    1982-01-01

    Analysis of risks, environmental effects, process feasibility, and costs for disposal of immobilized high-level wastes in geologic repositories indicates that the disposal system safety has a low sensitivity to the choice of the waste disposal form

  13. Bioreduction of chromate by immobilized cells of Halomonas sp

    Energy Technology Data Exchange (ETDEWEB)

    Murugavelh, S.; Mohanty, Kaustubha [Department of Chemical Engineering, Indian Institute of Technology Guwahati, Guwahati – 781039, Assam (India)

    2013-07-01

    In this work, the bioreduction of Cr(VI) by immobilized cells of Halomonas sp was reported. Ca alginate, acryl amide and agar were tested as the matrices for immobilization. Ca alginate was found to be the suitable matrix among the different matrices studied. Of the various dosages of inoculum studied 2 g/L was found to be the optimum. Glucose at 1 g L-1 was completely utilized by the immobilized Halomonas sp even in the presence of Cr(VI) at 40 mg L-1. The optimum pH for the bioreduction of Cr(VI) by immobilized Halomonas sp was found to be pH 6. The mechanical strength of the beads plays an essential role in the bioreduction process. Halomonas sp entrapped in a alginate matrix reported a maximum of 98.9 % of reduction for an initial Cr(VI) concentration of 10 mg L-1. The alginate beads can be reused for 3 times with slight drop in the percentage reduction. The presence of other metals decreased the bioreduction percentage.

  14. Catalysis of Rice Straw Hydrolysis by the Combination of Immobilized Cellulase from Aspergillus niger on β-Cyclodextrin-Fe3O4 Nanoparticles and Ionic Liquid

    Science.gov (United States)

    Huang, Po-Jung; Chang, Ken-Lin; Chen, Shui-Tein

    2015-01-01

    Cellulase from Aspergillus niger was immobilized onto β-cyclodextrin-conjugated magnetic particles by silanization and reductive amidation. The immobilized cellulase gained supermagnetism due to the magnetic nanoparticles. Ninety percent of cellulase was immobilized, but the activity of immobilized cellulase decreased by 10%. In this study, ionic liquid (1-butyl-3-methylimidazolium chloride) was introduced into the hydrolytic process because the original reaction was a solid-solid reaction. The activity of immobilized cellulase was improved from 54.87 to 59.11 U g immobilized cellulase−1 at an ionic liquid concentration of 200 mM. Using immobilized cellulase and ionic liquid in the hydrolysis of rice straw, the initial reaction rate was increased from 1.629 to 2.739 g h−1 L−1. One of the advantages of immobilized cellulase is high reusability—it was usable for a total of 16 times in this study. Compared with free cellulase, magnetized cellulase can be recycled by magnetic field and the activity of immobilized cellulase was shown to remain at 85% of free cellulase without denaturation under a high concentration of glucose (15 g L−1). Therefore, immobilized cellulase can hydrolyze rice straw continuously compared with free cellulase. The amount of harvested glucose can be up to twentyfold higher than that from the hydrolysis by free cellulase. PMID:25874210

  15. Catalysis of Rice Straw Hydrolysis by the Combination of Immobilized Cellulase from Aspergillus niger on β-Cyclodextrin-Fe3O4 Nanoparticles and Ionic Liquid

    Directory of Open Access Journals (Sweden)

    Po-Jung Huang

    2015-01-01

    Full Text Available Cellulase from Aspergillus niger was immobilized onto β-cyclodextrin-conjugated magnetic particles by silanization and reductive amidation. The immobilized cellulase gained supermagnetism due to the magnetic nanoparticles. Ninety percent of cellulase was immobilized, but the activity of immobilized cellulase decreased by 10%. In this study, ionic liquid (1-butyl-3-methylimidazolium chloride was introduced into the hydrolytic process because the original reaction was a solid-solid reaction. The activity of immobilized cellulase was improved from 54.87 to 59.11 U g immobilized cellulase−1 at an ionic liquid concentration of 200 mM. Using immobilized cellulase and ionic liquid in the hydrolysis of rice straw, the initial reaction rate was increased from 1.629 to 2.739 g h−1 L−1. One of the advantages of immobilized cellulase is high reusability—it was usable for a total of 16 times in this study. Compared with free cellulase, magnetized cellulase can be recycled by magnetic field and the activity of immobilized cellulase was shown to remain at 85% of free cellulase without denaturation under a high concentration of glucose (15 g L−1. Therefore, immobilized cellulase can hydrolyze rice straw continuously compared with free cellulase. The amount of harvested glucose can be up to twentyfold higher than that from the hydrolysis by free cellulase.

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

    Science.gov (United States)

    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

  17. Novel glucose fiber sensor combining ThFBG with GOD

    Science.gov (United States)

    Li, Mengmeng; Zhou, Ciming; Fan, Dian; Ou, Yiwen

    2016-10-01

    We propose a novel glucose fiber optic sensor combining a thinned cladding fiber Bragg grating (ThFBG) with glucose oxidase (GOD). By immobilizing GOD on the surface of a ThFBG, the fabricated sensor can obtain a high specificity to glucose. Because of the evanescent field, the sensor is very sensitive to the ambient refractive index change arising from the catalytic reaction between glucose and GOD. A four-level fiber model was simulated and verified the precision of the sensing principle. Two methods, glutaraldehyde crosslinking method (GCM) and 3-aminopropyl triethoxysilane covalent coupling method (ATCCM), were experimentally utilized to immobilize GOD. And sensor fabricated with the method ATCCM shows a measurement range of 0-0.82 mg/mL which is better than the sensor fabricated with the method GCM with measurement range of 0-0.67 mg/mL under the same condition. By using ATCCM to immobilize GOD with different concentrations, three sensors were fabricated and used for glucose measurement by monitoring the Bragg wavelength (λb) shifts, the results indicate a good linear relationship between wavelength shift and glucose concentration within a specific range, and the measurement range increases as GOD concentration increases. The highest sensitivity of sensor reaches up to 0.0549 nm/(mg.mL-1). The proposed sensor has distinct advantages in sensing structure, cost and specificity.

  18. PVA-Glutaraldehyde as support for lectin immobilization and affinity chromatography

    Directory of Open Access Journals (Sweden)

    Moacyr Jesus Barreto de Melo Rêgo

    2016-12-01

    Full Text Available Immobilized lectins are a powerful biotechnological tool for separation and isolation of glycoconjugates. In the present study, polyvinyl alcohol (PVA and glutaraldehyde (GA were used as a support for Concanavalin A (Con A covalent immobilization and for entrapment of Parkia pendula seed gum (PpeG. Con A immobilization yielded approximately 30% and 0.6 M glucose solution was the minimum concentration able to elute fetuin from column. PVA-GA-PpeG column was efficiently recognized by pure P. pendula lectin (PpeL. These findings indicate that PVA-GA interpenetrated network showed to be an efficient support for lectin covalent immobilization and as affinity chromatography matrix after trapping of PpeG.

  19. Neuroscience of glucose homeostasis

    NARCIS (Netherlands)

    La Fleur, S E; Fliers, E; Kalsbeek, A

    2014-01-01

    Plasma glucose concentrations are homeostatically regulated and maintained within strict boundaries. Several mechanisms are in place to increase glucose output when glucose levels in the circulation drop as a result of glucose utilization, or to decrease glucose output and increase tissue glucose

  20. Status of plutonium ceramic immobilization processes and immobilization forms

    Energy Technology Data Exchange (ETDEWEB)

    Ebbinghaus, B.B.; Van Konynenburg, R.A. [Lawrence Livermore National Lab., CA (United States); Vance, E.R.; Jostsons, A. [Australian Nuclear Science and Technology Organization, Menai (Australia)] [and others

    1996-05-01

    Immobilization in a ceramic followed by permanent emplacement in a repository or borehole is one of the alternatives currently being considered by the Fissile Materials Disposition Program for the ultimate disposal of excess weapons-grade plutonium. To make Pu recovery more difficult, radioactive cesium may also be incorporated into the immobilization form. Valuable data are already available for ceramics form R&D efforts to immobilize high-level and mixed wastes. Ceramics have a high capacity for actinides, cesium, and some neutron absorbers. A unique characteristic of ceramics is the existence of mineral analogues found in nature that have demonstrated actinide immobilization over geologic time periods. The ceramic form currently being considered for plutonium disposition is a synthetic rock (SYNROC) material composed primarily of zirconolite (CaZrTi{sub 2}O{sub 7}), the desired actinide host phase, with lesser amounts of hollandite (BaAl{sub 2}Ti{sub 6}O{sub 16}) and rutile (TiO{sub 2}). Alternative actinide host phases are also being considered. These include pyrochlore (Gd{sub 2}Ti{sub 2}O{sub 7}), zircon (ZrSiO{sub 4}), and monazite (CePO{sub 4}), to name a few of the most promising. R&D activities to address important technical issues are discussed. Primarily these include moderate scale hot press fabrications with plutonium, direct loading of PuO{sub 2} powder, cold press and sinter fabrication methods, and immobilization form formulation issues.

  1. Status of plutonium ceramic immobilization processes and immobilization forms

    International Nuclear Information System (INIS)

    Ebbinghaus, B.B.; Van Konynenburg, R.A.; Vance, E.R.; Jostsons, A.

    1996-01-01

    Immobilization in a ceramic followed by permanent emplacement in a repository or borehole is one of the alternatives currently being considered by the Fissile Materials Disposition Program for the ultimate disposal of excess weapons-grade plutonium. To make Pu recovery more difficult, radioactive cesium may also be incorporated into the immobilization form. Valuable data are already available for ceramics form R ampersand D efforts to immobilize high-level and mixed wastes. Ceramics have a high capacity for actinides, cesium, and some neutron absorbers. A unique characteristic of ceramics is the existence of mineral analogues found in nature that have demonstrated actinide immobilization over geologic time periods. The ceramic form currently being considered for plutonium disposition is a synthetic rock (SYNROC) material composed primarily of zirconolite (CaZrTi 2 O 7 ), the desired actinide host phase, with lesser amounts of hollandite (BaAl 2 Ti 6 O 16 ) and rutile (TiO 2 ). Alternative actinide host phases are also being considered. These include pyrochlore (Gd 2 Ti 2 O 7 ), zircon (ZrSiO 4 ), and monazite (CePO 4 ), to name a few of the most promising. R ampersand D activities to address important technical issues are discussed. Primarily these include moderate scale hot press fabrications with plutonium, direct loading of PuO 2 powder, cold press and sinter fabrication methods, and immobilization form formulation issues

  2. Covalent immobilization of β-glucosidase on magnetic particles for lignocellulose hydrolysis.

    Science.gov (United States)

    Alftrén, Johan; Hobley, Timothy John

    2013-04-01

    β-Glucosidase hydrolyzes cellobiose to glucose and is an important enzyme in the consortium used for hydrolysis of cellulosic and lignocellulosic feedstocks. In the present work, β-glucosidase was covalently immobilized on non-porous magnetic particles to enable re-use of the enzyme. It was found that particles activated with cyanuric chloride and polyglutaraldehyde gave the highest bead-related immobilized enzyme activity when tested with p-nitrophenyl-β-D-glucopyranoside (104.7 and 82.2 U/g particles, respectively). Furthermore, the purified β-glucosidase preparation from Megazyme gave higher bead-related enzyme activities compared to Novozym 188 (79.0 and 9.8 U/g particles, respectively). A significant improvement in thermal stability was observed for immobilized enzyme compared to free enzyme; after 5 h (at 65 °C), 36 % of activity remained for the former, while there was no activity in the latter. The performance and recyclability of immobilized β-glucosidase on more complex substrate (pretreated spruce) was also studied. It was shown that adding immobilized β-glucosidase (16 U/g dry matter) to free cellulases (8 FPU/g dry matter) increased the hydrolysis yield of pretreated spruce from ca. 44 % to ca. 65 %. In addition, it was possible to re-use the immobilized β-glucosidase in the spruce and retain activity for at least four cycles. The immobilized enzyme thus shows promise for lignocellulose hydrolysis.

  3. Nanoporous gold assembly of glucose oxidase for electrochemical biosensing

    DEFF Research Database (Denmark)

    Xiao, Xinxin; Ulstrup, Jens; Li, Hui

    2014-01-01

    Nanoporous gold (NPG) is composed of three-dimensional (3D) bicontinuous nanostructures with large surface area. Nano-channels inside NPG provide an ideal local environment for immobilization of enzyme molecules with expected stabilization of the protein molecules. In this work, glucose oxidase (...

  4. 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...

  5. Fabrication of Amperometric Glucose Sensor Using Glucose Oxidase-Cellulose Nanofiber Aqueous Solution.

    Science.gov (United States)

    Yasuzawa, Mikito; Omura, Yuya; Hiura, Kentaro; Li, Jiang; Fuchiwaki, Yusuke; Tanaka, Masato

    2015-01-01

    Cellulose nanofiber aqueous solution, which remained virtually transparent for more than one week, was prepared by using the clear upper layer of diluted cellulose nanofiber solution produced by wet jet milling. Glucose oxidase (GOx) was easily dissolved in this solution and GOx-immobilized electrode was easily fabricated by simple repetitious drops of GOx-cellulose solution on the surface of a platinum-iridium electrode. Glucose sensor properties of the obtained electrodes were examined in phosphate buffer solution of pH 7.4 at 40°C. The obtained electrode provided a glucose sensor response with significantly high response speed and good linear relationship between glucose concentration and response current. After an initial decrease of response sensitivity for a few days, relatively constant sensitivity was obtained for about 20 days. Nevertheless, the influence of electroactive compounds such as ascorbic acid, uric acid and acetoaminophen were not negletable.

  6. Immobilization needs and technology programs

    International Nuclear Information System (INIS)

    Gray, L.W.; Kan, T.; Shaw, H.; Armantrout, G.

    1995-01-01

    In the aftermath of the Cold War, the US and Russia agreed to large reductions in nuclear weapons. To aid in the selection of long-term management options, DOE has undertaken a multifaceted study to select options for storage and disposition of plutonium in keeping with US policy that plutonium must be subjected to the highest standards of safety, security, and accountability. One alternative being considered is immobilization. To arrive at a suitable immobilization form, we first reviewed published information on high-level waste immobilization technologies and identified 72 possible plutonium immobilization forms to be prescreened. Surviving forms were further screened using multi-attribute utility analysis to determine the most promising technology families. Promising immobilization families were further evaluated to identify chemical, engineering, environmental, safety, and health problems that remain to be solved prior to making technical decisions as to the viability of using the form for long- term disposition of plutonium. From this evaluation, a detailed research and development plan has been developed to provide answers to these remaining questions

  7. Improvement in glucose biosensing response of electrochemically grown polypyrrole nanotubes by incorporating crosslinked glucose oxidase.

    Science.gov (United States)

    Palod, Pragya Agar; Singh, Vipul

    2015-10-01

    In this paper a novel enzymatic glucose biosensor has been reported in which platinum coated alumina membranes (Anodisc™s) have been employed as templates for the growth of polypyrrole (PPy) nanotube arrays using electrochemical polymerization. The PPy nanotube arrays were grown on Anodisc™s of pore diameter 100 nm using potentiostatic electropolymerization. In order to optimize the polymerization time, immobilization of glucose oxidase (GOx) was first performed using physical adsorption followed by measuring its biosensing response which was examined amperometrically for increasing concentrations of glucose. In order to further improve the sensing performance of the biosensor fabricated for optimum polymerization duration, enzyme immobilization was carried out using cross-linking with glutaraldehyde and bovine serum albumin (BSA). Approximately six fold enhancement in the sensitivity was observed in the fabricated electrodes. The biosensors also showed a wide range of linear operation (0.2-13 mM), limit of detection of 50 μM glucose concentration, excellent selectivity for glucose, notable reliability for real sample detection and substantially improved shelf life. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Immobilization of cellulase by radiation polymerization

    International Nuclear Information System (INIS)

    Kumakura, M.; Kaetsu, I.

    1983-01-01

    Immobilization of cellulase by radiation polymerization at low temperatures was studied. The enzymatic activity of immobilized cellulase pellets varied with the monomer, enzyme concentration, and the thickness of immobilized cellulase pellets. The optimum monomer concentration in the immobilization of cellulase was 30-50% at the pellet thickness of 1.0 mm, in which the enzymatic activity was 50%. The enzymatic activity of immobilized cellulase pellets was examined using various substrates such as cellobiose, carboxymethylcellulose, and paper pretreated by radiation. It was found that irradiated paper can be hydrolyzed by immobilized cellulase pellets. (author)

  9. Amine functionalized TiO2-carbon nanotube composite: synthesis, characterization and application to glucose biosensing

    Science.gov (United States)

    Tasviri, Mahboubeh; Rafiee-Pour, Hossain-Ali; Ghourchian, Hedayatollah; Gholami, Mohammad Reza

    2011-12-01

    The synthesis of amine functionalized TiO2-coated multiwalled carbon nanotubes (NH2-TiO2-CNTs) using sol-gel method was investigated. The synthesized nanocomposite was characterized with XRD, FTIR spectroscopy, BET test and SEM imaging. The results demonstrated a unique nanostructure with no destruction of the CNTs' shape. In addition, the presence of amine groups on the composite surface was confirmed by FTIR. This nanocomposite was used for one-step immobilization of glucose oxidase (GOx) to sense glucose. The result of cyclic voltammetry showed a pair of well-defined and quasi-reversible peaks for direct electron transfer of GOx in the absence of glucose. Also, the result of electrochemical impedance spectroscopy indicated that GOx was successfully immobilized on the surface of NH2-TiO2-CNTs. Furthermore, good amperometric response showed that immobilized GOx on the NH2-TiO2-CNTs exhibits exceptional bioelectrocatalytic activity toward glucose oxidation.

  10. Enzymes immobilization on Fe 3O 4-gold nanoparticles

    Science.gov (United States)

    Kalska-Szostko, B.; Rogowska, M.; Dubis, A.; Szymański, K.

    2012-01-01

    In the present study Fe3O4 magnetic nanoparticles were synthesized by coprecipitation of Fe2+ and Fe3+ from chlorides. In the next step magnetite-gold core-shell nanoparticles were synthesized from HAuCl4 using an ethanol as a reducing agent. Finally, magnetic nanoparticles were functionalized by hexadecanethiol. The immobilization of biological molecules (trypsin and glucose oxidase) to the thiol-modified and unmodified magnetite-gold nanoparticles surface was tested. The resulting nanoparticles were characterized by infrared spectroscopy, differential scanning calorimetry, Mössbauer spectroscopy and transmission electron microscopy.

  11. 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

  12. 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

  13. Immobile Complex Verbs in Germanic

    DEFF Research Database (Denmark)

    Vikner, Sten

    2005-01-01

    the V° requirements or the V* requirements. Haider (1993, p. 62) and Koopman (1995), who also discuss such immobile verbs, only account for verbs with two prefix-like parts (e.g., German uraufführen ‘to perform (a play) for the first time' or Dutch herinvoeren ‘to reintroduce'), not for the more...... frequent type with only one prefix-like part (e.g., German bauchreden/Dutch buikspreken ‘to ventriloquize'). This analysis will try to account not only for the data discussed in Haider (1993) and Koopman (1995) but also for the following: - why immobile verbs include verbs with only one prefix-like part...... are immobile, - why such verbs are not found in Germanic VO-languages such as English and Scandinavian....

  14. 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...

