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Glucose Dehydrogenase (glucose + dehydrogenase)
Selected AbstractsBehavior of PQQ Glucose Dehydrogenase on Prussian Blue-Modified Carbon ElectrodeELECTROANALYSIS, Issue 13 2008Valdas Laurinavicius Abstract Glucose sensitive biosensor containing pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase immobilized on Prussian blue (PB)-modified graphite electrode was designed. Properties of the biosensor were investigated in the cathodic and anodic response detection regions. It was shown, that anodic response of the biosensor is sum of two signals: direct electron transport from reduced PQQ to the electrode and by formation of the PQQ-oxygen-PB-carbon ternary complex. Cathodic response of the biosensor is based on the oxidation of the reduced PQQ by PB-oxygen-PB complex. Electrochemical regeneration of the enzyme does not produce free hydrogen peroxide. [source] Inactivation of pqq genes of Enterobacter intermedium 60-2G reduces antifungal activity and induction of systemic resistanceFEMS MICROBIOLOGY LETTERS, Issue 1 2008Song Hee Han Abstract Enterobacter intermedium 60-2G, a phosphate solubilizing bacterium, has the ability to induce systemic resistance in plants against soft rot pathogen Erwinia carotovora. Glucose dehydrogenase, an enzyme that utilizes pyrroloquinoline quinone (PQQ) as a cofactor, is required for the synthesis of gluconic acid by E. intermedium 60-2G. Here, we report that the pqqA and pqqB genes are required for phosphate solubilization and induced systemic resistance against a soft rot pathogen in tobacco. Mutations in either the pqqA or pqqB gene abolished the production of 2-ketogluconic acid and eliminated the ability of E. intermedium to solubilize hydroxyapatite. Addition of gluconic acid to the growth media restored the ability of the pqqA mutant to produce 2-ketogluconic acid. Interestingly, both pqqA and pqqB mutants of E. intermedium lost their ability to inhibit the growth of the rice pathogen Magnaporthe grisea KI-409. Additionally, induced systemic resistance against the soft rot pathogen was attenuated in the pqq mutants. These functions were restored by complementation with the wild-type pqq gene cluster. Our findings suggest that PQQ plays an important function in beneficial traits including phosphate solubilization, antifungal activity, and induced systemic resistance of E. intermedium, possibly by acting as a cofactor for several enzymes including glucose dehydrogenase. [source] Mineral phosphate solubilization by rhizosphere bacteria and scope for manipulation of the direct oxidation pathway involving glucose dehydrogenaseJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2010B. Sashidhar Summary Microbial biodiversity in the soil plays a significant role in metabolism of complex molecules, helps in plant nutrition and offers countless new genes, biochemical pathways, antibiotics and other metabolites, useful molecules for agronomic productivity. Phosphorus being the second most important macro-nutrient required by the plants, next to nitrogen, its availability in soluble form in the soils is of great importance in agriculture. Microbes present in the soil employ different strategies to make use of unavailable forms of phosphate and in turn also help plants making phosphate available for plant use. Azotobacter, a free-living nitrogen fixer, is known to increase the fertility of the soil and in turn the productivity of different crops. The glucose dehydrogenase gene, the first enzyme in the direct oxidation pathway, contributes significantly to mineral phosphate solubilization ability in several Gram-negative bacteria. It is possible to enhance further the biofertilizer potential of plant growth-promoting rhizobacteria by introducing the genes involved mineral phosphate solubilization without affecting their ability to fix nitrogen or produce phytohormones for dual benefit to agricultural crops. Glucose dehydrogenases from Gram-negative bacteria can be engineered to improve their ability to use different substrates, function at higher temperatures and EDTA tolerance, etc., through site-directed mutagenesis. [source] An Aptamer-Based Bound/Free Separation System for Protein DetectionELECTROANALYSIS, Issue 11 2009Mieko Fukasawa Abstract Aptamer hybridizes with its complementary strand. However, the complementary strand has difficulties to hybridize with the aptamer bound to a target because the aptamer forms higher-order structures. Exploiting this property, we developed simple bound/free separation systems for thrombin and IgE detection. The complementary strand was immobilized onto beads and the aptamer was labeled with pyrroquinoline quinone glucose dehydrogenase (PQQGDH). In the absence of a target, the aptamer is trapped by beads, whereas in the presence of a target, the aptamer bound to the target is not trapped. Thus the aptamer-target complexes can be recovered easily and detected by PQQGDH activity. This system allow the detection of 270,pM thrombin and 1,nM IgE. [source] DNA Aptamers that Bind to PQQGDH