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. Immobilization of acid digestion residue

    International Nuclear Information System (INIS)

    Greenhalgh, W.O.; Allen, C.R.

    1983-01-01

    Acid digestion treatment of nuclear waste is similar to incineration processes and results in the bulk of the waste being reduced in volume and weight to some residual solids termed residue. The residue is composed of various dispersible solid materials and typically contains the resultant radioactivity from the waste. This report describes the immobilization of the residue in portland cement, borosilicate glass, and some other waste forms. Diagrams showing the cement and glass virtification parameters are included in the report as well as process steps and candidate waste product forms. Cement immobilization is simplest and probably least expensive; glass vitrification exhibits the best overall volume reduction ratio

  20. 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.

  1. Enhancement of L(+)-Lactic Acid Production of Immobilized Rhizopus Oryzae Implanted by Ion Beams

    International Nuclear Information System (INIS)

    Fan Yonghong; Yang Yingge; Zheng Zhiming; Li Wen; Wang Peng; Yao Liming; Yu Zengliang

    2008-01-01

    Immobilized Rhizopus oryzae culturing may be a solution to the inhibited production of L(+)-lactic acid in submerged fermentation, which is caused by aggregated mycelia floc. In the present study, a R. oryzae mutant (RL6041) with a 90% conversion rate of glucose into L-lactic acid was obtained by N + implantation under the optimized conditions of a beam energy of 15 keV and a dose of 2.6 x 10 15 ions/cm 2 . Using polyurethane foam as the immobilization matrix, the optimal L-lactic acid production conditions were determined as 4 mm polyurethane foam, 150 r/min, 50 g/L ∼ 80 g/L of initial glucose, 38 deg. C and pH 6.0. 15-cycle repeated productions of L-lactic acid by immobilized RL6041 were performed under the optimized culturing conditions and over 80% of the glucose was converted into L-lactic acid in 30 hours on average. The results show that immobilized RL6041 is a promising candidate for continuous L-lactic acid production.

  2. Carbon Nanotube Modified Screen Printed Electrodes: Pyranose Oxidase Immobilization Platform for Amperometric Enzyme Sensors

    Directory of Open Access Journals (Sweden)

    Dilek ODACI DEMIRKOL

    2017-03-01

    Full Text Available Here, a novel enzymatic biosensor was developed using multiwalled carbon nanotube including screen printed electrodes (MWCNT-SPE. Pyranose oxidase (PyOx was immobilized on the electrode surface by way of gelatin membrane and then cross-linked using glutaraldehyde. Glucose was detected at -0.7 V (vs. Ag/AgCl by watching consumed oxygen in enzymatic reaction after addition substrate. After optimization of pH and enzyme loading, the linearity was found in the range of 0.1–1.0 mM of glucose. After that, the effect of MCNT on the current was tested. Also the enzymatic biosensor including glucose oxidase instead of pyranose oxidase was prepared and the biosensor response followed for glucose. Furthermore, this system was tested for glucose analysis in soft drinks.

  3. Radiation immobilization of catalase and its application

    International Nuclear Information System (INIS)

    Wang Guanghui; Ha Hongfei; Wang Xia; Wu Jilan

    1988-01-01

    Catalase was immobilized by a chemical method on porous polyacrylamide particles produced by radiation polymerization of acrylamide monomer at low temperature (-78 0 C). Activity of immobilized catalase was enhanced distinctly by joining a chemical arm to the support. The method of recovery of catalase activity on immobilized polymer was found by soaking it in certain buffer. The treatment of H 2 O 2 both in aqueous solution and alcoholic solution by using the immobilized catalase was performed. (author)

  4. Immobilization and characterization of inulinase from Ulocladium

    Indian Academy of Sciences (India)

    Ulocladium atrum inulinase was immobilized on different composite membranes composed of chitosan/nonwoven fabrics. Km values of free and immobilized U. atrum inulinase on different composite membranes were calculated. The enzyme had optimum pH at 5.6 for free and immobilized U. atrum inulinase on polyester ...

  5. Immobilization of Mitochondria on Graphene

    Science.gov (United States)

    2013-08-29

    poly-L-lysine has also been reported for immobilization of yeast mitochondria. Coating was performed by repetitive washing of cover slips with 0.02...of Poly-L-lysine Applications of PLL PLL is a production of bacterial fermentation and is used as a food preservative. In biology, PLL is used in

  6. An Efficient, Recyclable, and Stable Immobilized Biocatalyst Based on Bioinspired Microcapsules-in-Hydrogel Scaffolds.

    Science.gov (United States)

    Zhang, Shaohua; Jiang, Zhongyi; Shi, Jiafu; Wang, Xueyan; Han, Pingping; Qian, Weilun

    2016-09-28

    Design and preparation of high-performance immobilized biocatalysts with exquisite structures and elucidation of their profound structure-performance relationship are highly desired for green and sustainable biotransformation processes. Learning from nature has been recognized as a shortcut to achieve such an impressive goal. Loose connective tissue, which is composed of hierarchically organized cells by extracellular matrix (ECM) and is recognized as an efficient catalytic system to ensure the ordered proceeding of metabolism, may offer an ideal prototype for preparing immobilized biocatalysts with high catalytic activity, recyclability, and stability. Inspired by the hierarchical structure of loose connective tissue, we prepared an immobilized biocatalyst enabled by microcapsules-in-hydrogel (MCH) scaffolds via biomimetic mineralization in agarose hydrogel. In brief, the in situ synthesized hybrid microcapsules encapsulated with glucose oxidase (GOD) are hierarchically organized by the fibrous framework of agarose hydrogel, where the fibers are intercalated into the capsule wall. The as-prepared immobilized biocatalyst shows structure-dependent catalytic performance. The porous hydrogel permits free diffusion of glucose molecules (diffusion coefficient: ∼6 × 10(-6) cm(2) s(-1), close to that in water) and retains the enzyme activity as much as possible after immobilization (initial reaction rate: 1.5 × 10(-2) mM min(-1)). The monolithic macroscale of agarose hydrogel facilitates the easy recycling of the immobilized biocatalyst (only by using tweezers), which contributes to the nonactivity decline during the recycling test. The fiber-intercalating structure elevates the mechanical stability of the in situ synthesized hybrid microcapsules, which inhibits the leaching and enhances the stability of the encapsulated GOD, achieving immobilization efficiency of ∼95%. This study will, therefore, provide a generic method for the hierarchical organization of (bio

  7. 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.

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

    Science.gov (United States)

    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.

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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

  16. Glucose and cardiovascular risk

    NARCIS (Netherlands)

    Fuchs, M.; Hoekstra, J. B. L.; Mudde, A. H.

    2002-01-01

    The American Diabetes Association and the World Health Organisation have recently redefined the spectrum of abnormal glucose tolerance. The criteria for diabetes mellitus were sharpened and impaired fasting glucose (IFG) and impaired glucose tolerance (IGT) were classified as intermediate stages

  17. Potential Applications of Carbohydrases Immobilization in the Food Industry

    Science.gov (United States)

    Contesini, Fabiano Jares; de Alencar Figueira, Joelise; Kawaguti, Haroldo Yukio; de Barros Fernandes, Pedro Carlos; de Oliveira Carvalho, Patrícia; Nascimento, Maria da Graça; Sato, Hélia Harumi

    2013-01-01

    Carbohydrases find a wide application in industrial processes and products, mainly in the food industry. With these enzymes, it is possible to obtain different types of sugar syrups (viz. glucose, fructose and inverted sugar syrups), prebiotics (viz. galactooligossacharides and fructooligossacharides) and isomaltulose, which is an interesting sweetener substitute for sucrose to improve the sensory properties of juices and wines and to reduce lactose in milk. The most important carbohydrases to accomplish these goals are of microbial origin and include amylases (α-amylases and glucoamylases), invertases, inulinases, galactosidases, glucosidases, fructosyltransferases, pectinases and glucosyltransferases. Yet, for all these processes to be cost-effective for industrial application, a very efficient, simple and cheap immobilization technique is required. Immobilization techniques can involve adsorption, entrapment or covalent bonding of the enzyme into an insoluble support, or carrier-free methods, usually based on the formation of cross-linked enzyme aggregates (CLEAs). They include a broad variety of supports, such as magnetic materials, gums, gels, synthetic polymers and ionic resins. All these techniques present advantages and disadvantages and several parameters must be considered. In this work, the most recent and important studies on the immobilization of carbohydrases with potential application in the food industry are reviewed. PMID:23344046

  18. An In-Line Photonic Biosensor for Monitoring of Glucose Concentrations

    Directory of Open Access Journals (Sweden)

    Ala'aldeen Al-Halhouli

    2014-08-01

    Full Text Available This paper presents two PDMS photonic biosensor designs that can be used for continuous monitoring of glucose concentrations. The first design, the internally immobilized sensor, consists of a reactor chamber, micro-lenses and self-alignment structures for fiber optics positioning. This sensor design allows optical detection of glucose concentrations under continuous glucose flow conditions of 33 µL/h based on internal co-immobilization of glucose oxidase (GOX and horseradish peroxidase (HRP on the internal PDMS surface of the reactor chamber. For this design, two co-immobilization methods, the simple adsorption and the covalent binding (PEG methods were tested. Experiments showed successful results when using the covalent binding (PEG method, where glucose concentrations up to 5 mM with a coefficient of determination (R2 of 0.99 and a limit of detection of 0.26 mM are detectable. The second design is a modified version of the internally immobilized sensor, where a microbead chamber and a beads filling channel are integrated into the sensor. This modification enabled external co-immobilization of enzymes covalently onto functionalized silica microbeads and allows binding a huge amount of HRP and GOX enzymes on the microbeads surfaces which increases the interaction area between immobilized enzymes and the analyte. This has a positive effect on the amount and rate of chemical reactions taking place inside the chamber. The sensor was tested under continuous glucose flow conditions and was found to be able to detect glucose concentrations up to 10 mM with R2 of 0.98 and a limit of detection of 0.7 mM. Such results are very promising for the application in photonic LOC systems used for online analysis.

  19. Luminol, horseradish peroxidase, and glucose oxidase ternary functionalized graphene oxide for ultrasensitive glucose sensing.

    Science.gov (United States)

    Li, Fang; Ma, Wenjing; Liu, Jiachang; Wu, Xiang; Wang, Yan; He, Jianbo

    2018-01-01

    Luminol, horseradish peroxidase (HRP), and glucose oxidase (GOx) ternary functionalized graphene oxide (HRP/GOx-luminol-GO) with excellent chemiluminescence (CL) activity and specific enzymatic property was prepared via a simple and general strategy for the first time. In this approach, luminol functionalized GO (luminol-GO) was prepared by gently stirring GO with luminol. Then HRP and GOx were further co-immobilized onto the surface of luminol-GO by storing HRP and GOx with luminol-GO at 4 °C overnight, to form HRP/GOx-luminol-GO bionanocomposites. The synthesized HRP/GOx-luminol-GO could react with H 2 O 2 generated from GOx catalyzed glucose oxidization reaction, to produce strong CL emission in the presence of co-immobilized HRP. Thus, we developed an ultrasensitive, homogeneous, reagentless, selective, and simple CL sensing system for glucose detection. The resulting biosensors exhibited ultra-wide linear range from 5.0 nM to 5.0 mM, and an ultra-low detection limit of 1.2 nM, which was more than 3 orders of magnitude lower than previously reported methods. Furthermore, the sensing system was successfully applied for the detection of glucose in human blood samples.

  20. 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...

  1. 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.

  2. Biodiesel production with immobilized lipase: A review.

    Science.gov (United States)

    Tan, Tianwei; Lu, Jike; Nie, Kaili; Deng, Li; Wang, Fang

    2010-01-01

    Fatty acid alkyl esters, also called biodiesel, are environmentally friendly and show great potential as an alternative liquid fuel. Biodiesel is produced by transesterification of oils or fats with chemical catalysts or lipase. Immobilized lipase as the biocatalyst draws high attention because that process is "greener". This article reviews the current status of biodiesel production with immobilized lipase, including various lipases, immobilization methods, various feedstocks, lipase inactivation caused by short chain alcohols and large scale industrialization. Adsorption is still the most widely employed method for lipase immobilization. There are two kinds of lipase used most frequently especially for large scale industrialization. One is Candida antartica lipase immobilized on acrylic resin, and the other is Candida sp. 99-125 lipase immobilized on inexpensive textile membranes. However, to further reduce the cost of biodiesel production, new immobilization techniques with higher activity and stability still need to be explored. Copyright 2010 Elsevier Inc. All rights reserved.

  3. Immobilization of Saccharomyces Cerevisiae in Rice Hulls for Ethanol Production

    Directory of Open Access Journals (Sweden)

    Edita Martini

    2011-05-01

    Full Text Available The whole cell immobilization in ethanol fermentation can be done by using natural carriers or through synthetic carriers. All of these methods have the same purpose of retaining high cell concentrations within a certain defined region of space which leads to higher ethanol productivity. Lignocellulosic plant substance represents one of highly potential sources in ethanol production. Some studies have found that cellulosic substances substances can also be used as a natural carrier in cell immobilization by re-circulating pre-culture medium into a reactor. In this experiment, rice hulls without any treatment were used to immobilize Saccharomyces cerevisiae through semi solid state incubation combined with re-circulating pre-culture medium. The scanning electron microscopy (SEM pictures of the carrier show that the yeast cells are absorbed and embedded to the rice hull pore. In liquid batch fermentation system with an initial sugar concentration of 50 g/L, nearly 100% total sugar was consumed after 48 hours. This resulted in an ethanol yield of 0.32 g ethanol/g glucose, which is 62.7% of the theoretical value. Ethanol productivity of 0.59 g/(L.h is 2.3 fold higher than that of free cells which is 0.26 g/(L.h. An effort to reuse the immobilized cells in liquid fermentation system showed poor results due to cell desorption in the first batch which led to high sugar concentration inhibitory effect in the second batch fermentation. This might be solved by using semi solid fermentation process in the future work.

  4. Utilization of ionizing radiation to obtention of polymeric supports for the enzyme immobilization with clinical potential use

    International Nuclear Information System (INIS)

    Rodas, Andrea Cecilia Dorion

    1997-01-01

    In the development of polymers with biological activity, it was studied the grafting of acrylic acid monomer onto polyethylene and polypropylene pellets by mutual radiation grafting technique. The effect of dose rate, irradiation total dose, and monomer concentration were studied. With the Pp pellets the best grafting yield occurred at dose rate of 0.25 kGy/h and with the PE pellets the dose rate was lower. The irradiation dose from 8 to 10 kGy was sufficient to obtain the highest grafting degree, and the AA concentration of 40% v/v was suitable. The graft of poly (acrylic acid) was chemically modified for the immobilization of two enzymes, the glucose oxidase and the urease. For both enzymes the increasing of grafting degree onto the pellets, increased the enzyme immobilization yield. The immobilized glucose oxidase showed the best activity when immobilized onto Pp-A A supports with grafting degree around 2%. The optimum p H and temperature profiles, and the Km and Vmax for free and immobilized enzyme were determined. The supports grafted with A A were not suitable for the chemical immobilization of urease. (author)

  5. Immobilization of iodine in concrete

    Science.gov (United States)

    Clark, Walter E.; Thompson, Clarence T.

    1977-04-12

    A method for immobilizing fission product radioactive iodine recovered from irradiated nuclear fuel comprises combining material comprising water, Portland cement and about 3-20 wt. % iodine as Ba(IO.sub.3).sub.2 to provide a fluid mixture and allowing the fluid mixture to harden, said Ba(IO.sub.3).sub.2 comprising said radioactive iodine. An article for solid waste disposal comprises concrete prepared by this method. BACKGROUND OF THE INVENTION This invention was made in the course of, or under a contract with the Energy Research and Development Administration. It relates in general to reactor waste solidification and more specifically to the immobilization of fission product radioactive iodine recovered from irradiated nuclear fuel for underground storage.

  6. Contaminant immobilization via microbial activity

    International Nuclear Information System (INIS)

    1991-11-01

    The aim of this study was to search the literature to identify biological techniques that could be applied to the restoration of contaminated groundwaters near uranium milling sites. Through bioremediation it was hypothesized that the hazardous heavy metals could be immobilized in a stable, low-solubility form, thereby halting their progress in the migrating groundwater. Three basic mechanisms were examined: reduction of heavy metals by microbially produced hydrogen sulfide; direct microbial mediated reduction; and biosorption

  7. Immobilization of iodine in concrete

    International Nuclear Information System (INIS)

    Clark, W.E.; Thompson, C.T.

    1977-01-01

    A method for immobilizing fission product radioactive iodine recovered from irradiated nuclear fuel comprises combining material comprising water, Portland cement and about 3 to 20 wt percent iodine as Ba(IO 3 ) 2 to provide a fluid mixture and allowing the fluid mixture to harden, said Ba(IO 3 ) 2 comprising said radioactive iodine. An article for solid waste disposal comprises concrete prepared by this method. 10 claims, 2 figures

  8. Covalent Immobilization of β-Glucosidase on Magnetic Particles for Lignocellulose Hydrolysis

    DEFF Research Database (Denmark)

    Alftrén, Johan; Hobley, Timothy John

    2013-01-01

    β-Glucosidase hydrolyzes cellobiose to glucose and is an important enzyme in the consortium used for hydrolysis of cellulosic and lignocellulosic feedstocks. In the present work, β-glucosidase was covalently immobilized on non-porous magnetic particles to enable re-use of the enzyme. It was found...... that particles activated with cyanuric chloride and polyglutaraldehyde gave the highest bead-related immobilized enzyme activity when tested with p-nitrophenyl-β-D-glucopyranoside (104.7 and 82.2 U/g particles, respectively). Furthermore, the purified β-glucosidase preparation from Megazyme gave higher bead......-related enzyme activities compared to Novozym 188 (79.0 and 9.8 U/g particles, respectively). A significant improvement in thermal stability was observed for immobilized enzyme compared to free enzyme; after 5 h (at 65 °C), 36 % of activity remained for the former, while there was no activity in the latter...

  9. The development, characterization, and application of biomimetic nanoscale enzyme immobilization

    Science.gov (United States)

    Haase, Nicholas R.

    The utilization of enzymes is of interest for applications such as biosensors and biofuel cells. Immobilizing enzymes provides a means to develop these applications. Previous immobilization efforts have been accomplished by exposing surfaces on which silica-forming molecules are present to solutions containing an enzyme and a silica precursor. This approach leads to the enzyme being entrapped in a matrix three orders of magnitude larger than the enzyme itself, resulting in low retention of enzyme activity. The research herein introduces a method for the immobilization of enzymes during the layer-by-layer buildup of Si-O and Ti-O coatings which are nanoscale in thickness. This approach is an application of a peptide-induced mineral deposition method developed in the Sandhage and Kroger groups, and it involves the alternating exposure of a surface to solutions containing the peptide protamine and then an aqueous precursor solution of silicon- or titanium-oxide at near-neutral pH. A method has been developed that enables in situ immobilization of enzymes in the protamine/mineral oxide coatings. Depending on the layer and mineral (silica or titania) within which the enzyme is incorporated, the resulting multilayer biocatalytic hybrid materials retain 20 -- 100% of the enzyme activity. Analyses of kinetic properties of the immobilized enzyme, coupled with characterization of physical properties of the mineral-bearing layers (thickness, porosity, pore size distribution), indicates that the catalytic activities of the enzymes immobilized in the different layers are largely determined by substrate diffusion. The enzyme was also found to be substantially stabilized against heat-induced denaturation and largely protected from proteolytic attack. These functional coatings are then developed for use as antimicrobial materials. Glucose oxidase, which catalyzes production of the cytotoxic agent hydrogen peroxide, was immobilized with silver nanoparticles, can release

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Continuous Ethanol Production Using Immobilized-Cell/Enzyme Biocatalysts in Fluidized-Bed Bioreactor (FBR)

    Energy Technology Data Exchange (ETDEWEB)

    Nghiem, NP

    2003-11-16

    The immobilized-cell fluidized-bed bioreactor (FBR) was developed at Oak Ridge National Laboratory (ORNL). Previous studies at ORNL using immobilized Zymomonas mobilis in FBR at both laboratory and demonstration scale (4-in-ID by 20-ft-tall) have shown that the system was more than 50 times as productive as industrial benchmarks (batch and fed-batch free cell fermentations for ethanol production from glucose). Economic analysis showed that a continuous process employing the FBR technology to produce ethanol from corn-derived glucose would offer savings of three to six cents per gallon of ethanol compared to a typical batch process. The application of the FBR technology for ethanol production was extended to investigate more complex feedstocks, which included starch and lignocellulosic-derived mixed sugars. Economic analysis and mathematical modeling of the reactor were included in the investigation. This report summarizes the results of these extensive studies.

  17. Effect of irradiation on immobilized enzymes compared with that on enzymes in solution

    International Nuclear Information System (INIS)

    Schachinger, L.; Schippel, C.; Altmann, E.; Diepold, B.; Yang, C.; Jaenike, M.; Hochhaeuser, E.

    1985-01-01

    Glucose oxidase and catalase were immobilized by attaching them to nylon fibers that had been treated with triethyloxonium-tetrafluoroborate, diaminohexane and glutaraldialdehyde according to Morris, Campell and Hornby (1975). This method assures that the enzymes are bound to a side chain of the polyamide structure. Enzyme activity (as measured by the O 2 -uptake and by microcalorimetry) was found to be unchanged after 2 years. The apparent Ksub(m)-constants of the immobilized enzymes with glucose were the same as those for enzymes in solution. GOD and catalase immobilized in poly(acrylamide) gel had the same Ksub(m)-value. Despite the high stability during storage, the radiation induced inactivation of enzymes immobilized on gel or chromosorb, an inorganic carrier, was of the same order of magnitude as that of the dissolved enzymes. The enzymes bound to nylon fibers showed a higher radiation sensitivity. This might have been caused by an additional attack on the binding site of the carrier. (orig.)

  18. Layer by layer assembly of glucose oxidase and thiourea onto glassy carbon electrode: Fabrication of glucose biosensor

    International Nuclear Information System (INIS)

    Salimi, Abdollah; Noorbakhsh, Abdollah

    2011-01-01

    Highlights: → Although various enzymes immobilization have been approve for the construction of glucose biosensor, a layer by layer (LBL) technique has attracted more attention due to simplicity of the procedure, wide choice of materials that can be used, controllability of film thickness and unique mechanical properties. → In this paper, we described a novel and simple strategy for developing an amperometric glucose biosensor based on layer-by-layer self assembly of glucose oxidase on the glassy carbon electrode modified by thiourea. → Thiourea has two amino groups that the one can be immobilized on the activated glassy carbon electrode and the other can be used for the coupling of glucose oxidase enzyme. → The biosensor exhibited good performance for electrocatalytic oxidation of glucose, such as high sensitivity, low detection limit, short response time and wide concentration range. → Finally, the new method is strongly recommended for immobilization of many other enzymes or proteins containing carbaldehyde or carboxylic groups for fabricating third generation biosensors and bioelectronics devices. - Abstract: For the first time a novel, simple and facile approach is described to construct highly stable glucose oxidase (GOx) multilayer onto glassy carbon (GC) electrode using thiourea (TU) as a covalent attachment cross-linker. The layer by layer (LBL) attachment process was confirmed by cyclic voltammetry, electrochemical impedance spectroscopy and Fourier transform infrared reflection spectroscopy (FT-IR-RS) techniques. Immobilized GOx shows excellent electrocatalytic activity toward glucose oxidation using ferrocenemethanol as artificial electron transfer mediator and biosensor response was directly correlated to the number of bilayers. The surface coverage of active GOx per bilayer, heterogeneous electron transfer rate constant (k s ) and Michaelis-Menten constant (K M ), of immobilized GOx were 1.50 x 10 -12 mol cm -2 , 9.2 ± 0.5 s -1 and 3.42(±0

  19. Layer by layer assembly of glucose oxidase and thiourea onto glassy carbon electrode: Fabrication of glucose biosensor

    Energy Technology Data Exchange (ETDEWEB)

    Salimi, Abdollah, E-mail: absalimi@yahoo.com [Department of Chemistry, University of Kurdistsn, P.O. Box 416, Sanandaj (Iran, Islamic Republic of); Research Center for Nanotechnology, University of Kurdistan, P.O. Box 416, Sanandaj (Iran, Islamic Republic of); Noorbakhsh, Abdollah [Department of Chemistry, University of Kurdistsn, P.O. Box 416, Sanandaj (Iran, Islamic Republic of); Department of Nanotechnology Engenering, Faculty of Advanced Science and Technology, University of Isfahan, 81746-73441 (Iran, Islamic Republic of)

    2011-07-01

    Highlights: > Although various enzymes immobilization have been approve for the construction of glucose biosensor, a layer by layer (LBL) technique has attracted more attention due to simplicity of the procedure, wide choice of materials that can be used, controllability of film thickness and unique mechanical properties. > In this paper, we described a novel and simple strategy for developing an amperometric glucose biosensor based on layer-by-layer self assembly of glucose oxidase on the glassy carbon electrode modified by thiourea. > Thiourea has two amino groups that the one can be immobilized on the activated glassy carbon electrode and the other can be used for the coupling of glucose oxidase enzyme. > The biosensor exhibited good performance for electrocatalytic oxidation of glucose, such as high sensitivity, low detection limit, short response time and wide concentration range. > Finally, the new method is strongly recommended for immobilization of many other enzymes or proteins containing carbaldehyde or carboxylic groups for fabricating third generation biosensors and bioelectronics devices. - Abstract: For the first time a novel, simple and facile approach is described to construct highly stable glucose oxidase (GOx) multilayer onto glassy carbon (GC) electrode using thiourea (TU) as a covalent attachment cross-linker. The layer by layer (LBL) attachment process was confirmed by cyclic voltammetry, electrochemical impedance spectroscopy and Fourier transform infrared reflection spectroscopy (FT-IR-RS) techniques. Immobilized GOx shows excellent electrocatalytic activity toward glucose oxidation using ferrocenemethanol as artificial electron transfer mediator and biosensor response was directly correlated to the number of bilayers. The surface coverage of active GOx per bilayer, heterogeneous electron transfer rate constant (k{sub s}) and Michaelis-Menten constant (K{sub M}), of immobilized GOx were 1.50 x 10{sup -12} mol cm{sup -2}, 9.2 {+-} 0.5 s{sup -1

  20. Fabrication and characterization of spiral interdigitated electrodes based biosensor for salivary glucose detection

    Science.gov (United States)

    Adelyn, P. Y. P.; Hashim, U.; Arshad, M. K. Md; Voon, C. H.; Liu, Wei-Wen; Kahar, S. M.; Huda, A. R. N.; Lee, H. Cheun

    2017-03-01

    This work introduces the non-invasive glucose monitoring technique by using the Complementary Metal Oxide Semiconductor (CMOS) technologically fabricated spiral Interdigitated Electrodes (IDE) based biosensor. Scanning Electron Microscopy (SEM) image explores the morphology of spiral IDE while Energy Dispersive X-Ray (EDX) determines the elements induced in spiral IDE. Oral saliva of two patients are collected and tested on the spiral IDE sensor with electrical characterization as glucose detection results. However, both patients exhibit their glucose level characteristics inconsistently. Therefore, this work could be extended and enhanced by adding Glutaraldehyde in between 3-Aminoproply)triethoxysilane (APTES) modified and glucose oxidase (GOD) enzyme immobilized layer with FTIR validation for bonding attachment.

  1. Titanium dioxide–cellulose hybrid nanocomposite and its glucose biosensor application

    International Nuclear Information System (INIS)

    Maniruzzaman, Mohammad; Jang, Sang-Dong; Kim, Jaehwan

    2012-01-01

    Highlights: ► An organic–inorganic hybrid nanocomposite was fabricated by blending TiO 2 nanoparticles and cellulose solution. ► The hybrid nanocomposite has advantages of biodegradability and bio-compatibility of cellulose and physical properties of TiO 2 . ► Enzyme glucose oxidase (GOx) was immobilized into the hybrid nanocomposite and covalent bonding between TiO 2 and GOx was confirmed by X-ray photoelectron analysis. ► Linear response of the glucose biosensor was obtained in the range of 1–10 mM. - Abstract: This paper investigates the fabrication of titanium dioxide (TiO 2 )–cellulose hybrid nanocomposite and its possibility for a conductometric glucose biosensor. TiO 2 nanoparticles were blended with cellulose solution prepared by dissolving cotton pulp with lithium chloride/N,N-dimethylacetamide solvent to fabricate TiO 2 –cellulose hybrid nanocomposite. The enzyme, glucose oxidase (GOx) was immobilized into this hybrid nanocomposite by physical adsorption method. The successful immobilization of glucose oxidase into TiO 2 –cellulose hybrid nanocomposite via covalent bonding between TiO 2 and GOx was confirmed by X-ray photoelectron analysis. The linear response of the glucose biosensor is obtained in the range of 1–10 mM. This study demonstrates that TiO 2 –cellulose hybrid nanocomposite can be a potential candidate for an inexpensive, flexible and disposable glucose biosensor.

  2. Properties of immobilized papain by radiation polymerization

    International Nuclear Information System (INIS)

    Kumakura, Minoru; Kaetsu, Isao

    1984-01-01

    Papain was immobilized by the radiation polymerization of various monomers at low temperatures and the effects of the polymer matrix on the enzyme activity and thermal stability of the immobilized enzymes were studied. The activity of the immobilized enzymes prepared from monofunctional (acrylate and methacrylate) monomers was higher than that from bifunctional (bismethacrylate) monomers and that from polyoxyethylene dimethacrylate monomers increased with an increase in the number of oxyethylene units. The thermal stability of the immobilized enzymes prepared from hydrophilic monomers was higher than that from hydrophobic monomers and increased markedly with increasing monomer concentration. (author)

  3. Immobilization of Lactobacillus rhamnosus in mesoporous silica-based material: An efficiency continuous cell-recycle fermentation system for lactic acid production.

    Science.gov (United States)

    Zhao, Zijian; Xie, Xiaona; Wang, Zhi; Tao, Yanchun; Niu, Xuedun; Huang, Xuri; Liu, Li; Li, Zhengqiang

    2016-06-01

    Lactic acid bacteria immobilization methods have been widely used for lactic acid production. Until now, the most common immobilization matrix used is calcium alginate. However, Ca-alginate gel disintegrated during lactic acid fermentation. To overcome this deficiency, we developed an immobilization method in which Lactobacillus rhamnosus cells were successfully encapsulated into an ordered mesoporous silica-based material under mild conditions with a high immobilization efficiency of 78.77% by using elemental analysis. We also optimized the cultivation conditions of the immobilized L. rhamnosus and obtained a high glucose conversion yield of 92.4%. Furthermore, L. rhamnosus encapsulated in mesoporous silica-based material exhibited operational stability during repeated fermentation processes and no decrease in lactic acid production up to 8 repeated batches. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  4. Nanoporous cerium oxide thin film for glucose biosensor.

    Science.gov (United States)

    Saha, Shibu; Arya, Sunil K; Singh, S P; Sreenivas, K; Malhotra, B D; Gupta, Vinay

    2009-03-15

    Nanoporous cerium oxide (CeO(2)) thin film deposited onto platinum (Pt) coated glass plate using pulsed laser deposition (PLD) has been utilized for immobilization of glucose oxidase (GOx). Atomic force microscopy studies reveal the formation of nanoporous surface morphology of CeO(2) thin film. Response studies carried out using differential pulsed voltammetry (DPV) and optical measurements show that the GOx/CeO(2)/Pt bio-electrode shows linearity in the range of 25-300 mg/dl of glucose concentration. The low value of Michaelis-Menten constant (1.01 mM) indicates enhanced enzyme affinity of GOx to glucose. The observed results show promising application of the nanoporous CeO(2) thin film for glucose sensing application without any surface functionalization or mediator.

  5. Glucose sensing based on Pt-MWCNT and MWCNT

    Science.gov (United States)

    Aryasomayajula, Lavanya; Xie, Jining; Wang, Shouyan; Varadan, Vijay K.

    2007-04-01

    It is known that multi walled carbon nanotubes (MWCNTs) is an excellent materials for biosensing applications and with the introduction of Pt nanoparticles (Pt-MWCNTs) of about 3nm in diameter in MWCNTs greatly increases the current sensitivity and also the signal to noise ratio. We fabricated the CNT- based glucose sensor by immobilization the bio enzyme, glucose oxidase (GoX), on the Pt-MWCNT and electrode were prepared. The sensor has been tested effectively for both the abnormal blood glucose levels- greater than 6.9 mM and less than 3.5 mM which are the prediabetic and diabetic glucose levels, respectively. The current signal obtained from the Pt-MWCNT was much higher compared to the MWCNT based sensors.

  6. Hexavalent chromate reduction during growth and by immobilized cells of arthrobacter sp. suk 1205

    International Nuclear Information System (INIS)

    Dey, S.; Paul, A.K.

    2017-01-01

    The chromate reducing actinomycetes, Arthrobacter sp. SUK 1205, isolated from chromite mine overburden of Odisha, India exhibited significant chromate reduction during growth with characteristic formation of pale green insoluble precipitate. Reduction of chromate increased with increase in inoculum density but the reduction potential declined as and when Cr(VI) concentration in the medium was increased. Chromate reducing efficiency was promoted when glycerol and glucose were used as electron donors and pH and temperature were maintained at 7.0 and 35 degree C, respectively. The reduction process was inhibited by several metal ions and metabolic inhibitors but not by Cu(II), Mn(II) and DNP. Among the matrices tested for whole cell immobilization, Ca-alginate immobilized whole cells were found to be most effective and were comparable with non-immobilized cells. Minimal salts (MS) medium was the most effective base for Cr(VI) reduction studies with immobilized cells. Under such conditions, the immobilized cells retained their enzymatic activity at least for 4 consecutive cycles indicating the potential of Arthrobacter sp. SUK 1205 in bioremediation of environmental chromium pollution. (author)

  7. Hydrolysis of whey lactose by immobilized β-Galactosidase

    Directory of Open Access Journals (Sweden)

    Marcela Panaro Mariotti

    2008-12-01

    Full Text Available Hydrolysis of whey lactose to glucose and galactose by immobilized galactosidase comes as an alternative to enlarge the possibilities of commercial use of this feedstock. To be applied at industrial scale, the process should be performed continuously .This work aimed to study the hydrolysis of whey lactose by an immobilized enzyme reactor. b-Galactosidase from Aspergillus oryzae was immobilized on silica and activity and stability were evaluated. The best immobilization results were attained by using glutaraldehyde as support's activator and enzyme stabilizer. The optimized enzyme proportion for immobilization was 15-20 mg g-1 of support. Treatments of whey were performed (microfiltration, thermal treatment and ultrafiltration, seeking the elimination of sludge, and the effects on operating the fixed bed reactor were evaluated. Ultrafiltration was the best treatment towards a proper substrate solution for feeding the reactor.A hidrólise de lactose de soro de leite, resultando em glicose e galactose, apresenta-se como uma alternativa para ampliar as possibilidades de uso comercial deste insumo. Para ser aplicado em escala industrial, o processo deve ser operado de modo contínuo. Reporta-se o estudo de um sistema objetivando hidrólise de lactose de soro de leite através de um reator com enzima imobilizada. b-Galactosidase de Aspergillus oryzae foi imobilizada em sílica, sendo avaliadas a estabilidade e atividade. Os melhores resultados de imobilização foram obtidos usando glutaraldeído como ativante do suporte e estabilizador da enzima. A proporção otimizada entre enzima e suporte foi 15-20 mg.g-1. Foram estudadas formas de tratamento do soro (microfiltração, tratamento térmico e ultrafiltração, objetivando eliminação de material suspenso, e avaliando os efeitos na operação de reator de leito fixo. A ultrafiltração foi o melhor tratamento, na busca de uma solução de substrato apropriada para o reator contínuo.

  8. Production of Biodiesel Using Immobilized Lipase and the Characterization of Different Co-Immobilizing Agents and Immobilization Methods

    Directory of Open Access Journals (Sweden)

    Kang Zhao

    2016-08-01

    Full Text Available Lipase from Candida sp. 99–125 is widely employed to catalyzed transesterification and can be used for biodiesel production. In this study, the lipase was immobilized by combined adsorption and entrapment to catalyze biodiesel production from waste cooking oil (WCO via transesterification, and investigating co-immobilizing agents as additives according to the enzyme activity. The addition of the mixed co-immobilizing agents has positive effects on the activities of the immobilized lipase. Three different immobilizing methods were compared by the conversion ratio of biodiesel and structured by Atom Force Microscopy (AFM and Scanning Electron Microscopy (SEM, respectively. It was found that entrapment followed by adsorption was the best method. The effect of the co-immobilizing agent amount, lipase dosage, water content, and reuse ability of the immobilized lipase was investigated. By comparison with previous research, this immobilized lipase showed good reuse ability: the conversion ratio excesses 70% after 10 subsequent reactions, in particular, was better than Novozym435 and TLIM on waste cooking oil for one unit of lipase.

  9. 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.

  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. Technetium Immobilization Forms Literature Survey

    Energy Technology Data Exchange (ETDEWEB)

    Westsik, Joseph H.; Cantrell, Kirk J.; Serne, R. Jeffrey; Qafoku, Nikolla

    2014-05-01

    Of the many radionuclides and contaminants in the tank wastes stored at the Hanford site, technetium-99 (99Tc) is one of the most challenging to effectively immobilize in a waste form for ultimate disposal. Within the Hanford Tank Waste Treatment and Immobilization Plant (WTP), the Tc will partition between both the high-level waste (HLW) and low-activity waste (LAW) fractions of the tank waste. The HLW fraction will be converted to a glass waste form in the HLW vitrification facility and the LAW fraction will be converted to another glass waste form in the LAW vitrification facility. In both vitrification facilities, the Tc is incorporated into the glass waste form but a significant fraction of the Tc volatilizes at the high glass-melting temperatures and is captured in the off-gas treatment systems at both facilities. The aqueous off-gas condensate solution containing the volatilized Tc is recycled and is added to the LAW glass melter feed. This recycle process is effective in increasing the loading of Tc in the LAW glass but it also disproportionally increases the sulfur and halides in the LAW melter feed which increases both the amount of LAW glass and either the duration of the LAW vitrification mission or the required supplemental LAW treatment capacity.

  12. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... symptoms include the following: High blood glucose High levels of sugar in the urine Frequent urination Increased ... you should check and what your blood glucose levels should be. Checking your blood and then treating ...

  13. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... blood glucose High levels of sugar in the urine Frequent urination Increased thirst Part of managing your ... glucose is above 240 mg/dl, check your urine for ketones. If you have ketones, do not ...