as an Electrochemical Labeling ToolELECTROANALYSIS, Issue 11 2009Yuko Osawa Abstract We screened DNA aptamers that bind to pyrroquinoline quinone glucose dehydrogenase (PQQGDH) for the development of an electrochemical labeling tool. PQQGDH is an excellent enzyme for the signal amplification of biosensors. We focused on DNA aptamers as labeling agents and tried to select those DNA aptamers that bind to PQQGDH without affecting its enzymatic activity. After 7 rounds of screening, one aptamer was obtained: ,PGa4'. It bound to PQQGDH with specificity and showed no effect on the glucose dehydrogenase (GDH) activity. Moreover, beads labeled with PQQGDH via PGa4 generated an electrical current upon glucose addition. Therefore, we believe that the PGa4 aptamer against PQQGDH may become a powerful labeling tool for electrochemical biosensors. [source] Behavior of PQQ Glucose Dehydrogenase on Prussian Blue-Modified Carbon ElectrodeELECTROANALYSIS, Issue 13 2008Valdas Laurinavicius Abstract Glucose sensitive biosensor containing pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase immobilized on Prussian blue (PB)-modified graphite electrode was designed. Properties of the biosensor were investigated in the cathodic and anodic response detection regions. It was shown, that anodic response of the biosensor is sum of two signals: direct electron transport from reduced PQQ to the electrode and by formation of the PQQ-oxygen-PB-carbon ternary complex. Cathodic response of the biosensor is based on the oxidation of the reduced PQQ by PB-oxygen-PB complex. Electrochemical regeneration of the enzyme does not produce free hydrogen peroxide. [source] Enhancement of the NAD(P)(H) Pool in Escherichia coli for BiotransformationENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 4 2007F. Heuser Abstract In pyridine nucleotide-dependent, reductive whole cell biotransformation with resting cells of Escherichia coli, the availability of intracellular NAD(P)(H) is a pivotal point for an efficient and highly productive substrate conversion. The question whether an increase of the intracellular NAD(P)(H) concentration could increase the productivity was discussed controversially in the past. This is the first report on an E. coli strain with an increased NAD(P)(H) pool which was tested in a reductive biotransformation system for an increased productivity. Biotransformation was performed with a strain overexpressing a gene encoding an (R)-specific alcohol dehydrogenase for the stereospecific, NADPH-dependent reduction of methyl acetoacetate (MAA) to (R)-methyl-3-hydroxybutanoate (MHB). Cofactor regeneration was implemented via glucose oxidation by coexpression of a gene encoding glucose dehydrogenase. The specific MHB productivity (mmol mg,1 cell dry weight,1h,1) enabled a comparison between the E. coli,BL21(DE3) wild-type and a genetically modified strain. The enhancement of the NAD(P)(H) pool was achieved by genetic manipulation of the NAD(H) biosynthetic pathways. After simultaneous overexpression of the pncB and nadE genes, encoding nicotinic acid phosphoribosyltransferase and NAD synthetase, measurements of the total NAD(P)(H) pool, sizes showed a 7-fold and 2-fold increased intracellular concentration of NAD(H) and NADP(H), respectively. However, the implementation of an E.,coli strain carrying a genomically integrated pncB gene with an upstream T7,promoter for biotransformation did not result in reproducible increased specific cell productivity. [source] Inactivation of pqq genes of Enterobacter intermedium 60-2G reduces antifungal activity and induction of systemic resistanceFEMS MICROBIOLOGY LETTERS, Issue 1 2008Song Hee Han Abstract Enterobacter intermedium 60-2G, a phosphate solubilizing bacterium, has the ability to induce systemic resistance in plants against soft rot pathogen Erwinia carotovora. Glucose dehydrogenase, an enzyme that utilizes pyrroloquinoline quinone (PQQ) as a cofactor, is required for the synthesis of gluconic acid by E. intermedium 60-2G. Here, we report that the pqqA and pqqB genes are required for phosphate solubilization and induced systemic resistance against a soft rot pathogen in tobacco. Mutations in either the pqqA or pqqB gene abolished the production of 2-ketogluconic acid and eliminated the ability of E. intermedium to solubilize hydroxyapatite. Addition of gluconic acid to the growth media restored the ability of the pqqA mutant to produce 2-ketogluconic acid. Interestingly, both pqqA and pqqB mutants of E. intermedium lost their ability to inhibit the growth of the rice pathogen Magnaporthe grisea KI-409. Additionally, induced systemic resistance against the soft rot pathogen was attenuated in the pqq mutants. These functions were restored by complementation with the wild-type pqq gene cluster. Our findings suggest that PQQ plays an important function in beneficial traits including phosphate solubilization, antifungal activity, and induced systemic resistance of E. intermedium, possibly by acting as a cofactor for several enzymes including glucose dehydrogenase. [source] Biocatalytic aldehyde reduction using tailor-made whole-cell catalysts: a novel synthesis of the aroma chemical cinnamyl alcohol,FLAVOUR AND FRAGRANCE JOURNAL, Issue 3 2007Francoise Chamouleau Abstract A biocatalytic method for the synthesis of the aroma chemical cinnamyl alcohol by means of a wholecell-catalysed reduction of cinnamyl aldehyde has been developed. As a biocatalyst, recombinant whole cells overexpressing an alcohol dehydrogenase from Lactobacillus kefir and a glucose dehydrogenase from Thermoplasma acidophilum have been used in combination with d- glucose as co-substrate. The reduction process proceeds with a conversion of 98%, even at a high substrate input of 166 g/l cinnamyl aldehyde, and gives a yield of 77% of the desired product, cinnamyl alcohol. In addition to high product concentrations, further advantages of this approach are the use of a low-cost whole-cell catalyst, the high purity of the product, as well as the fact that there is no need for the addition of external co-factor in the biotransformation step. Copyright © 2007 John Wiley & Sons, Ltd. [source] Enantioselective Reduction of 4-Fluoroacetophenone at High Substrate Concentration using a Tailor-Made Recombinant Whole-Cell CatalystADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 4-5 2007Harald Gröger Abstract A practical and highly efficient biocatalytic synthesis of optically active (R)-4-fluorophenylethan-1-ol has been developed based on reduction of the corresponding 4-fluoroacetophenone in the presence of a tailor-made recombinant whole-cell biocatalyst, containing an alcohol dehydrogenase and a glucose dehydrogenase. The reaction proceeds in a pure aqueous solvent media at a substrate concentration of ca. 0.5,M, and gives the desired product with high conversion (>95,%), good yield (87,%) and with an excellent enantioselectivity of >99,% ee. In addition, activity tests further showed that also the analogous 2- and 3-fluoroacetophenones are promising substrates. [source] Mineral phosphate solubilization by rhizosphere bacteria and scope for manipulation of the direct oxidation pathway involving glucose dehydrogenaseJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2010B. Sashidhar Summary Microbial biodiversity in the soil plays a significant role in metabolism of complex molecules, helps in plant nutrition and offers countless new genes, biochemical pathways, antibiotics and other metabolites, useful molecules for agronomic productivity. Phosphorus being the second most important macro-nutrient required by the plants, next to nitrogen, its availability in soluble form in the soils is of great importance in agriculture. Microbes present in the soil employ different strategies to make use of unavailable forms of phosphate and in turn also help plants making phosphate available for plant use. Azotobacter, a free-living nitrogen fixer, is known to increase the fertility of the soil and in turn the productivity of different crops. The glucose dehydrogenase gene, the first enzyme in the direct oxidation pathway, contributes significantly to mineral phosphate solubilization ability in several Gram-negative bacteria. It is possible to enhance further the biofertilizer potential of plant growth-promoting rhizobacteria by introducing the genes involved mineral phosphate solubilization without affecting their ability to fix nitrogen or produce phytohormones for dual benefit to agricultural crops. Glucose dehydrogenases from Gram-negative bacteria can be engineered to improve their ability to use different substrates, function at higher temperatures and EDTA tolerance, etc., through site-directed mutagenesis. [source] Stereoselective synthesis of L-[15N] amino acids with glucose dehydrogenase and galactose mutarotase as NADH regenerating systemJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 4 2008Maria Chiriac Abstract We have developed an efficient stereospecific enzymatic synthesis of L-[15N]-valine, L-[15N]-leucine, L-[15N]-norvaline, L-[15N]-norleucine and L-[15N]-isoleucine from the corresponding ,-keto acids by coupling the reactions catalysed by leucine dehydrogenase and glucose dehydrogenase/galactose mutarotase. Giving high yields of L-amino acids, the procedure is economical and easy to perform and to monitor at a synthetically useful scale (1,10,g). Copyright © 2008 John Wiley & Sons, Ltd. [source] Determination of enzyme mechanisms by molecular dynamics: Studies on quinoproteins, methanol dehydrogenase, and soluble glucose dehydrogenasePROTEIN SCIENCE, Issue 8 2004Swarnalatha Y. Reddy Abstract Molecular dynamics (MD) simulations have been carried out to study the enzymatic mechanisms of quinoproteins, methanol dehydrogenase (MDH), and soluble glucose dehydrogenase (sGDH). The mechanisms of reduction of the orthoquinone cofactor (PQQ) of MDH and sGDH involve concerted base-catalyzed proton abstraction from the hydroxyl moiety of methanol or from the 1-hydroxyl of glucose, and hydride equivalent transfer from the substrate to the quinone carbonyl carbon C5 of PQQ. The products of methanol and glucose oxidation