  14. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... can often lower your blood glucose level by exercising. However, if your blood glucose is above 240 ... ketones. If you have ketones, do not exercise. Exercising when ketones are present may make your blood ...

  15. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Complications DKA (Ketoacidosis) & Ketones Kidney Disease (Nephropathy) Gastroparesis Mental Health Step On Up Treatment & Care Blood Glucose ... glucose) Dawn Phenomenon Checking for Ketones Tight Diabetes Control donate en -- A Future Without Diabetes - a-future- ...

  16. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Español Hyperglycemia (High Blood Glucose) Hyperglycemia is the technical term for high blood glucose (blood sugar). High ... We Are Research Leaders We Support Your Doctor Student Resources Patient Access to Research Research Resources Practice ...

  17. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Blood Pressure Physical Activity High Blood Glucose My Health Advisor Tools To Know Your Risk Alert Day ... DKA (Ketoacidosis) & Ketones Kidney Disease (Nephropathy) Gastroparesis Mental Health Step On Up Treatment & Care Blood Glucose Testing ...

  18. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Your Carbs Count Glycemic Index Low-Calorie Sweeteners Sugar and Desserts Fitness Exercise & Type 1 Diabetes Get ... the technical term for high blood glucose (blood sugar). High blood glucose happens when the body has ...

  19. Studies Regarding the Membranous Support of a Glucose Biosensor Based on Gox

    Directory of Open Access Journals (Sweden)

    Otilia Bizerea-Spiridon

    2010-05-01

    Full Text Available To obtain glucose biosensors based on glucose oxidase (GOx, the enzyme can be immobilized on the sensitive surface of a glass electrode by different techniques: deposition on membranous support (cellophane or other macromolecular material or entrapment in a matrix. Deposition on membranous support also involves cross-linking with glutaraldehyde or entrapment in silica gel, following the sol-gel procedure. The aim of this preliminary work was to study the influence of cellophane replacement with a PVA based membranous support on the glucose biosensor performance. The data obtained at pH measurements of buffer solutions with cellophane and PVA membranous supports respectively, show that the PVA based membrane assures superior performances of the biosensor for low glucose concentrations determination (about 10-4 M. These results allow the transition to an improved immobilization technique, namely the enzyme entrapment in membranous material.

  20. Electro chemiluminescence Biosensor Based on Thioglycolic Acid-Capped Cd Se QDs for Sensing Glucose

    International Nuclear Information System (INIS)

    Jung, E. Y.; Ye, J. H.; Choi, S. H.; Jung, S. H.

    2016-01-01

    In order to detect low level glucose concentration, an electro chemiluminescence (ECL) biosensor based on TGA-capped Cd Se quantum dots (QDs) was fabricated by the immobilization of Cd Se QDs after modifying the surface of a glassy carbon electrode (GCE) with 4-amino thiophenol diazonium salts by the electrochemical method. For the detection of glucose concentration, glucose oxidase (GOD) was immobilized onto the fabricated Cd Se QDs-modified electrode. The fabricated ECL biosensor based on TGA-capped Cd Se QDs was characterized using a scanning electron microscope (SEM), UV-vis spectrophotometry, transmission electron microscopy (TEM), a fluorescence spectrometer (PL), and cyclic voltammetry (CV). The fabricated ECL biosensor based on TGA-capped Cd Se QDs is suitable for the detection of glucose concentrations in real human blood samples.

  1. Electrochemiluminescence Biosensor Based on Thioglycolic Acid-Capped CdSe QDs for Sensing Glucose

    Directory of Open Access Journals (Sweden)

    Eun-Young Jung

    2016-01-01

    Full Text Available In order to detect low level glucose concentration, an electrochemiluminescence (ECL biosensor based on TGA-capped CdSe quantum dots (QDs was fabricated by the immobilization of CdSe QDs after modifying the surface of a glassy carbon electrode (GCE with 4-aminothiophenol diazonium salts by the electrochemical method. For the detection of glucose concentration, glucose oxidase (GOD was immobilized onto the fabricated CdSe QDs-modified electrode. The fabricated ECL biosensor based on TGA-capped CdSe QDs was characterized using a scanning electron microscope (SEM, UV-vis spectrophotometry, transmission electron microscopy (TEM, a fluorescence spectrometer (PL, and cyclic voltammetry (CV. The fabricated ECL biosensor based on TGA-capped CdSe QDs is suitable for the detection of glucose concentrations in real human blood samples.

  2. Reusable urine glucose sensor based on functionalized graphene oxide conjugated Au electrode with protective layers

    Directory of Open Access Journals (Sweden)

    Hye Youn Kim

    2014-09-01

    Full Text Available An electrochemical based system with multiple layers coated on a functionalized graphene oxide Au electrode was developed to measure glucose concentration in urine in a more stable way. Two types of gold printed circuit boards were fabricated and graphene oxide was immobilized on their surface by chemical adsorption. Multiple layers, composed of a couple of polymers, were uniformly coated on the surface electrode. This device exhibited higher electrochemical responses against glucose, a greater resistivity in the presence of interferential substances in urine, and durable stabilities for longer periods of time than conventional units. The efficiency in current level according to the order and ratio of solution was evaluated during the immobilization of the layer. The fabricated electrodes were then also evaluated using hyperglycemic clinical samples and compared with the patterns of blood glucose measured with commercially available glucose meters. Our findings show that not only was their pattern similar but this similarity is well correlated.

  3. Enzymatic activity of Glucose Oxidase from Aspergillus niger IPBCC.08.610 On Modified Carbon Paste Electrode as Glucose Biosensor

    Science.gov (United States)

    Rohmayanti, T.; Ambarsari, L.; Maddu, A.

    2017-03-01

    Glucose oxidase (GOx) has been developed as glucose sensor for measuring blood glucose level because of its specificity to glucose oxidation. This research aimed to determine kinetic parameters of GOx activity voltametrically and further test its potential as a glucose biosensor. GOx, in this research, was produced by local fungi Aspergillus niger IPBCC.08.610 which was isolated from local vine in Tarakan, East Borneo, Indonesia. GOx was immobilized with glutaraldehyde, which cross-linked onto modified carbon paste electrode (MCPE) nanofiber polyaniline. Intracellular GOx activity was higher than extracellular ones. Immobilized GOx used glutaraldehyde 2.5% and dripped on the surface of MCPE nanofiber polyaniline. MCPE have a high conductance in copper with the diameter of 3 mm. The concentration of glucose in the lowest concentration of 0.2 mM generated a current value of 0.413 mA while 2 mM of glucose induced a current of 3,869 mA value. Km and Imax of GOx in MCPE activities polyaniline nanofiber were 2.88 mM and 3.869 mA,respectively, with turnover (Kcat) of 13 s-1. Sensitivity was 1.09 mA/mM and response time to produce a maximum peak current was 25 seconds. Km value was then converted into units of mg/dL and obtained 56.4 mg/dL. GOximmo-IPB|MCPE electrode is potential to be able to detect blood glucose level in a normal condition and hypoglycemia conditions

  4. [Blood glucose self monitoring].

    Science.gov (United States)

    Wascher, Thomas C; Stechemesser, Lars

    2016-04-01

    Self monitoring of blood glucose contributes to the integrated management of diabetes mellitus. It, thus, should be available for all patients with diabetes mellitus type-1 and type-2. Self monitoring of blood glucose improves patients safety, quality of life and glucose control. The current article represents the recommendations of the Austrian Diabetes Association for the use of blood glucose self monitoring according to current scientific evidence.

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

    Science.gov (United States)

    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.

  6. 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

  7. Optimization of Adsorptive Immobilization of Alcohol Dehydrogenases

    NARCIS (Netherlands)

    Trivedi, Archana; Heinemann, Matthias; Spiess, Antje C.; Daussmann, Thomas; Büchs, Jochen

    2005-01-01

    In this work, a systematic examination of various parameters of adsorptive immobilization of alcohol dehydrogenases (ADHs) on solid support is performed and the impact of these parameters on immobilization efficiency is studied. Depending on the source of the enzymes, these parameters differently

  8. Plutonium Immobilization Can Loading Equipment Review

    International Nuclear Information System (INIS)

    Kriikku, E.; Ward, C.; Stokes, M.; Randall, B.; Steed, J.; Jones, R.; Hamilton, L.

    1998-05-01

    This report lists the operations required to complete the Can Loading steps on the Pu Immobilization Plant Flow Sheets and evaluates the equipment options to complete each operation. This report recommends the most appropriate equipment to support Plutonium Immobilization Can Loading operations

  9. Strong and Reversible Monovalent Supramolecular Protein Immobilization

    NARCIS (Netherlands)

    Young, Jacqui F.; Nguyen, Hoang D.; Yang, Lanti; Huskens, Jurriaan; Jonkheijm, Pascal; Brunsveld, Luc

    2010-01-01

    Proteins with an iron clasp: Site-selective incorporation of a ferrocene molecule into a protein allows for easy, strong, and reversible supramolecular protein immobilization through a selective monovalent interaction of the ferrocene with a cucurbit[7]uril immobilized on a gold surface. The

  10. Drug immobilization of walrus (Odobenus rosmarus)

    Science.gov (United States)

    DeMaster, D.P.; Faro, J.B.; Estes, J.A.; Taggart, James; Zabel, C.

    1981-01-01

    Five out of nine walrus (Odobenus rosmarus) were successfully immobilized at Round Island, Alaska, in May of 1978 by combinations of phencyclidine hydrochloride and acepromazine hydrochloride. A crossbow was an effective delivery technique. Walruses that had recently hauled out were more suitable for immobilization than well-rested animals. Care was taken to prevent walruses from overheating or suffocating.

  11. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Carbohydrate Counting Make Your Carbs Count Glycemic Index Low-Calorie Sweeteners Sugar and Desserts Fitness Exercise & Type ... Checking Your Blood Glucose A1C and eAG Hypoglycemia (Low blood glucose) Hyperglycemia (High blood glucose) Dawn Phenomenon ...

  12. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... how often you should check and what your blood glucose levels should be. Checking your blood and then treating ... I Treat Hyperglycemia? You can often lower your blood glucose level by exercising. However, if your blood glucose is ...

  13. Electrocatalytic glucose sensor

    Energy Technology Data Exchange (ETDEWEB)

    Gebhardt, U; Luft, G; Mund, K; Preidel, W; Richter, G J

    1983-01-01

    An artificial pancreas consists of an insulin depot, a dosage unit and a glucose sensor. The measurement of the actual glucose concentration in blood is still an unsolved problem. Two methods are described for an electrocatalytic glucose sensor. Under the interfering action of amino acids and urea in-vitro measurements show an error of between 10% and 20%.

  14. Intrinsic kinetic parameters of substrate utilization by immobilized anaerobic sludge.

    Science.gov (United States)

    Zaiat, M; Vieira, L G; Foresti, E

    1997-01-20

    This article presents a method for evaluating the intrinsic kinetic parameters of the specific substrate utilization rate (r) equation and discusses the results obtained for anaerobic sludge-bed samples taken from a horizontal-flow anaerobic immobilized sludge (HAIS) reactor. This method utilizes a differential reactor filled with polyurethane foam matrices containing immobilized anaerobic sludge which is subjected to a range of feeding substrate flow rates. The range of liquid superficial velocities thus obtained are used for generating data of observed specific substrate utilization rates (r(obs)) under a diversity of external mass transfer resistance conditions. The r(obs) curves are then adjusted to permit their extrapolation for the condition of no external mass transfer resistance, and the values determined are used as a test for the condition of absence of limitation of internal mass transfer. The intrinsic parameters r(max), the maximum specific substrate utilization rate, and K(s), the half-velocity coefficient, are evaluated from the r values under no external mass transfer resistance and no internal mass transfer limitation. The application of such a method for anaerobic sludge immobilized in polyurethane foam particles treating a glucose substrate at 30 degrees C resulted in intrinsic r(max) and K(s), respectively, of 0.330 mg chemical oxygen demand (COD) . mg(-1) volatile suspended solids (VSS) . h(-1) and 72 mg COD . L(-1). In comparison with the values found in the literature, intrinsic r(max) is significantly high and intrinsic K(s) is relatively low. (c) 1997 John Wiley & Sons, Inc.

  15. Thermal Stabilization of Enzymes Immobilized within Carbon Paste Electrodes.

    Science.gov (United States)

    Wang, J; Liu, J; Cepra, G

    1997-08-01

    In this note we report on the remarkable thermal stabilization of enzymes immobilized in carbon paste electrodes. Amperometric biosensors are shown for the first time to withstand a prolonged high-temperature (>50 °C) stress. Nearly full activity of glucose oxidase is retained over periods of up to 4 months of thermal stress at 60-80 °C. Dramatic improvements in the thermostability are observed for polyphenol oxidase, lactate oxidase, alcohol oxidase, horseradish peroxidase, and amino acid oxidase. Such resistance to heat-induced denaturation is attributed to the conformational rigidity of these biocatalysts within the highly hydrophobic (mineral oil or silicone grease) pasting liquid. While no chemical stabilizer is needed for attaining such protective action, it appears that low humidity (i.e., low water content) is essential for minimizing the protein mobility. Besides their implications for electrochemical biosensors, such observations should lead to a new generation of thermoresistant enzyme reactors based on nonpolar semisolid supports.

  16. Immobilized fluid membranes for gas separation

    Science.gov (United States)

    Liu, Wei; Canfield, Nathan L; Zhang, Jian; Li, Xiaohong Shari; Zhang, Jiguang

    2014-03-18

    Provided herein are immobilized liquid membranes for gas separation, methods of preparing such membranes and uses thereof. In one example, the immobilized membrane includes a porous metallic host matrix and an immobilized liquid fluid (such as a silicone oil) that is immobilized within one or more pores included within the porous metallic host matrix. The immobilized liquid membrane is capable of selective permeation of one type of molecule (such as oxygen) over another type of molecule (such as water). In some examples, the selective membrane is incorporated into a device to supply oxygen from ambient air to the device for electrochemical reactions, and at the same time, to block water penetration and electrolyte loss from the device.

  17. An Integrated Glucose Sensor with an All-Solid-State Sodium Ion-Selective Electrode for a Minimally Invasive Glucose Monitoring System

    Directory of Open Access Journals (Sweden)

    Junko Kojima

    2015-06-01

    Full Text Available We developed a minimally invasive glucose monitoring system that uses a microneedle to permeate the skin surface and a small hydrogel to accumulate interstitial fluid glucose. The measurement of glucose and sodium ion levels in the hydrogel is required for estimating glucose levels in blood; therefore, we developed a small, enzyme-fixed glucose sensor with a high-selectivity, all-solid-state, sodium ion-selective electrode (ISE integrated into its design. The glucose sensor immobilized glucose oxidase showed a good correlation between the glucose levels in the hydrogels and the reference glucose levels (r > 0.99, and exhibited a good precision (coefficient of variation = 2.9%, 0.6 mg/dL. In the design of the sodium ISEs, we used the insertion material Na0.33MnO2 as the inner contact layer and DD16C5 exhibiting high Na+/K+ selectivity as the ionophore. The developed sodium ISE exhibited high selectivity (\\( \\log \\,k^{pot}_{Na,K} = -2.8\\ and good potential stability. The sodium ISE could measure 0.4 mM (10−3.4 M sodium ion levels in the hydrogels containing 268 mM (10−0.57 M KCl. The small integrated sensor (ϕ < 10 mm detected glucose and sodium ions in hydrogels simultaneously within 1 min, and it exhibited sufficient performance for use as a minimally invasive glucose monitoring system.

  18. Active coping with stress suppresses glucose metabolism in the rat hypothalamus.

    Science.gov (United States)

    Ono, Yumie; Lin, Hsiao-Chun; Tzen, Kai-Yuan; Chen, Hui-Hsing; Yang, Pai-Feng; Lai, Wen-Sung; Chen, Jyh-Horng; Onozuka, Minoru; Yen, Chen-Tung

    2012-03-01

    We used 18F-fluorodeoxyglucose small-animal positron-emission tomography to determine whether different styles of coping with stress are associated with different patterns of neuronal activity in the hypothalamus. Adult rats were subjected to immobilization (IMO)-stress or to a non-immobilized condition for 30 min, in random order on separate days, each of which was followed by brain-scanning. Some rats in the immobilized condition were allowed to actively cope with the stress by chewing a wooden stick during IMO, while the other immobilized rats were given nothing to chew on. Voxel-based statistical analysis of the brain imaging data shows that chewing counteracted the stress-induced increased glucose uptake in the hypothalamus to the level of the non-immobilized condition. Region-of-interest analysis of the glucose uptake values further showed that chewing significantly suppressed stress-induced increased glucose uptake in the paraventricular hypothalamic nucleus and the anterior hypothalamic area but not in the lateral hypothalamus. Together with the finding that the mean plasma corticosterone concentration at the termination of the IMO was also significantly suppressed when rats had an opportunity to chew a wooden stick, our results showed that active coping by chewing inhibited the activation of the hypothalamic-pituitary-adrenal axis to reduce the endocrine stress response.

  19. Plutonium Immobilization Can Loading Concepts

    International Nuclear Information System (INIS)

    Kriikku, E.; Ward, C.; Stokes, M.; Randall, B.; Steed, J.; Jones, R.; Hamilton, L.; Rogers, L.; Fiscus, J.; Dyches, G.

    1998-05-01

    The Plutonium Immobilization Facility will encapsulate plutonium in ceramic pucks and seal the pucks inside welded cans. Remote equipment will place these cans in magazines and the magazines in a Defense Waste Processing Facility (DWPF) canister. The DWPF will fill the canister with glass for permanent storage. This report discusses five can loading conceptual designs and the lists the advantages and disadvantages for each concept. This report identifies loading pucks into cans and backfilling cans with helium as the top priority can loading development areas. The can loading welder and cutter are very similar to the existing Savannah River Site (SRS) FB-Line bagless transfer welder and cutter and thus they are a low priority development item

  20. Immobilization of organic liquid wastes

    International Nuclear Information System (INIS)

    Greenhalgh, W.O.

    1985-01-01

    This report describes a portland cement immobilization process for the disposal treatment of radioactive organic liquid wastes which would be generated in a a FFTF fuels reprocessing line. An incineration system already on-hand was determined to be too costly to operate for the 100 to 400 gallons per year organic liquid. Organic test liquids were dispersed into an aqueous phosphate liquid using an emulsifier. A total of 109 gallons of potential and radioactive aqueous immiscible organic liquid wastes from Hanford 300 Area operations were solidified with portland cement and disposed of as solid waste during a 3-month test program with in-drum mixers. Waste packing efficiencies varied from 32 to 40% and included pump oils, mineral spirits, and TBP-NPH type solvents

  1. Uranium Immobilization in Wetland Soils

    Science.gov (United States)

    Jaffe, Peter R.; Koster van Groos, Paul G.; Li, Dien; Chang, Hyun-Shik; Seaman, John C.; Kaplan, Daniel I.; Peacock, Aaron D.; Scheckel, Kirk

    2014-05-01

    stronger for the mesocosms with the higher Fe(II) load. Analysis via XANES showed that a fraction (up to ~1/3) of uranium was reduced to U(IV), for mesocosms operated under low iron loading, indicating that iron cycling in the rhizosphere also results in uranium reduction and immobilization. For mesocosms operating under the higher iron loading, the fraction of uranium immobilized as U(IV) was much lower, indicating that uranium co-precipitation with iron might have been the dominant immobilization process. In parallel to these mesocosm experiments, dialysis samplers have been deployed at the Savannah River National Laboratory near a creek with uranium contamination, to determine dissolved species, including Fe(II) and U(VI) in these wetland soils and their seasonal variability. The results show that there is a strong seasonal variability in dissolved iron and uranium, indicating a strong immobilization during the growing season, which is consistent with the mesocosm experimental results that the rhizosphere iron and uranium cycling are closely linked.

  2. ZnS nanoparticles electrodeposited onto ITO electrode as a platform for fabrication of enzyme-based biosensors of glucose

    International Nuclear Information System (INIS)

    Du, Jian; Yu, Xiuping; Wu, Ying; Di, Junwei

    2013-01-01

    The electrochemical and photoelectrochemical biosensors based on glucose oxidase (GOD) and ZnS nanoparticles modified indium tin oxide (ITO) electrode were investigated. The ZnS nanoparticles were electrodeposited directly on the surface of ITO electrode. The enzyme was immobilized on ZnS/ITO electrode surface by sol–gel method to fabricate glucose biosensor. GOD could electrocatalyze the reduction of dissolved oxygen, which resulted in a great increase of the reduction peak current. The reduction peak current decreased linearly with the addition of glucose, which could be used for glucose detection. Moreover, ZnS nanoparticles deposited on ITO electrode surface showed good photocurrent response under illumination. A photoelectrochemical biosensor for the detection of glucose was also developed by monitoring the decreases in the cathodic peak photocurrent. The results indicated that ZnS nanoparticles deposited on ITO substrate were a good candidate material for the immobilization of enzyme in glucose biosensor construction. - Highlights: ► ZnS nanoparticles were electrodeposited directly on ITO surface. ► The direct electron transfer of GOD immobilized on ZnS surface was obtained. ► The enzyme electrode was used to the determination of glucose in the presence of oxygen. ► The response of photoelectrochemical biosensor towards glucose was more sensitive

  3. ZnS nanoparticles electrodeposited onto ITO electrode as a platform for fabrication of enzyme-based biosensors of glucose

    Energy Technology Data Exchange (ETDEWEB)

    Du, Jian; Yu, Xiuping; Wu, Ying; Di, Junwei, E-mail: djw@suda.edu.cn

    2013-05-01

    The electrochemical and photoelectrochemical biosensors based on glucose oxidase (GOD) and ZnS nanoparticles modified indium tin oxide (ITO) electrode were investigated. The ZnS nanoparticles were electrodeposited directly on the surface of ITO electrode. The enzyme was immobilized on ZnS/ITO electrode surface by sol–gel method to fabricate glucose biosensor. GOD could electrocatalyze the reduction of dissolved oxygen, which resulted in a great increase of the reduction peak current. The reduction peak current decreased linearly with the addition of glucose, which could be used for glucose detection. Moreover, ZnS nanoparticles deposited on ITO electrode surface showed good photocurrent response under illumination. A photoelectrochemical biosensor for the detection of glucose was also developed by monitoring the decreases in the cathodic peak photocurrent. The results indicated that ZnS nanoparticles deposited on ITO substrate were a good candidate material for the immobilization of enzyme in glucose biosensor construction. - Highlights: ► ZnS nanoparticles were electrodeposited directly on ITO surface. ► The direct electron transfer of GOD immobilized on ZnS surface was obtained. ► The enzyme electrode was used to the determination of glucose in the presence of oxygen. ► The response of photoelectrochemical biosensor towards glucose was more sensitive.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. 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)

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. 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

  17. 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.

  18. Analytical modeling of glucose biosensors based on carbon nanotubes.

    Science.gov (United States)

    Pourasl, Ali H; Ahmadi, Mohammad Taghi; Rahmani, Meisam; Chin, Huei Chaeng; Lim, Cheng Siong; Ismail, Razali; Tan, Michael Loong Peng

    2014-01-15

    In recent years, carbon nanotubes have received widespread attention as promising carbon-based nanoelectronic devices. Due to their exceptional physical, chemical, and electrical properties, namely a high surface-to-volume ratio, their enhanced electron transfer properties, and their high thermal conductivity, carbon nanotubes can be used effectively as electrochemical sensors. The integration of carbon nanotubes with a functional group provides a good and solid support for the immobilization of enzymes. The determination of glucose levels using biosensors, particularly in the medical diagnostics and food industries, is gaining mass appeal. Glucose biosensors detect the glucose molecule by catalyzing glucose to gluconic acid and hydrogen peroxide in the presence of oxygen. This action provides high accuracy and a quick detection rate. In this paper, a single-wall carbon nanotube field-effect transistor biosensor for glucose detection is analytically modeled. In the proposed model, the glucose concentration is presented as a function of gate voltage. Subsequently, the proposed model is compared with existing experimental data. A good consensus between the model and the experimental data is reported. The simulated data demonstrate that the analytical model can be employed with an electrochemical glucose sensor to predict the behavior of the sensing mechanism in biosensors.

  19. Direct electrochemistry of glucose oxidase and a biosensor for glucose based on a glass carbon electrode modified with MoS2 nanosheets decorated with gold nanoparticles

    International Nuclear Information System (INIS)

    Su, Shao; Sun, Haofan; Xu, Fei; Yuwen, Lihui; Wang, Lianhui; Fan, Chunhai

    2014-01-01

    An electrochemical glucose biosensor was developed by immobilizing glucose oxidase (GOx) on a glass carbon electrode that was modified with molybdenum disulfide (MoS 2 ) nanosheets that were decorated with gold nanoparticles (AuNPs). The electrochemical performance of the modified electrode was investigated by cyclic voltammetry, and it is found that use of the AuNPs-decorated MoS 2 nanocomposite accelerates the electron transfer from electrode to the immobilized enzyme. This enables the direct electrochemistry of GOx without any electron mediator. The synergistic effect the MoS 2 nanosheets and the AuNPs result in excellent electrocatalytic activity. Glucose can be detected in the concentration range from 10 to 300 μM, and down to levels as low as 2.8 μM. The biosensor also displays good reproducibility and long-term stability, suggesting that it represents a promising tool for biological assays. (author)

  20. Hydrolysis of whey lactose by immobilized β-galactosidase in a bioreactor with a spirally wound membrane.

    Science.gov (United States)

    Vasileva, Nastya; Ivanov, Yavor; Damyanova, Stanka; Kostova, Iliana; Godjevargova, Tzonka

    2016-01-01

    The β-galactosidase was covalently immobilized onto a modified polypropylene membrane, using glutaraldehyde. The optimal conditions for hydrolysis of lactose (4.7%) by immobilized β-galactosidase in a batch process were determined 13.6 U enzyme activity, 40°C, pH 6.8 and 10h. The obtained degree of hydrolysis was compared with results received by a free enzyme. It was found, that the lactose hydrolysis by an immobilized enzyme was 1.6 times more effective than the lactose hydrolysis by a free enzyme. It was determined that the stability of the immobilized enzyme was 2 times higher in comparison with the stability of free enzyme. The obtained immobilized system β-galactosidase/polypropylene membrane was applied to produce glucose-galactose syrup from waste whey. The whey characteristics and the different preliminary treatments of the whey were investigated. Then the whey lactose hydrolysis in a bioreactor by an immobilized enzyme on a spirally wound membrane was performed. The optimal membrane surface and the optimal flow rate of the whey through the membrane module were determined, respectively 100 cm(2) and 1.0 mL min(-1). After 10h, the degree of lactose hydrolysis was increased to 91%. The operation stability was studied. After 20th cycle the yield of bioreactor was 69.7%. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Radioactive seed immobilization techniques for interstitial brachytherapy

    International Nuclear Information System (INIS)

    Yan, K.; Podder, T.; Buzurovic, I.; Hu, Y.; Dicker, A.; Valicenti, R.; Yu, Y.; Messing, E.; Rubens, D.; Sarkar, N.; Ng, W.

    2008-01-01

    In prostate brachytherapy, seeds can detach from their deposited sites and move locally in the pelvis or migrate to distant sites including the pulmonary and cardiac regions. Undesirable consequences of seed migration include inadequate dose coverage of the prostate and tissue irradiation effects at the site of migration. Thus, it is clinically important to develop seed immobilization techniques. We first analyze the possible causes for seed movement, and propose three potential techniques for seed immobilization: (1) surgical glue, (2) laser coagulation and (3) diathermy coagulation. The feasibility of each method is explored. Experiments were carried out using fresh bovine livers to investigate the efficacy of seed immobilization using surgical glue. Results have shown that the surgical glue can effectively immobilize the seeds. Evaluation of the radiation dose distribution revealed that the non-immobilized seed movement would change the planned isodose distribution considerably; while by using surgical glue method to immobilize the seeds, the changes were negligible. Prostate brachytherapy seed immobilization is necessary and three alternative mechanisms are promising for addressing this issue. Experiments for exploring the efficacy of the other two proposed methods are ongoing. Devices compatible with the brachytherapy procedure will be designed in future. (orig.)

  2. Insulin resistance for glucose metabolism in disused soleus muscle of mice

    Science.gov (United States)

    Seider, M. J.; Nicholson, W. F.; Booth, F. W.

    1981-01-01

    Results of this study on mice provide the first direct evidence of insulin resistance for glucose metabolism in skeletal muscle that has undergone a previous period of reduced muscle usage. This lack of responsiveness to insulin developed in one day and in the presence of hypoinsulinemia. Future studies will utilize the model of hindlimb immobilization to determine the causes of these changes.

  3. Engineering of Class II Cellobiose Dehydrogenases for Improved Glucose Sensitivity and Reduced Maltose Affinity

    DEFF Research Database (Denmark)

    Ortiz, Roberto; Rahman, Mahbubur; Zangrilli, Beatrice

    2017-01-01

    The front cover artwork is provided by Prof. Lo Gorton from Lund University (Sweden) and his co-workers. The image shows mutated cellobiose dehydrogenase (CDH) immobilized on a graphite electrode and how preferentially glucose is oxidized by this enzyme. Read the full text of the Article at 10.1002...

  4. Factors affecting N immobilisation/mineralisation kinetics for cellulose-, glucose- and straw-amended sandy soils

    NARCIS (Netherlands)

    Vinten, A.J.A.; Whitmore, A.P.; Bloem, J.; Howard, R.; Wright, F.

    2002-01-01

    The kinetics of nitrogen immobilization/mineralization for cellulose-, glucose- and straw-amended sandy soils were investigated in a series of laboratory incubations. Three Scottish soils expected to exhibit a range of biological activity were used: aloamy sand, intensively cropped horticultural

  5. Amperometric glucose biosensor based on glucose oxidase dispersed in multiwalled carbon nanotubes/graphene oxide hybrid biocomposite

    Energy Technology Data Exchange (ETDEWEB)

    Palanisamy, Selvakumar; Cheemalapati, Srikanth; Chen, Shen-Ming, E-mail: smchen78@ms15.hinet.net

    2014-01-01

    An amperometric glucose biosensor based on enhanced and fast direct electron transfer (DET) of glucose oxidase (GOx) at enzyme dispersed multiwalled carbon nanotubes/graphene oxide (MWCNT/GO) hybrid biocomposite was developed. The fabricated hybrid biocomposite was characterized by transmission electron microscopy (TEM), Raman and infrared spectroscopy (IR). The TEM image of hybrid biocomposite reveals that a thin layer of GOx was covered on the surface of MWCNT/GO hybrid composite. IR results validate that the hybrid biocomposite was formed through the electrostatic interactions between GOx and MWCNT/GO hybrid composite. Further, MWCNT/GO hybrid composite has also been characterized by TEM and UV–visible spectroscopy. A pair of well-defined redox peak was observed for GOx immobilized at the hybrid biocomposite electrode than that immobilized at the MWCNT modified electrode. The electron transfer rate constant (K{sub s}) of GOx at the hybrid biocomposite was calculated to be 11.22 s{sup −1}. The higher K{sub s} value revealed that fast DET of GOx occurred at the electrode surface. Moreover, fabricated biosensor showed a good sensitivity towards glucose oxidation over a linear range 0.05–23.2 mM. The limit of detection (LOD) was estimated to be 28 μM. The good features of the proposed biosensor could be used for the accurate detection of glucose in the biological samples. - Highlights: • An amperometric glucose biosensor has been developed at MWCNT/GO hybrid biocomposite. • Enhanced and fast direct electron transfer kinetics of glucose oxidase has been achieved at hybrid biocomposite. • Hybrid biocomposite has been characterized by TEM, IR and Raman spectroscopy. • Highly sensitive and selective for glucose determination.

  6. Amperometric glucose biosensor based on glucose oxidase dispersed in multiwalled carbon nanotubes/graphene oxide hybrid biocomposite

    International Nuclear Information System (INIS)

    Palanisamy, Selvakumar; Cheemalapati, Srikanth; Chen, Shen-Ming

    2014-01-01

    An amperometric glucose biosensor based on enhanced and fast direct electron transfer (DET) of glucose oxidase (GOx) at enzyme dispersed multiwalled carbon nanotubes/graphene oxide (MWCNT/GO) hybrid biocomposite was developed. The fabricated hybrid biocomposite was characterized by transmission electron microscopy (TEM), Raman and infrared spectroscopy (IR). The TEM image of hybrid biocomposite reveals that a thin layer of GOx was covered on the surface of MWCNT/GO hybrid composite. IR results validate that the hybrid biocomposite was formed through the electrostatic interactions between GOx and MWCNT/GO hybrid composite. Further, MWCNT/GO hybrid composite has also been characterized by TEM and UV–visible spectroscopy. A pair of well-defined redox peak was observed for GOx immobilized at the hybrid biocomposite electrode than that immobilized at the MWCNT modified electrode. The electron transfer rate constant (K s ) of GOx at the hybrid biocomposite was calculated to be 11.22 s −1 . The higher K s value revealed that fast DET of GOx occurred at the electrode surface. Moreover, fabricated biosensor showed a good sensitivity towards glucose oxidation over a linear range 0.05–23.2 mM. The limit of detection (LOD) was estimated to be 28 μM. The good features of the proposed biosensor could be used for the accurate detection of glucose in the biological samples. - Highlights: • An amperometric glucose biosensor has been developed at MWCNT/GO hybrid biocomposite. • Enhanced and fast direct electron transfer kinetics of glucose oxidase has been achieved at hybrid biocomposite. • Hybrid biocomposite has been characterized by TEM, IR and Raman spectroscopy. • Highly sensitive and selective for glucose determination

  7. Surface cell immobilization within perfluoroalkoxy microchannels

    Energy Technology Data Exchange (ETDEWEB)

    Stojkovič, Gorazd; Krivec, Matic [Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana (Slovenia); Vesel, Alenka [Jožef Stefan Institute, Jamova cesta 39, 1000 Ljubljana (Slovenia); Marinšek, Marjan [Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana (Slovenia); Žnidaršič-Plazl, Polona, E-mail: polona.znidarsic@fkkt.uni-lj.si [Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana (Slovenia)

    2014-11-30

    Graphical abstract: - Highlights: • A very efficient approach for immobilization of cells into microreactors is presented. • It is applicable to various materials, including PFA and cyclic olefin (co)polymers. • It was used to immobilize different prokaryotic and eukaryotic microbes. • Cells were immobilized on the surface in high density and showed good stability. • Mechanisms of APTES interactions with target materials are proposed. - Abstract: Perfluoroalkoxy (PFA) is one of the most promising materials for the fabrication of cheap, solvent resistant and reusable microfluidic chips, which have been recently recognized as effective tools for biocatalytic process development. The application of biocatalysts significantly depends on efficient immobilization of enzymes or cells within the reactor enabling long-term biocatalyst use. Functionalization of PFA microchannels by 3-aminopropyltriethoxysilane (ATPES) and glutaraldehyde was used for rapid preparation of microbioreactors with surface-immobilized cells. X-ray photoelectron spectroscopy and scanning electron microscopy were used to accurately monitor individual treatment steps and to select conditions for cell immobilization. The optimized protocol for Saccharomyces cerevisiae immobilization on PFA microchannel walls comprised ethanol surface pretreatment, 4 h contacting with 10% APTES aqueous solution, 10 min treatment with 1% glutaraldehyde and 20 min contacting with cells in deionized water. The same protocol enabled also immobilization of Escherichia coli, Pseudomonas putida and Bacillus subtilis cells on PFA surface in high densities. Furthermore, the developed procedure has been proved to be very efficient also for surface immobilization of tested cells on other materials that are used for microreactor fabrication, including glass, polystyrene, poly (methyl methacrylate), polycarbonate, and two olefin-based polymers, namely Zeonor{sup ®} and Topas{sup ®}.

  8. Biodegradation of different petroleum hydrocarbons by free and immobilized microbial consortia.

    Science.gov (United States)

    Shen, Tiantian; Pi, Yongrui; Bao, Mutai; Xu, Nana; Li, Yiming; Lu, Jinren

    2015-12-01

    The efficiencies of free and immobilized microbial consortia in the degradation of different types of petroleum hydrocarbons were investigated. In this study, the biodegradation rates of naphthalene, phenanthrene, pyrene and crude oil reached about 80%, 30%, 56% and 48% under the optimum environmental conditions of free microbial consortia after 7 d. We evaluated five unique co-metabolic substances with petroleum hydrocarbons, α-lactose was the best co-metabolic substance among glucose, α-lactose, soluble starch, yeast powder and urea. The orthogonal biodegradation analysis results showed that semi-coke was the best immobilized carrier followed by walnut shell and activated carbon. Meanwhile, the significance of various factors that contribute to the biodegradation of semi-coke immobilized microbial consortia followed the order of: α-lactose > semi-coke > sodium alginate > CaCl2. Moreover, the degradation rate of the immobilized microbial consortium (47%) was higher than that of a free microbial consortium (26%) under environmental conditions such as the crude oil concentration of 3 g L(-1), NaCl concentration of 20 g L(-1), pH at 7.2-7.4 and temperature of 25 °C after 5 d. SEM and FTIR analyses revealed that the structure of semi-coke became more porous and easily adhered to the microbial consortium; the functional groups (e.g., hydroxy and phosphate) were identified in the microbial consortium and were changed by immobilization. This study demonstrated that the ability of microbial adaptation to the environment can be improved by immobilization which expands the application fields of microbial remediation.

  9. Immobilization of cellulase using porous polymer matrix

    International Nuclear Information System (INIS)

    Kumakura, M.; Kaetsu, I.

    1984-01-01

    A new method is discussed for the immobilization of cellulase using porous polymer matrices, which were obtained by radiation polymerization of hydrophilic monomers. In this method, the immobilized enzyme matrix was prepared by enzyme absorbtion in the porous polymer matrix and drying treatment. The enzyme activity of the immobilized enzyme matrix varied with monomer concentration, cooling rate of the monomer solution, and hydrophilicity of the polymer matrix, takinn the change of the nature of the porous structure in the polymer matrix. The leakage of the enzymes from the polymer matrix was not observed in the repeated batch enzyme reactions

  10. Immobilization of Peroxidase onto Magnetite Modified Polyaniline

    Directory of Open Access Journals (Sweden)

    Eduardo Fernandes Barbosa

    2012-01-01

    Full Text Available The present study describes the immobilization of horseradish peroxidase (HRP on magnetite-modified polyaniline (PANImG activated with glutaraldehyde. After the optimization of the methodology, the immobilization of HRP on PANImG produced the same yield (25% obtained for PANIG with an efficiency of 100% (active protein. The optimum pH for immobilization was displaced by the effect of the partition of protons produced in the microenvironment by the magnetite. The tests of repeated use have shown that PANImG-HRP can be used for 13 cycles with maintenance of 50% of the initial activity.