are formaldehyde and glucolactone, respectively. The immediate product of PQQ reduction, PQQH, [,HC5(O,) ,C4( = O) ,] and PQQH [,HC5(OH) ,C4( = O) ,] converts to the hydroquinone PQQH2 [,C5(OH) = C4(OH) ,]. The main focus is on MD structures of MDH , PQQ , methanol, MDH , PQQH,, MDH , PQQH, sGDH , PQQ , glucose, sGDH , PQQH, (glucolactone, and sGDH , PQQH. The reaction PQQ , PQQH, occurs with Glu 171,CO2, and His 144,Im as the base species in MDH and sGDH, respectively. The general-base-catalyzed hydroxyl proton abstraction from substrate concerted with hydride transfer to the C5 of PQQ is assisted by hydrogen-bonding to the C5 = O by Wat1 and Arg 324 in MDH and by Wat89 and Arg 228 in sGDH. Asp 297,COOH would act as a proton donor for the reaction PQQH, , PQQH, if formed by transfer of the proton from Glu 171,COOH to Asp 297,CO2, in MDH. For PQQH , PQQH2, migration of H5 to the C4 oxygen may be assisted by a weak base like water (either by crystal water Wat97 or bulk solvent, hydrogen-bonded to Glu 171,CO2, in MDH and by Wat89 in sGDH). [source] Laser-improved protein crystallization screeningACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Neela Yennawar Screening of proteins for crystallization under laser irradiation was investigated using six proteins: ribonuclease B, glucose dehydrogenase, lysozyme, sorbitol dehydrogenase, fructose dehydrogenase and myoglobin. Shining 532,nm green circularly polarized laser light with a picosecond pulse and 6,mW power for 30,s on newly set-up protein drops showed a marked improvement in the number of screen conditions amenable for crystal growth compared with control drops under identical conditions but without laser exposure. For glucose dehydrogenase and sorbitol dehydrogenase, larger and better quality crystals were formed and the resolution of X-ray diffraction was improved. The speed of crystallization increased in the case of ribonuclease B, lysozyme and sorbitol dehydrogenase. During laser irradiation, the amount of precipitation in the screened drops increased, indicating a transient decrease in protein solubility. At the optimized laser settings, there was no deleterious effect of the laser on crystal growth or on the protein. In the cases of ribonuclease B and lysozyme the crystal packing did not change owing to the laser exposure. [source] Pyrroloquinoline quinone-dependent carbohydrate dehydrogenase: Activity enhancement and the role of artificial electron acceptorsBIOTECHNOLOGY JOURNAL, Issue 8 2010Juozas Kulys Professor Abstract Pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (PQQ-GDH) offers a variety of opportunities for applications, e.g. in highly sensitive biosensors and electrosynthetic reactions. Here we report on the acceleration (up to 4.9 x 104 -fold) of enzymatic ferricyanide reduction by artificial redox mediators (enhancers). The reaction mechanism includes reduction of the PQQ-GDH by glucose followed by oxidation of the reduced PQQ cofactor with either ferricyanide or a redox mediator. A synergistic effect occurs through the oxidation of a reduced mediator by ferricyanide. Using kinetic description of the coupled reaction, the second order rate constant for the reaction of an oxidized mediator with the reduced enzyme cofactor (kox) can be calculated. For different mediators this value is 2.2 x 106,1.6 x 108 M -1s -1 at pH 7.2 and 25°C. However, no correlation of the rate constant with the midpoint redox potential of the mediator could be established. For low-potential mediators the synergistic effect is proportional to the ratio of kox(med)/kox(ferricyanide), whereas for the high-potential mediators the effect depends on both this ratio and the concentration of the oxidized mediator, which can be calculated from the Nernst equation. The described effect can be applied in various ways, e.g. for substrate reactivity determination, electrosynthetic PQQ cofactor regeneration or building of new highly sensitive biosensors. [source] Preliminary crystallographic studies of glucose dehydrogenase from the promiscuous Entner,Doudoroff pathway in the hyperthermophilic archaeon Sulfolobus solfataricusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2005Alex Theodossis The hyperthermophilic archaeon Sulfolobus solfataricus grows optimally above 353,K and can metabolize glucose and its C4 epimer galactose via a non-phosphorylative variant of the Entner,Doudoroff pathway involving catalytically promiscuous enzymes that can operate with both sugars. The initial oxidation step is catalysed by glucose dehydrogenase (SsGDH), which can utilize both NAD and NADP as cofactors. The enzyme operates with glucose and galactose at similar catalytic efficiency, while its substrate profile also includes a range of other five- and six-carbon sugars. Crystals of the 164,kDa SsGDH homotetramer have been grown under a variety of conditions. The best crystals to date diffract to 1.8,Å on a synchrotron source, have orthorhombic symmetry and belong to space group P21212. Attempts are being made to solve the structure by MAD and MR. [source] | |||||||||||||