  11. Preparation and characterization of immobilized lipase on magnetic hydrophobic microspheres

    DEFF Research Database (Denmark)

    Guo, Zheng; Bai, Shu; Sun, Yan

    2003-01-01

    H for the immobilized CCL were determined. Activity amelioration of the immobilized CCL for the hydrolysis of olive oil was observed, indicating an interfacial activation of the enzyme after immobilization. Moreover, the immobilized CCL showed enhanced thermal stability and good durability in the repeated use after...

  12. Amperometric glucose biosensor based on layer-by-layer films of microperoxidase-11 and liposome-encapsulated glucose oxidase.

    Science.gov (United States)

    Graça, J S; de Oliveira, R F; de Moraes, M L; Ferreira, M

    2014-04-01

    An important step in several bioanalytical applications is the immobilization of biomolecules. Accordingly, this procedure must be carefully chosen to preserve their biological structure and fully explore their properties. For this purpose, we combined the versatility of the layer-by-layer (LbL) method for the immobilization of biomolecules with the protective behavior of liposome-encapsulated systems to fabricate a novel amperometric glucose biosensor. To obtain the biosensing unit, an LbL film of the H2O2 catalyst polypeptide microperoxidase-11 (MP-11) was assembled onto an indium-tin oxide (ITO) electrode followed by the deposition of a liposome-encapsulated glucose oxidase (GOx) layer. The biosensor response toward glucose detection showed a sensitivity of 0.91±0.09 (μA/cm2)/mM and a limit of detection (LOD) of 8.6±1.1 μM, demonstrating an improved performance compared to similar biosensors with a single phospholipid-liposome or even containing a non-encapsulated GOx layer. Finally, glucose detection was also performed in a zero-lactose milk sample to demonstrate the potential of the biosensor for food analysis. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Amperometric glucose biosensor based on glucose oxidase dispersed in multiwalled carbon nanotubes/graphene oxide hybrid biocomposite.

    Science.gov (United States)

    Palanisamy, Selvakumar; Cheemalapati, Srikanth; Chen, Shen-Ming

    2014-01-01

    An amperometric glucose biosensor based on enhanced and fast direct electron transfer (DET) of glucose oxidase (GOx) at enzyme dispersed multiwalled carbon nanotubes/graphene oxide (MWCNT/GO) hybrid biocomposite was developed. The fabricated hybrid biocomposite was characterized by transmission electron microscopy (TEM), Raman and infrared spectroscopy (IR). The TEM image of hybrid biocomposite reveals that a thin layer of GOx was covered on the surface of MWCNT/GO hybrid composite. IR results validate that the hybrid biocomposite was formed through the electrostatic interactions between GOx and MWCNT/GO hybrid composite. Further, MWCNT/GO hybrid composite has also been characterized by TEM and UV-visible spectroscopy. A pair of well-defined redox peak was observed for GOx immobilized at the hybrid biocomposite electrode than that immobilized at the MWCNT modified electrode. The electron transfer rate constant (Ks) of GOx at the hybrid biocomposite was calculated to be 11.22s(-1). The higher Ks value revealed that fast DET of GOx occurred at the electrode surface. Moreover, fabricated biosensor showed a good sensitivity towards glucose oxidation over a linear range 0.05-23.2mM. The limit of detection (LOD) was estimated to be 28μM. The good features of the proposed biosensor could be used for the accurate detection of glucose in the biological samples. © 2013.

  14. Post-stress recovery of pituitary-adrenal hormones and glucose, but not the response during exposure to the stressor, is a marker of stress intensity in highly stressful situations.

    Science.gov (United States)

    Márquez, Cristina; Belda, Xavier; Armario, Antonio

    2002-02-01

    Acute immobilization in male rats elicited the same ACTH, corticosterone and glucose response as foot shock when measured immediately after stress. However, post-stress recovery of plasma ACTH, corticosterone and glucose levels were delayed in immobilized versus shocked rats. Similarly, stress-induced anorexia was much greater in the former animals. All these data suggest that post-stress speed of recovery of some physiological variables is positively related to stressor intensity and could be used to evaluate it.

  15. An improved glucose/O2 membrane-less biofuel cell through glucose oxidase purification.

    Science.gov (United States)

    Gao, Feng; Courjean, Olivier; Mano, Nicolas

    2009-10-15

    A key objective in any bioelectrochemical systems is to improve the current densities and mass transport limitation. Most of the work is focused on increasing the specific surface of the electrodes or improving the electron transfer between enzymes and electrodes. However, nothing is said about the comparison of purified and non-purified enzyme and their effects on the biosensor efficiency. To illustrate the effect of the enzyme purity, we studied the widely used commercial Glucose Oxidase (GOx) from Aspergillus niger that we are using in our miniature membrane-less biofuel cell. Our results indicate that even if additional compounds contained in the lyophilized enzyme powder do not interfere with its intrinsic catalytic properties, they could prevent a good electron transfer between the enzyme and the electrode surface. By introducing a purified glucose oxidase into a bioelectrocatalyst immobilized on an electrode surface, we show that we can increase the interaction between the enzyme and the redox polymer, forming a better homogenous, leather like gel. At 5mM glucose concentration and under oxygen atmosphere, the current is three-fold higher when using a purified enzyme than it is when using a non-purified enzyme. Built with this novel anode, we showed that a miniature implantable membrane-less glucose-O(2) biofuel cell could produce, under air, twice the power density that is usually obtained when using a non-purified GOx.

  16. Measuring brain glucose phosphorylation with labeled glucose

    International Nuclear Information System (INIS)

    Brondsted, H.E.; Gjedde, A.

    1988-01-01

    This study tested whether glucose labeled at the C-6 position generates metabolites that leave brain so rapidly that C-6-labeled glucose cannot be used to measure brain glucose phosphorylation (CMRGlc). In pentobarbital-anesthetized rats, the parietal cortex uptake of [ 14 C]glucose labeled in the C-6 position was followed for times ranging from 10 s to 60 min. We subtracted the observed radioactivity from the radioactivity expected with no loss of labeled metabolites from brain by extrapolation of glucose uptake in an initial period when loss was negligible. The observed radioactivity was a monoexponentially declining function of the total radioactivity expected in the absence of metabolite loss. The constant of decline was 0.0077.min-1 for parietal cortex. Metabolites were lost from the beginning of the experiment. However, with correction for the loss of labeled metabolites, it was possible to determine an average CMRGlc between 4 and 60 min of circulation of 64 +/- 4 (SE; n = 49) mumol.hg-1.min-1

  17. Glucose from a cellulose-containing raw material

    Energy Technology Data Exchange (ETDEWEB)

    Feniksova, R V

    1977-01-01

    Glucose was produced by mixing the raw material with an enzyme and water, enzymic hydrolysis of raw material, and separation of the hydrolyzate. To increase the yield of glucose, the hydrolysis took place in 2 stages: at the 1st stage, the hydrolysis was continued until the mixture of glucose and oligosaccharides was obtained under the influence of an active enzyme (1 to 20 units/g of cellulose present in the raw material); at the 2nd stage, cellulase immobilized on an insoluble carrier (by covalent bonds) was used for the hydrolysis of oligosaccharides. The hydromodulus of the suspension of cellulose-containing raw material was maintained 5 to 30. A carrier nonhydrolyzable by cellulase, for instance a homogenious macroporous Aerosil gel, a porous glass, or porous ceramics, was used.

  18. Antimicrobial activity of immobilized lactoferrin and lactoferricin.

    Science.gov (United States)

    Chen, Renxun; Cole, Nerida; Dutta, Debarun; Kumar, Naresh; Willcox, Mark D P

    2017-11-01

    Lactoferrin and lactoferricin were immobilized on glass surfaces via two linkers, 4-azidobenzoic acid (ABA) or 4-fluoro-3-nitrophenyl azide (FNA). The resulting surfaces were characterized by X-ray photoelectron spectroscopy (XPS) and contact angle measurements. The antimicrobial activity of the surfaces was determined using Pseudomonas aeruginosa and Staphylococcus aureus strains by fluorescence microscopy. Lactoferrin and lactoferricin immobilization was confirmed by XPS showing significant increases (p lactoferricin immobilized on glass significantly (p lactoferricin were successfully immobilized on glass surfaces and showed promising antimicrobial activity against pathogenic bacteria. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2612-2617, 2017. © 2016 Wiley Periodicals, Inc.

  19. Immobilization of Rocky Flats Graphite Fines Residue

    International Nuclear Information System (INIS)

    Rudisill, T.S.

    1999-01-01

    The development of the immobilization process for graphite fines has proceeded through a series of experimental programs. The experimental procedures and results from each series of experiments are discussed in this report

  20. Immobilization and characterization of inulinase from Ulocladium ...

    Indian Academy of Sciences (India)

    2014-10-20

    Oct 20, 2014 ... The enzyme had optimum pH at 5.6 for free and immobilized U. atrum inulinase on polyester ... ceutical industry because of their beneficial effects in ..... Hanover LWJ 1993 Manufacturing, composing and applications of.

  1. Plutonium Immobilization Bagless Transfer Can Size Evaluation

    International Nuclear Information System (INIS)

    Kriikku, E.; Stokes, M.; Rogers, L.; Ward, C.

    1998-02-01

    This report identifies and documents the most appropriate bagless transfer can size to support Plutonium Immobilization Can Loading operations. Also, this report considers can diameter, can wall thickness, and can length

  2. Modeling intrinsic kinetics in immobilized photocatalytic microreactors

    NARCIS (Netherlands)

    Visan, Aura; Rafieian Boroujeni, Damon; Ogieglo, Wojciech; Lammertink, Rob G.H.

    2014-01-01

    The article presents a simple model for immobilized photocatalytic microreactors following a first order reaction rate with either light independency or light dependency described by photon absorption carrier generation semiconductor physics. Experimental data obtained for various residence times,

  3. A simplified technique for nasoendotracheal tube immobilization.

    OpenAIRE

    Berardo, N.; Leban, S. G.; Williams, F. A.

    1989-01-01

    A simplified technique for immobilization of a nasoendotracheal tube is described in which a wide strap of open cell, hypoallergenic, foam-backed fabric is secured to the patient's head with a Velcro fastener.

  4. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Complications Neuropathy Foot Complications DKA (Ketoacidosis) & Ketones Kidney Disease (Nephropathy) Gastroparesis Mental Health Step On Up Treatment & Care Blood Glucose Testing Medication Doctors, Nurses & More ...

  5. Biostable glucose permeable polymer

    DEFF Research Database (Denmark)

    2017-01-01

    A new biostable glucose permeable polymer has been developed which is useful, for example, in implantable glucose sensors. This biostable glucose permeable polymer has a number of advantageous characteristics and, for example, does not undergo hydrolytic cleavage and degradation, thereby providing...... a composition that facilitates long term sensor stability in vivo. The versatile characteristics of this polymer allow it to be used in a variety of contexts, for example to form the body of an implantable glucose sensor. The invention includes the polymer composition, sensor systems formed from this polymer...

  6. Hydrolysis of inulin from Jerusalem artichoke by inulinase immobilized on aminoethylcellulose

    Energy Technology Data Exchange (ETDEWEB)

    Kim, W.Y.; Byun, S.M.; Uhm, T.B.

    1982-01-01

    Purified inulinase (I, EC 3.2.1.7) of Kluyveromyces fragilis was immobilized on 2-aminoethylcellulose by treatment with 2% glutaraldehyde in 0.05M phosphate buffer, pH 7.0, for 2 hours at room temperature. The immobilized enzyme preparation had 39.3 units I activity/dried matrix, with 53.4% recovery yield of activity, and showed good operational stability in the presence of substrate, inulin or the tuber extract of Jerusalem artichoke. Optimum pH and temperature were 5.5 and 45 degrees, respectively. In a batch reactor, the conversion was 90% (D-fructose/D-glucose = 76/24) and 34 mg D-fructose/mL was produced from the artichoke tuber extract by the immobilized I in 20 hours. In column reactor packed with 28 mL immobilized I, the following conditions were optimal: height/diameter ratio of column 10.3 space time 3.8 hours temperature 40 degrees. Operation under these conditions gave 90% conversion of a 7% inulin solution and the productivity was 102 mmol/L/h.

  7. Immobilization technology for krypton in amorphous zeolite

    International Nuclear Information System (INIS)

    Takusagawa, Atsushi; Ishiyama, Keiichi

    1989-01-01

    Radioactive krypton recovered from the offgas of a reprocessing plant requires long-term storage on the order of 100 years. Immobilization technology for krypton into amorphous zeolite 5A is considered one of the best methods for long-term storage. In this report, conditions for immobilization treatment and stability of amorphous zeolite 5A loaded krypton against heat, radiation and water are discussed, and a treatment system using this technology is described. (author)

  8. Immobilization Technologies in Probiotic Food Production

    Directory of Open Access Journals (Sweden)

    Gregoria Mitropoulou

    2013-01-01

    Full Text Available Various supports and immobilization/encapsulation techniques have been proposed and tested for application in functional food production. In the present review, the use of probiotic microorganisms for the production of novel foods is discussed, while the benefits and criteria of using probiotic cultures are analyzed. Subsequently, immobilization/encapsulation applications in the food industry aiming at the prolongation of cell viability are described together with an evaluation of their potential future impact, which is also highlighted and assessed.

  9. Characterization of an immobilized cell, trickle bed reactor during long term butanol (ABE) fermentation.

    Science.gov (United States)

    Park, C H; Okos, M R; Wankat, P C

    1990-06-20

    Acetone-butanol-ethanol (ABE) fermentation was performed continuously in an immobilized cell, trickle bed reactor for 54 days without, degeneration by maintaining the pH above 4.3. Column clogging was minimized by structured packing of immobilization matrix. The reactor contained two serial glass columns packed with Clostridium acetobutylicum adsorbed on 12- and 20-in.-long polyester sponge strips at total flow rates between 38 and 98.7 mL/h. Cells were initially grown at 20 g/L glucose resulting in low butanol (1.15 g/L) production encouraging cell growth. After the initial cell growth phase a higher glucose concentration (38.7 g/L) improved solvent yield from 13.2 to 24.1 wt%, and butanol production rate was the best. Further improvement in solvent yield and butanol production rate was not observed with 60 g/L of glucose. However, when the fresh nutrient supply was limited to only the first column, solvent yield increased to 27.3 wt% and butanol selectivity was improved to 0.592 as compared to 0.541 when fresh feed was fed to both columns. The highest butanol concentration of 5.2 g/L occurred at 55% conversion of the feed with 60 g/L glucose. Liquid product yield of immobilized cells approached the theoretical value reported in the literature. Glucose and product concentration profiles along the column showed that the columns can be divided into production and inhibition regions. The length of each zone was dependent upon the feed glucose concentration and feed pattern. Unlike batch fermentation, there was no clear distinction between acid and solvent production regions. The pH dropped, from 6.18-6.43 to 4.50-4.90 in the first inch of the reactor. The pH dropped further to 4.36-4.65 by the exit of the column. The results indicate that the strategy for long term stable operation with high solvent yield requires a structured packing of biologically stable porous matrix such as polyester sponge, a pH maintenance above 4.3, glucose concentrations up to 60 g/L and

  10. The glucose oxidase-peroxidase assay for glucose

    Science.gov (United States)

    The glucose oxidase-peroxidase assay for glucose has served as a very specific, sensitive, and repeatable assay for detection of glucose in biological samples. It has been used successfully for analysis of glucose in samples from blood and urine, to analysis of glucose released from starch or glycog...

  11. Ceramification: A plutonium immobilization process

    Energy Technology Data Exchange (ETDEWEB)

    Rask, W.C. [Dept. of Energy, Golden, CO (United States); Phillips, A.G. [Rocky Flats Environmental Technology Site, Golden, CO (United States)

    1996-05-01

    This paper describes a low temperature technique for stabilizing and immobilizing actinide compounds using a combination process/storage vessel of stainless steel, in which measured amounts of actinide nitrate solutions and actinide oxides (and/or residues) are systematically treated to yield a solid article. The chemical ceramic process is based on a coating technology that produces rare earth oxide coatings for defense applications involving plutonium. The final product of this application is a solid, coherent actinide oxide with process-generated encapsulation that has long-term environmental stability. Actinide compounds can be stabilized as pure materials for ease of re-use or as intimate mixtures with additives such as rare earth oxides to increase their degree of proliferation resistance. Starting materials for the process can include nitrate solutions, powders, aggregates, sludges, incinerator ashes, and others. Agents such as cerium oxide or zirconium oxide may be added as powders or precursors to enhance the properties of the resulting solid product. Additives may be included to produce a final product suitable for use in nuclear fuel pellet production. The process is simple and reduces the time and expense for stabilizing plutonium compounds. It requires a very low equipment expenditure and can be readily implemented into existing gloveboxes. The process is easily conducted with less associated risk than proposed alternative technologies.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  17. 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.

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

    Science.gov (United States)

    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.

  19. 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).

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  2. 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.

  3. 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.

  4. Biotechnological production of vanillin using immobilized enzymes.

    Science.gov (United States)

    Furuya, Toshiki; Kuroiwa, Mari; Kino, Kuniki

    2017-02-10

    Vanillin is an important and popular plant flavor, but the amount of this compound available from plant sources is very limited. Biotechnological methods have high potential for vanillin production as an alternative to extraction from plant sources. Here, we report a new approach using immobilized enzymes for the production of vanillin. The recently discovered oxygenase Cso2 has coenzyme-independent catalytic activity for the conversion of isoeugenol and 4-vinylguaiacol to vanillin. Immobilization of Cso2 on Sepabeads EC-EA anion-exchange carrier conferred enhanced operational stability enabling repetitive use. This immobilized Cso2 catalyst allowed 6.8mg yield of vanillin from isoeugenol through ten reaction cycles at a 1mL scale. The coenzyme-independent decarboxylase Fdc, which has catalytic activity for the conversion of ferulic acid to 4-vinylguaiacol, was also immobilized on Sepabeads EC-EA. We demonstrated that the immobilized Fdc and Cso2 enabled the cascade synthesis of vanillin from ferulic acid via 4-vinylguaiacol with repetitive use of the catalysts. This study is the first example of biotechnological production of vanillin using immobilized enzymes, a process that provides new possibilities for vanillin production. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Radiation technology for immobilization of bioactive materials

    International Nuclear Information System (INIS)

    1988-12-01

    Within the framework of the Agency's coordinated research programme on ''Application of Radiation Technology in Immobilization of Bioactive Materials'', the third and final research coordination meeting was held at Beijing University, Beijing, People's Republic of China, 15-18 June 1987. The present publication compiles all presentations made at the meeting. Fundamental processes for the immobilization of enzymes, antibodies, cells and drugs were developed and established using gamma radiation, electron beams and plasma discharge. Applications of various biofunctional components, immobilized by radiation techniques in different processes, were studied. A range of backbone polymers has been examined together with various monomers. Coupling procedures have been developed which are relevant to our particular requirements. Enzymes of various types and characteristics have been immobilized with considerable efficiency. The immobilized biocatalysts have been shown to possess significant activity and retention of activity on storage. There appears to be a high degree of specificity associated with the properties of the immobilised biocatalysts, their activity and the ease of their preparation. Novel additives which lower the total radiation dose in grafting have been discovered and their value in immobilization processes assessed. Potential applications include: medical (diagnostic, therapeutic), and industrial processes (fermentation, bioseparation, etc.). Refs, figs and tabs

  6. 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.

  7. Preparation of Glucose Sensor Using Polydimethylsiloxane / Polypyrrole Complex

    Science.gov (United States)

    Yasuzawa, Mikito; Inoue, Shigeru; Imai, Shinji

    New glucose oxidase (GOD) immobilized glucose sensors were prepared by the electropolymerization of 1-(6-D-gluconamidohexyl) pyrrole (GHP) on the platinum wire electrode precoated with the mixture solution of pyrrole derivative GHP, polydimethylsiloxane (PDS) and Nafion. The addition of Nafion into the precoating mixture solution was essential to obtain suitable sensor sensitivity. However, the sensitivity was about the half of that of the electrode without PDS precoating. Although, the introduction of Nafion was effective to improve the long-term stability of the enzyme-immobilized electrode, the electrode prepared using Nafion, PDS and GHP performed excellent long-term stability even at the measurement and storage temperatures of 40°C. Relatively constant response current was obtained over 30 days under the condition of 40°C and over 9 months measured at 25°C. Moreover, the GOD-immobilized GHP polymer film prepared on the electrode precoated with GHP, PDS and Nafion solution, was found to have excellent hemocompatibility from the result of platelet rich plasma contacting test.

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

    Science.gov (United States)

    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.

  9. 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...

  10. 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...

  11. Methyl Red Decolorization Efficiency of a Korea Strain of Aspergillus sp. Immobilized into Different Polymeric Matrices.

    Science.gov (United States)

    Kim, Beom-Su; Blaghen, Mohamed; Lee, Kang-Min

    2017-07-01

      Intensive research studies have revealed that fungal decolorization of dye wastewater is a promising replacement for the current process of dye wastewater decolorization. The authors isolated an Aspergillus sp. from the effluent of a textile industry area in Korea and assessed the effects of a variety of operational parameters on the decolorization of methyl red (MR) by this strain of Aspergillus sp. This Aspergillus sp. was then immobilized by entrapment in several polymeric matrices and the effects of operational conditions on MR decolorization were investigated again. The optimal decolorization activity of this Aspergillus sp. was observed in 1% glucose at a temperature of 37 °C and pH of 6.0. Furthermore, stable decolorization efficiency was observed when fungal biomass was immobilized into alginate gel during repeated batch experiment. These results suggest that the Aspergillus sp. isolated in Korea could be used to treat industrial wastewaters containing MR dye.

  12. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... by Mail Close www.diabetes.org > Living With Diabetes > Treatment and Care > Blood Glucose Testing Share: Print Page ... and-how-tos, . In this section Living With Diabetes Treatment and Care Blood Glucose Testing Checking Your Blood ...

  13. Blood Glucose Determination

    DEFF Research Database (Denmark)

    Lippi, Giuseppe; Nybo, Mads; Cadamuro, Janne

    2018-01-01

    The measurement of fasting plasma glucose may be biased by a time-dependent decrease of glucose in blood tubes, mainly attributable to blood cell metabolism when glycolysis is not rapidly inhibited or blood cells cannot be rapidly separated from plasma. Although glycolysis inhibitors such as sodium...

  14. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... On Up Treatment & Care Blood Glucose Testing Medication Doctors, Nurses & More Oral Health & Hygiene Women A1C Insulin Pregnancy 8 Tips for ... is checking your blood glucose often. Ask your doctor how often you should ... associated with hyperglycemia. How Do I Treat Hyperglycemia? ...

  15. Parallel synthesis of libraries of anodic and cathodic functionalized electrodeposition paints as immobilization matrix for amperometric biosensors.

    Science.gov (United States)

    Ngounou, Bertrand; Aliyev, Elchin H; Guschin, Dmitrii A; Sultanov, Yusif M; Efendiev, Ayaz A; Schuhmann, Wolfgang

    2007-09-01

    The integration of flexible anchoring groups bearing imidazolyl or pyridyl substituents into the structure of electrodeposition paints (EDP) is the basis for the parallel synthesis of a library containing 107 members of different cathodic and anodic EDPs with a high variation in polymer properties. The obtained EDPs were used as immobilization matrix for biosensor fabrication using glucose oxidase as a model enzyme. Amperometric glucose sensors based on the different EDPs showed a wide variation in their sensor characteristics with respect to the apparent Michaelis-Menten constant (KM(app)) representing the linear measuring range and the maximum current (Imax(app)). Based on these results first assumptions concerning the impact of different side chains in the EDP on the expected biosensor properties could be obtained allowing for an improved rational optimization of EDPs used as immobilization matrix in amperometric biosensors.

  16. Brain Glucose Metabolism Controls Hepatic Glucose and Lipid Production

    OpenAIRE

    Lam, Tony K.T.

    2007-01-01

    Brain glucose-sensing mechanisms are implicated in the regulation of feeding behavior and hypoglycemic-induced hormonal counter-regulation. This commentary discusses recent findings indicating that the brain senses glucose to regulate both hepatic glucose and lipid production.

  17. Smart point-of-care systems for molecular diagnostics based on nanotechnology: whole blood glucose analysis

    Science.gov (United States)

    Devadhasan, Jasmine P.; Kim, Sanghyo

    2015-07-01

    Complementary metal oxide semiconductor (CMOS) image sensors are received great attention for their high efficiency in biological applications. The present work describes a CMOS image sensor-based whole blood glucose monitoring system through a point-of-care (POC) approach. A simple poly-ethylene terephthalate (PET) film chip was developed to carry out the enzyme kinetic reaction at various concentrations of blood glucose. In this technique, assay reagent was adsorbed onto amine functionalized silica (AFSiO2) nanoparticles in order to achieve glucose oxidation on the PET film chip. The AFSiO2 nanoparticles can immobilize the assay reagent with an electrostatic attraction and eased to develop the opaque platform which was technically suitable chip to analyze by the camera module. The oxidized glucose then produces a green color according to the glucose concentration and is analyzed by the camera module as a photon detection technique. The photon number decreases with increasing glucose concentration. The simple sensing approach, utilizing enzyme immobilized AFSiO2 nanoparticle chip and assay detection method was developed for quantitative glucose measurement.

  18. Self-assembly of glucose oxidase on reduced graphene oxide-magnetic nanoparticles nanocomposite-based direct electrochemistry for reagentless glucose biosensor.

    Science.gov (United States)

    Pakapongpan, Saithip; Poo-Arporn, Rungtiva P

    2017-07-01

    A novel approach of the immobilization of a highly selective and stable glucose biosensor based on direct electrochemistry was fabricated by a self-assembly of glucose oxidase (GOD) on reduced graphene oxide (RGO) covalently conjugated to magnetic nanoparticles (Fe 3 O 4 NPs) modified on a magnetic screen-printed electrode (MSPE). The RGO-Fe 3 O 4 nanocomposite has remarkable enhancement in large surface areas, is favorable environment for enzyme immobilization, facilitates electron transfer between enzymes and electrode surfaces and possesses superparamagnetism property. The morphology and electrochemical properties of RGO-Fe 3 O 4 /GOD were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, cyclic voltammetry (CV) and amperometry. The modified electrode was a fast, direct electron transfer with an apparent electron transfer rate constant (k s ) of 13.78s -1 . The proposed biosensor showed fast amperometric response (3s) to glucose with a wide linear range from 0.05 to 1mM, a low detection limit of 0.1μM at a signal to noise ratio of 3 (S/N=3) and good sensitivity (5.9μA/mM). The resulting biosensor has high stability, good reproducibility, excellent selectivity and successfully applied detection potential at -0.45V. This mediatorless glucose sensing used the advantages of covalent bonding and self-assembly as a new approach for immobilizing enzymes without any binder. It would be worth noting that it opens a new avenue for fabricating excellent electrochemical biosensors. This is a new approach that reporting the immobilization of glucose oxidase on reduced graphene oxide (RGO) covalently conjugated to magnetic nanoparticles (Fe 3 O 4 NPs) by electrostatic interaction and modified screen printed electrode. We propose the reagentless with fabrication method without binder and adhesive agents for immobilized enzyme. Fe 3 O 4 NPs increasing surface area to enhance the immobilization and prevent

  19. Nanomaterials in glucose sensing

    CERN Document Server

    Burugapalli, Krishna

    2013-01-01

    The smartness of nano-materials is attributed to their nanoscale and subsequently unique physicochemical properties and their use in glucose sensing has been aimed at improving performance, reducing cost and miniaturizing the sensor and its associated instrumentation. So far, portable (handheld) glucose analysers were introduced for hospital wards, emergency rooms and physicians' offices; single-use strip systems achieved nanolitre sampling for painless and accurate home glucose monitoring; advanced continuous monitoring devices having 2 to 7 days operating life are in clinical and home use; and continued research efforts are being made to develop and introduce increasingly advanced glucose monitoring systems for health as well as food, biotechnology, cell and tissue culture industries. Nanomaterials have touched every aspect of biosensor design and this chapter reviews their role in the development of advanced technologies for glucose sensing, and especially for diabetes. Research shows that overall, nanomat...

  20. Influence of Acute High Glucose on Protein Abundance Changes in Murine Glomerular Mesangial Cells

    Directory of Open Access Journals (Sweden)

    Michelle T. Barati

    2016-01-01

    Full Text Available The effects of acute exposure to high glucose levels as experienced by glomerular mesangial cells in postprandial conditions and states such as in prediabetes were investigated using proteomic methods. Two-dimensional gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry methods were used to identify protein expression patterns in immortalized rat mesangial cells altered by 2 h high glucose (HG growth conditions as compared to isoosmotic/normal glucose control (NG⁎ conditions. Unique protein expression changes at 2 h HG treatment were measured for 51 protein spots. These proteins could be broadly grouped into two categories: (1 proteins involved in cell survival/cell signaling and (2 proteins involved in stress response. Immunoblot experiments for a protein belonging to both categories, prohibitin (PHB, supported a trend for increased total expression as well as significant increases in an acidic PHB isoform. Additional studies confirmed the regulation of proteasomal subunit alpha-type 2 and the endoplasmic reticulum chaperone and oxidoreductase PDI (protein disulfide isomerase, suggesting altered ER protein folding capacity and proteasomal function in response to acute HG. We conclude that short term high glucose induces subtle changes in protein abundances suggesting posttranslational modifications and regulation of pathways involved in proteostasis.

  1. Continuous bio-catalytic conversion of sugar mixture to acetone-butanol-ethanol by immobilized Clostridium acetobutylicum DSM 792.

    Science.gov (United States)

    Survase, Shrikant A; van Heiningen, Adriaan; Granström, Tom

    2012-03-01

    Continuous production of acetone, n-butanol, and ethanol (ABE) was carried out using immobilized cells of Clostridium acetobutylicum DSM 792 using glucose and sugar mixture as a substrate. Among various lignocellulosic materials screened as a support matrix, coconut fibers and wood pulp fibers were found to be promising in batch experiments. With a motive of promoting wood-based bio-refinery concept, wood pulp was used as a cell holding material. Glucose and sugar mixture (glucose, mannose, galactose, arabinose, and xylose) comparable to lignocellulose hydrolysate was used as a substrate for continuous production of ABE. We report the best solvent productivity among wild-type strains using column reactor. The maximum total solvent concentration of 14.32 g L(-1) was obtained at a dilution rate of 0.22 h(-1) with glucose as a substrate compared to 12.64 g L(-1) at 0.5 h(-1) dilution rate with sugar mixture. The maximum solvent productivity (13.66 g L(-1) h(-1)) was obtained at a dilution rate of 1.9 h(-1) with glucose as a substrate whereas solvent productivity (12.14 g L(-1) h(-1)) was obtained at a dilution rate of 1.5 h(-1) with sugar mixture. The immobilized column reactor with wood pulp can become an efficient technology to be integrated with existing pulp mills to convert them into wood-based bio-refineries.

  2. Reversible thermal denaturation of immobilized rhodanese

    International Nuclear Information System (INIS)

    Horowitz, P.; Bowman, S.

    1987-01-01

    For the first time, the enzyme rhodanese had been refolded after thermal denaturation. This was previously not possible because of the strong tendency for the soluble enzyme to aggregate at temperatures above 37 degrees C. The present work used rhodanese that was covalently coupled to a solid support under conditions that were found to preserve enzyme activity. Rhodanese was immobilized using an N-hydroxymalonimidyl derivative of Sepharose containing a 6-carbon spacer. The number of immobilized competent active sites was measured by using [ 35 S]SO 3 (2-) to form an active site persulfide that is the obligatory catalytic intermediate. Soluble enzyme was irreversibly inactivated in 10 min at 52 degrees C. The immobilized enzyme regained at least 30% of its original activity even after boiling for 20 min. The immobilized enzyme had a Km and Vmax that were each approximately 3 times higher than the corresponding values for the native enzyme. After preincubation at high temperatures, progress curves for the immobilized enzyme showed induction periods of up to 5 min before attaining apparently linear steady states. The pH dependence of the activity was the same for both the soluble and the immobilized enzyme. These results indicate significant stabilization of rhodanese after immobilization, and instabilities caused by adventitious solution components are not the sole reasons for irreversibility of thermal denaturation seen with the soluble enzyme. The results are consistent with models for rhodanese that invoke protein association as a major cause of inactivation of the enzyme. Furthermore, the induction period in the progress curves is consistent with studies which show that rhodanese refolding proceeds through intermediate states

  3. The modulatory role of alpha-melanocyte stimulating hormone administered spinally in the regulation of blood glucose level in d-glucose-fed and restraint stress mouse models.

    Science.gov (United States)

    Sim, Yun-Beom; Park, Soo-Hyun; Kim, Sung-Su; Lim, Su-Min; Jung, Jun-Sub; Suh, Hong-Won

    2014-08-01

    Alpha-melanocyte stimulating hormone (α-MSH) is known as a regulator of the blood glucose homeostasis and food intake. In the present study, the possible roles of α-MSH located in the spinal cord in the regulation of the blood glucose level were investigated in d-glucose-fed and immobilization stress (IMO) mouse models. We found in the present study that intrathecal (i.t.) injection with α-MSH alone did not affect the blood glucose level. However, i.t. administration with α-MSH reduced the blood glucose level in d-glucose-fed model. The plasma insulin level was increased in d-glucose-fed model and was further increased by α-MSH, whereas α-MSH did not affect plasma corticosterone level in d-glucose-fed model. In addition, i.t. administration with glucagon alone enhanced blood glucose level and, i.t. injection with glucagon also increased the blood glucose level in d-glucose-fed model. In contrasted to results observed in d-glucose-fed model, i.t. treatment with α-MSH caused enhancement of the blood glucose level in IMO model. The plasma insulin level was increased in IMO model. The increased plasma insulin level by IMO was reduced by i.t. treatment with α-MSH, whereas i.t. pretreatment with α-MSH did not affect plasma corticosterone level in IMO model. Taken together, although spinally located α-MSH itself does not alter the blood glucose level, our results suggest that the activation of α-MSH system located in the spinal cord play important modulatory roles for the reduction of the blood glucose level in d-glucose fed model whereas α-MSH is responsible for the up-regulation of the blood glucose level in IMO model. The enhancement of insulin release may be responsible for modulatory action of α-MSH in down-regulation of the blood glucose in d-glucose fed model whereas reduction of insulin release may be responsible for modulatory action of α-MSH in up-regulation of the blood glucose in IMO model. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Characteristics of Immobilized Urease on Grafted Alginate Bead Systems

    Directory of Open Access Journals (Sweden)

    Enas N. Danial

    2015-04-01

    Full Text Available This study evaluated the biological importance of immobilized urease enzyme over the free urease. The support material used for urease immobilization was alginate. Generally, the immobilization of urease in alginate gel showed a marked increase in Km and Vmax. However, the immobilized urease showed higher thermal stability than that of free enzyme. The rate of thermal inactivation of the immobilized enzyme decreased due to entrapment in gel matrix. Also, the activity of the immobilized urease was more stable in retention than that of the free enzyme during the storage in solution, although the activity of the immobilized enzyme was lower in comparison with the free enzyme. A stable immobilized system and long storage life are convenient for applications that would not be feasible with a soluble enzyme system. These results highlighted the technical and biochemical benefits of immobilized urease over the free enzyme.

  5. Glucose screening tests during pregnancy

    Science.gov (United States)

    Oral glucose tolerance test - pregnancy; OGTT - pregnancy; Glucose challenge test - pregnancy; Gestational diabetes - glucose screening ... screening test between 24 and 28 weeks of pregnancy. The test may be done earlier if you ...

  6. Hydrogel-based electrochemical sensor for non-invasive and continuous glucose monitoring

    Science.gov (United States)

    Park, Habeen; Lee, Ji-Young; Kim, Dong-Chul; Koh, Younggook; Cha, Junhoe

    2017-07-01

    Monitoring blood glucose level of diabetic patients is crucial in diabetes care from life threating complications. Selfmonitoring blood glucose (SMBG) that involves finger prick to draw blood samples into the measurement system is a widely-used method of routine measurement of blood glucose levels to date. SMBG includes, however, unavoidable pain problems resulting from the repetitive measurements. We hereby present a hydrogel-based electrochemical (H-EC) sensor to monitor the glucose level, non-invasively. Glucose oxidase (GOx) was immobilized in the disc-type hydroxyethyl methacrylate (HEMA) based hydrogel and kept intact in the hydrogel. Fast electron transfer mediated by Prussian blue (PB, hexacyanoferrate) generated efficient signal amplifications to facilitate the detection of the extracted glucose from the interstitial fluid. The linear response and the selectivity against glucose of the H-EC sensor were validated by chronoamperometry. For the practical use, the outcomes from the correlation of the extracted glucose concentration and the blood glucose value by on-body extraction, as well as the validation of the hydrogel-based electrochemical (H-EC) device, were applied to the on-body glucose monitoring.

  7. Biological acetate production from carbon dioxide by Acetobacterium woodii and Clostridium ljungdahlii: The effect of cell immobilization.

    Science.gov (United States)

    Cheng, Hai-Hsuan; Syu, Jyun-Cyuan; Tien, Shih-Yuan; Whang, Liang-Ming

    2018-08-01

    This study investigated the acetate production from gas mixture of hydrogen (H 2 ) and carbon dioxide (CO 2 ) in the ratio of 7:3 using two acetogens: Acetobacterium woodii and Clostridium ljungdahlii. Batch result shows A. woodii performed two-phase degradation with the presence of glucose that lactate was produced from glucose and was reutilized for the production of butyrate and few acetate, while only acetate was detected when providing gas mixture. C. ljungdahlii produced butyrate and ethanol along with acetate when glucose was introduced, while only ethanol and acetate were found by feeding gas mixture. The acetate-to-ethanol (A/E) ratio can be enhanced by cell immobilization, while GAC immobilization produced only acetate and the production rate reached 0.072 mmol/d under fed-batch operation. Acetate production rate increased from 18 to 28 mmol/L/d with GAC immobilization when gas flowrate increased from 100 to 300 mL/min in anaerobic fluidized membrane bioreactor (AFMBR), and a highest A/E ratio of 30 implies the possible application of acetate recovery from H 2 and CO 2 . Copyright © 2018 Elsevier Ltd. All rights reserved.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. Excess Weapons Plutonium Immobilization in Russia

    International Nuclear Information System (INIS)

    Jardine, L.; Borisov, G.B.

    2000-01-01

    The joint goal of the Russian work is to establish a full-scale plutonium immobilization facility at a Russian industrial site by 2005. To achieve this requires that the necessary engineering and technical basis be developed in these Russian projects and the needed Russian approvals be obtained to conduct industrial-scale immobilization of plutonium-containing materials at a Russian industrial site by the 2005 date. This meeting and future work will provide the basis for joint decisions. Supporting R and D projects are being carried out at Russian Institutes that directly support the technical needs of Russian industrial sites to immobilize plutonium-containing materials. Special R and D on plutonium materials is also being carried out to support excess weapons disposition in Russia and the US, including nonproliferation studies of plutonium recovery from immobilization forms and accelerated radiation damage studies of the US-specified plutonium ceramic for immobilizing plutonium. This intriguing and extraordinary cooperation on certain aspects of the weapons plutonium problem is now progressing well and much work with plutonium has been completed in the past two years. Because much excellent and unique scientific and engineering technical work has now been completed in Russia in many aspects of plutonium immobilization, this meeting in St. Petersburg was both timely and necessary to summarize, review, and discuss these efforts among those who performed the actual work. The results of this meeting will help the US and Russia jointly define the future direction of the Russian plutonium immobilization program, and make it an even stronger and more integrated Russian program. The two objectives for the meeting were to: (1) Bring together the Russian organizations, experts, and managers performing the work into one place for four days to review and discuss their work with each other; and (2) Publish a meeting summary and a proceedings to compile reports of all the

  16. Disposition of surplus fissile materials via immobilization

    International Nuclear Information System (INIS)

    Gray, L.W.; Kan, T.; Sutcliffe, W.G.; McKibben, J.M.; Danker, W.

    1995-01-01

    In the Cold War aftermath, the US and Russia have agreed to large reductions in nuclear weapons. To aid in the selection of long-term management options, the USDOE has undertaken a multifaceted study to select options for storage and disposition of surplus plutonium (Pu). One disposition alternative being considered is immobilization. Immobilization is a process in which surplus Pu would be embedded in a suitable material to produce an appropriate form for ultimate disposal. To arrive at an appropriate form, we first reviewed published information on HLW immobilization technologies to identify forms to be prescreened. Surviving forms were screened using multi-attribute utility analysis to determine promising technologies for Pu immobilization. We further evaluated the most promising immobilization families to identify and seek solutions for chemical, chemical engineering, environmental, safety, and health problems; these problems remain to be solved before we can make technical decisions about the viability of using the forms for long-term disposition of Pu. All data, analyses, and reports are being provided to the DOE Office of Fissile Materials Disposition to support the Record of Decision that is anticipated in Summer of 1996

  17. Production of cellulase from immobilized Trichoderma reesei

    International Nuclear Information System (INIS)

    Kasai, Noboru; Tamada, Masao; Kumakura, Minoru

    1989-05-01

    This report completed the results that obtained on the study of the enzyme activity in the culture of immobilized Trichoderma reesei cells in flask scale (100ml) and bench scale (30l). In the flask scale culture, the batch and repeated batch culture were carried out, and in the bench scale culture, the batch, repeated batch and continuous culture were done by using a culture equipment that is an unit process of the bench scale test plant for saccharification of cellulosic wastes. The enzyme activity of the immobilized cells was higher than that of the intact cells in the flask scale culture and it was confirmed that the enzyme activity was not decreased on the repeated batch culture of six times even. In the bench scale culture, it was found that a optimum culture condition of the immobilized cells was not different from that of the free cells and the immobilized cells gave the enzyme solution with a high enzyme activity in the culture condition of 450rpm stirring speed and air supply of 0.1v/v/m above. The technique of the repeated batch and continuous culture for long times in bench scale without contamination was established. The enzyme activity of the immobilized cells in continuous culture became to be 85 % to that in batch culture and it was found that the enzyme solution with high enzyme activity was continuously obtained in the continuous culture for long times. (author)

  18. Haloalkane hydrolysis with an immobilized haloalkane dehalogenase.

    Science.gov (United States)

    Dravis, B C; Swanson, P E; Russell, A J

    2001-11-20

    Haloalkane dehalogenase from Rhodococcus rhodochrous was covalently immobilized onto a polyethyleneimine impregnated gamma-alumina support. The dehalogenating enzyme was found to retain greater than 40% of its original activity after immobilization, displaying an optimal loading (max. activity/supported protein) of 70 to 75 mg/g with an apparent maximum (max. protein/support) of 156 mg/g. The substrate, 1,2,3-trichloropropane, was found to favorably partition (adsorb) onto the inorganic alumina carrier (10 to 20 mg/g), thereby increasing the local reactant concentration with respect to the catalyst's environment, whereas the product, 2,3-dichloropropan-1-ol, demonstrated no affinity. Additionally, the inorganic alumina support exhibited no adverse effects because of solvent/component incompatibilities or deterioration due to pH variance (pH 7.0 to 10.5). As a result of the large surface area to volume ratio of the support matrix and the accessibility of the bound protein, the immobilized biocatalyst was not subject to internal mass transfer limitations. External diffusional restrictions could be eliminated with simple agitation (mixing speed: 50 rpm; flux: 4.22 cm/min). The pH-dependence of the immobilized dehalogenase was essentially the same as that for the native enzyme. Finally, both the thermostability and resistance toward inactivation by organic solvent were improved by more than an order of magnitude after immobilization. Copyright 2001 John Wiley & Sons, Inc.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. Enzymes immobilization on Fe{sub 3}O{sub 4}-gold nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Kalska-Szostko, B., E-mail: kalska@uwb.edu.pl [Institute of Chemistry, University of Bialystok, Hurtowa 1, 15-399 Bialystok (Poland); Rogowska, M.; Dubis, A. [Institute of Chemistry, University of Bialystok, Hurtowa 1, 15-399 Bialystok (Poland); Szymanski, K. [Department of Physics, University of Bialystok, Lipowa 41, 15-424 Bialystok (Poland)

    2012-01-15

    In the present study Fe{sub 3}O{sub 4} magnetic nanoparticles were synthesized by coprecipitation of Fe{sup 2+} and Fe{sup 3+} from chlorides. In the next step magnetite-gold core-shell nanoparticles were synthesized from HAuCl{sub 4} using an ethanol as a reducing agent. Finally, magnetic nanoparticles were functionalized by hexadecanethiol. The immobilization of biological molecules (trypsin and glucose oxidase) to the thiol-modified and unmodified magnetite-gold nanoparticles surface was tested. The resulting nanoparticles were characterized by infrared spectroscopy, differential scanning calorimetry, Moessbauer spectroscopy and transmission electron microscopy.

  8. A Universal Protocol for Photochemical Covalent Immobilization of Intact Carbohydrates for the Preparation of Carbohydrate Microarrays

    Science.gov (United States)

    Wang, Huibin; Zhang, Yiming; Yuan, Xun; Chen, Yi; Yan, Mingdi

    2010-01-01

    A universal photochemical method has been established for the immobilization of intact carbohydrates and their analogues, and for the fabrication of carbohydrate microarrays. The method features the use of perfluorophenyl azide (PFPA)-modified substrates and the photochemical reaction of surface azido groups with printed carbohydrates. Various aldoses, ketoses, non-reducing sugars such as alditols and their derivatives can be directly arrayed on the PFPA-modified chips. The lectin-recognition ability of arrayed mannose, glucose and their oligo- and polysaccharides were confirmed using surface plasmon resonance imaging and laser-induced fluorescence imaging. PMID:21138274

  9. Direct electrochemistry of glucose oxidase and glucose biosensing on a hydroxyl fullerenes modified glassy carbon electrode.

    Science.gov (United States)

    Gao, Yun-Fei; Yang, Tian; Yang, Xiao-Lu; Zhang, Yu-Shuai; Xiao, Bao-Lin; Hong, Jun; Sheibani, Nader; Ghourchian, Hedayatollah; Hong, Tao; Moosavi-Movahedi, Ali Akbar

    2014-10-15

    Direct electrochemistry of glucose oxidase (GOD) was achieved when GOD-hydroxyl fullerenes (HFs) nano-complex was immobilized on a glassy carbon (GC) electrode and protected with a chitosan (Chit) membrane. The ultraviolet-visible absorption spectrometry (UV-vis), transmission electron microscopy (TEM), and circular dichroism spectropolarimeter (CD) methods were utilized for additional characterization of the GOD, GOD-HFs and Chit/GOD-HFs. Chit/HFs may preserve the secondary structure and catalytic properties of GOD. The cyclic voltammograms (CVs) of the modified GC electrode showed a pair of well-defined quasi-reversible redox peaks with the formal potential (E°') of 353 ± 2 mV versus Ag/AgCl at a scan rate of 0.05 V/s. The heterogeneous electron transfer constant (ks) was calculated to be 2.7 ± 0.2s(-1). The modified electrode response to glucose was linear in the concentrations ranging from 0.05 to 1.0mM, with a detection limit of 5 ± 1 μM. The apparent Michaelis-Menten constant (Km(app)) was 694 ± 8 μM. Thus, the modified electrode could be applied as a third generation biosensor for glucose with high sensitivity, selectivity and low detection limit. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Glucose oxidase-graphene-chitosan modified electrode for direct electrochemistry and glucose sensing

    International Nuclear Information System (INIS)

    Kang, Xinhuang; Wang, Jun; Wu, Hong; Aksay, Ilhan A.; Liu, Jun; Lin, Yuehe

    2009-01-01

    Direct electrochemistry of a glucose oxidase (GOD)/graphene/chitosan nanocomposite was studied. The immobilized enzyme retains its bioactivity, exhibits a surface confined, reversible two-proton and two-electron transfer reaction, and has good stability, activity and a fast heterogeneous electron transfer rate with the rate constant (k s ) of 2.83 s -1 . A much higher enzyme loading (1.12 x 10 -9 mol/cm 2 ) is obtained as compared to the bare glass carbon surface. This GOD/graphene/chitosan nanocomposite film can be used for sensitive detection of glucose. The biosensor exhibits a wider linearity range from 0.08 mM to 12 mM glucose with a detection limit of 0.02 mM and much higher sensitivity (37.93 (micro)A mM -1 cm -2 ) as compared with other nanostructured supports. The excellent performance of the biosensor is attributed to large surface-to-volume ratio and high conductivity of graphene, and good biocompatibility of chitosan, which enhances the enzyme absorption and promotes direct electron transfer between redox enzymes and the surface of electrodes.

  11. An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes

    Science.gov (United States)

    Mohamad, Nur Royhaila; Marzuki, Nur Haziqah Che; Buang, Nor Aziah; Huyop, Fahrul; Wahab, Roswanira Abdul

    2015-01-01

    The current demands of sustainable green methodologies have increased the use of enzymatic technology in industrial processes. Employment of enzyme as biocatalysts offers the benefits of mild reaction conditions, biodegradability and catalytic efficiency. The harsh conditions of industrial processes, however, increase propensity of enzyme destabilization, shortening their industrial lifespan. Consequently, the technology of enzyme immobilization provides an effective means to circumvent these concerns by enhancing enzyme catalytic properties and also simplify downstream processing and improve operational stability. There are several techniques used to immobilize the enzymes onto supports which range from reversible physical adsorption and ionic linkages, to the irreversible stable covalent bonds. Such techniques produce immobilized enzymes of varying stability due to changes in the surface microenvironment and degree of multipoint attachment. Hence, it is mandatory to obtain information about the structure of the enzyme protein following interaction with the support surface as well as interactions of the enzymes with other proteins. Characterization technologies at the nanoscale level to study enzymes immobilized on surfaces are crucial to obtain valuable qualitative and quantitative information, including morphological visualization of the immobilized enzymes. These technologies are pertinent to assess efficacy of an immobilization technique and development of future enzyme immobilization strategies. PMID:26019635

  12. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... and Learning About Prediabetes Type 2 Diabetes Risk Test Lower Your Risk Healthy Eating Overweight Smoking High Blood Pressure Physical Activity High Blood Glucose My Health Advisor Tools ...

  13. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Overweight Smoking High Blood Pressure Physical Activity High Blood Glucose My Health Advisor Tools To Know Your Risk Alert Day Diabetes Basics Home Symptoms Diagnosis America's Diabetes Challenge Type 1 Type 2 Facts About Type 2 Enroll ...

  14. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... to Give Close Are You at Risk? Home Prevention Diagnosing Diabetes and Learning About Prediabetes Type 2 ... Disease (Nephropathy) Gastroparesis Mental Health Step On Up Treatment & Care Blood Glucose Testing Medication Doctors, Nurses & More ...

  15. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Ketoacidosis) & Ketones Kidney Disease (Nephropathy) Gastroparesis Mental Health Step On Up Treatment & Care Blood Glucose Testing Medication ... Learning at Camp Find a Camp Fundraising Events Step Out Walk to Stop Diabetes Tour de Cure ...

  16. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... EXPO Volunteer Opportunities Sponsorship and Exhibit Opportunities Camp ... when ketones are present may make your blood glucose level go even higher. You'll need to work with your doctor ...

  17. CSF glucose test

    Science.gov (United States)

    ... in the space surrounding the spinal cord and brain. ... Abnormal results include higher and lower glucose levels. Abnormal results may be due to: Infection (bacterial or fungus) Inflammation of the central nervous system Tumor

  18. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Risk Test Lower Your Risk Healthy Eating Overweight Smoking High Blood Pressure Physical Activity High Blood Glucose ... Clinical Practice Guidelines Patient Education Materials Scientific Sessions Journals for Professionals Professional Books Patient Access to Research ...

  19. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Risk Test Lower Your Risk Healthy Eating Overweight Smoking High Blood Pressure Physical Activity High Blood Glucose ... Diabetes Meal Plans Create Your Plate Gluten Free Diets Meal Planning for Vegetarian Diets Cook with Heart- ...

  20. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Risk Test Lower Your Risk Healthy Eating Overweight Smoking High Blood Pressure Physical Activity High Blood Glucose ... Index Low-Calorie Sweeteners Sugar and Desserts Fitness Exercise & Type 1 Diabetes Get Started Safely Get And ...

  1. Nocturnal continuous glucose monitoring

    DEFF Research Database (Denmark)

    Bay, Christiane; Kristensen, Peter Lommer; Pedersen-Bjergaard, Ulrik

    2013-01-01

    Abstract Background: A reliable method to detect biochemical nocturnal hypoglycemia is highly needed, especially in patients with recurrent severe hypoglycemia. We evaluated reliability of nocturnal continuous glucose monitoring (CGM) in patients with type 1 diabetes at high risk of severe...

  2. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Disease (Nephropathy) Gastroparesis Mental Health Step On Up Treatment & Care Blood Glucose Testing Medication Doctors, Nurses & More ... us get closer to curing diabetes and better treatments for those living with diabetes. Other Ways to ...

  3. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... for energy. When your body breaks down fats, waste products called ketones are produced. Your body cannot ... glucose) Dawn Phenomenon Checking for Ketones Tight Diabetes Control donate en -- A Future Without Diabetes - a-future- ...

  4. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... breast cancer and AIDS combined. Your gift today will help us get closer to curing diabetes and ... blood and then treating high blood glucose early will help you avoid problems associated with hyperglycemia. How ...

  5. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Glucose Testing Medication Doctors, Nurses & More Oral Health & Hygiene Women A1C Insulin Pregnancy 8 Tips for Caregivers ... updated, this is the "take-you-by-the-hand" guide that will become a trusted friend and ...

  6. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Test Lower Your Risk Healthy Eating Overweight Smoking High Blood Pressure Physical Activity High Blood Glucose My Health Advisor Tools To Know Your Risk Alert Day Diabetes Basics Home Symptoms Diagnosis America's Diabetes Challenge Type ...

  7. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Know Your Rights Employment Discrimination Health Care Professionals Law Enforcement Driver's License For Lawyers Food & Fitness Home ... symptoms include the following: High blood glucose High levels of sugar in the urine Frequent urination Increased ...

  8. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Risk Test Lower Your Risk Healthy Eating Overweight Smoking High Blood Pressure Physical Activity High Blood Glucose ... Day in the Life of Diabetes Famous People Working to Stop Diabetes Common Terms Diabetes Statistics Infographics ...

  9. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Superfoods Non-starchy Vegetables Grains and Starchy Vegetables Fats Alcohol What Can I Drink? Fruit Dairy Food ... glucose for fuel, so your body breaks down fats to use for energy. When your body breaks ...

  10. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Care Blood Glucose Testing Medication Doctors, Nurses & More Oral ... someone new is diagnosed. Diabetes causes more deaths a year than breast cancer and AIDS combined. Your gift today will help ...

  11. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... and Care > Blood Glucose Testing Share: Print Page Text Size: A A A Listen En Español Hyperglycemia ( ... compact USB drives that can carry a person's full medical record for use in an emergency. How ...

  12. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... for Association Events Messaging Tools Recruiting Advocates Local Market Planning Training Webinars News & Events Advocacy News Call ... Care > Blood Glucose Testing Share: Print Page Text Size: A A A Listen En Español Hyperglycemia (High ...

  13. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... and Learning About Prediabetes Type 2 Diabetes Risk Test Lower Your Risk Healthy Eating Overweight Smoking High Blood Pressure Physical Activity High Blood Glucose My Health ...

  14. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Nephropathy) Gastroparesis Mental Health Step On Up Treatment & Care Blood Glucose Testing Medication Doctors, Nurses & More Oral ... 2 Diabetes Know Your Rights Employment Discrimination Health Care Professionals Law Enforcement Driver's License For Lawyers Food & ...

  15. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Complications DKA (Ketoacidosis) & Ketones Kidney Disease (Nephropathy) Gastroparesis Mental Health Step On Up Treatment & Care Blood Glucose Testing Medication Doctors, Nurses & More Oral Health & Hygiene Women A1C Insulin Pregnancy 8 Tips for Caregivers Health ...

  16. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... blood glucose level go even higher. You'll need to work with your doctor to find the ... lead to ketoacidosis. Ketoacidosis is life-threatening and needs immediate treatment. Symptoms include: Shortness of breath Breath ...

  17. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Diagnosing Diabetes and Learning About Prediabetes Type 2 Diabetes Risk Test Lower Your Risk Healthy Eating Overweight Smoking High Blood Pressure Physical Activity High Blood Glucose My Health Advisor ...

  18. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... your blood and then treating high blood glucose early will help you avoid problems associated with hyperglycemia. ... to detect hyperglycemia so you can treat it early — before it gets worse. If you're new ...

  19. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Risk Healthy Eating Overweight Smoking High Blood Pressure Physical Activity High Blood Glucose My Health Advisor Tools To ... Email: Sign Up Thank you for signing up ' + ' '); $('.survey-form').show(); }, success: function (data) { $('#survey-errors').remove(); $('. ...

  20. Hyperglycemia (High Blood Glucose)

    Medline Plus

    Full Text Available ... Complications DKA (Ketoacidosis) & Ketones Kidney Disease (Nephropathy) Gastroparesis Mental Health Step On Up Treatment & Care Blood Glucose Testing ... Living With Diabetes Recently Diagnosed Treatment & Care Complications Health ... EXPOs Awareness Programs Wellness Lives Here Become a Member American ...