Pyranose 2-oxidase Research Articles

ITRAQ-based quantitative secretome analysis of Phanerochaete chrysosporium

The basidiomycete fungi such as Phanerochaete chrysosporium secrete large amount of hydrolytic and oxidative enzymes and degrade lignocellulosic biomass. The lignin depolymerizing proteins were extensively studied, but cellulose, hemicellulose and pectin hydrolyzing enzymes were poorly explored. In this study P. chrysosporium was grown in cellulose, lignin and mixture of cellulose and lignin, and secretory proteins were quantified by isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics using liquid chromatography tandem mass spectrometry (LC-MS/MS). An iTRAQ quantified 117 enzymes comprising cellulose hydrolyzing endoglucanases, exoglucanases, beta-glucosidases; hemicelluloses hydrolyzing xylanases, acetylxylan esterases, mannosidases, mannanases; pectin-degrading enzymes polygalacturonase, rhamnogalacturonase, arabinose and lignin degrading protein belonging to oxidoreductase family. Under cellulose and cellulose with lignin culture conditions, enzymes such as endoglucanases, exoglucanases, β-glucosidases and cellobiose dehydrogenase were significantly upregulated and iTRAQ data suggested hydrolytic and oxidative cellulose degradation. When lignin was used as a major carbon source, enzymes such as copper radical oxidase, isoamyl oxidase, glutathione S-transferase, thioredoxin peroxidase, quinone oxidoreductase, aryl alcohol oxidase, pyranose 2-oxidase, aldehyde dehydrogenase, and alcohol dehydrogenase were expressed and significantly regulated. This study explored cellulose, hemicellulose, pectin and lignin degrading enzymes of P. chrysosporium that are valuable for lignocellulosic bioenergy.

A new set up for multi-analyte sensing: At-line bio-process monitoring

A multi-analyte sensing device is described, for simultaneous at-line monitoring of glucose, ethanol, pO 2-value and cell density. It consists of a dual biosensor, a modified microscope and a fiber optical pO 2-sensor that are integrated into a flow analysis (FA) system. The biosensor is based on a conventional thin layer flow-through cell equipped with a gold (Au) dual electrode (serial configuration). The biosensors with no cross-talking were produced by modifying the electrochemical transducers. Each Au surface was initially modified by self-assembled monolayer (SAM) of cysteamine. Alcohol oxidase (AOx) and pyranose oxidase (PyOx) were immobilized each onto a gold surface by means of PAMAM (polyamidoamine) dendrimer via glutaraldehyde cross-linking. The responses for glucose and ethanol were linear up to 0.5 mM. The operational stability of the biosensors was very promising, after 11 h continuous operation, only 6.0% of the initial activity was lost. The potential of the described biosensor was demonstrated by parallel determination of ethanol and glucose in yeast fermentation process. Simultaneously the cell density of the culture was monitored with an in situ microscope (ISM), which was integrated into the FA system. Both the used in situ microscope and the image processing algorithm used for the analysis of the acquired image data are described. Furthermore the pO 2-value was monitored using a fiber optical sensor, which was embedded in a flow cell. The multi-sensor device allows the at-line monitoring of several process values without the need for further sampling or time consuming offline measurements.

Hydrogen Peroxide Elimination from C4a-hydroperoxyflavin in a Flavoprotein Oxidase Occurs through a Single Proton Transfer from Flavin N5 to a Peroxide Leaving Group

C4a-hydroperoxyflavin is found commonly in the reactions of flavin-dependent monooxygenases, in which it plays a key role as an intermediate that incorporates an oxygen atom into substrates. Only recently has evidence for its involvement in the reactions of flavoprotein oxidases been reported. Previous studies of pyranose 2-oxidase (P2O), an enzyme catalyzing the oxidation of pyranoses using oxygen as an electron acceptor to generate oxidized sugars and hydrogen peroxide (H(2)O(2)), have shown that C4a-hydroperoxyflavin forms in P2O reactions before it eliminates H(2)O(2) as a product (Sucharitakul, J., Prongjit, M., Haltrich, D., and Chaiyen, P. (2008) Biochemistry 47, 8485-8490). In this report, the solvent kinetic isotope effects (SKIE) on the reaction of reduced P2O with oxygen were investigated using transient kinetics. Our results showed that D(2)O has a negligible effect on the formation of C4a-hydroperoxyflavin. The ensuing step of H(2)O(2) elimination from C4a-hydroperoxyflavin was shown to be modulated by an SKIE of 2.8 ± 0.2, and a proton inventory analysis of this step indicates a linear plot. These data suggest that a single-proton transfer process causes SKIE at the H(2)O(2) elimination step. Double and single mixing stopped-flow experiments performed in H(2)O buffer revealed that reduced flavin specifically labeled with deuterium at the flavin N5 position generated kinetic isotope effects similar to those found with experiments performed with the enzyme pre-equilibrated in D(2)O buffer. This suggests that the proton at the flavin N5 position is responsible for the SKIE and is the proton-in-flight that is transferred during the transition state. The mechanism of H(2)O(2) elimination from C4a-hydroperoxyflavin is consistent with a single proton transfer from the flavin N5 to the peroxide leaving group, possibly via the formation of an intramolecular hydrogen bridge.

Platinum Islands on SAMs as Template for Enzyme-Catalyzed Glucose Oxidation

Multilayer electrodes for highly enantioselective sugar conversion are obtained by using monoatomically high platinum islands as pattern for further electrode modification with pyranose-2-oxidase (P2Ox). We describe this kind of electrode for the glucose oxidation. The reaction is monitored via the H2O2 production by the enzyme as side product. As first layer, a self-assembled monolayer (SAM) is arranged on the Au(111) surface via dip coating in a solution of 4-mercaptopyridine. On top of the SAM, a controlled distribution of platinum islands is obtained by a combination of electroless and electrochemical deposition using a solution of K2PtCl4. The platinum islands have a diameter of about 6 nm. Thereon, poly(allylamine hydrochloride) (PAA) obtained by dip coating provides a linker for immobilization of the enzyme pyranose-2-oxidase. Our assumption is that only one enzyme molecule is bound to one platinum island. This well ordered surface structure is probably the reason for the very high conversion efficiency.

Regioselective Control of β- d-Glucose Oxidation by Pyranose 2-Oxidase Is Intimately Coupled to Conformational Degeneracy

Trametes multicolor pyranose 2-oxidase (P2O) is a flavoprotein oxidase that oxidizes d-glucose at C2 to 2-keto- d-glucose by a highly regioselective mechanism. In this work, fluorinated sugar substrates were used as mechanistic probes to investigate the basis of regioselectivity in P2O. Although frequently used to study the mechanisms of glycoside hydrolases, our work provides the first example of applying these probes to sugar oxidoreductases. Our previous structure of the P2O mutant H167A in complex with the slow substrate 2-deoxy-2-fluoro- d-glucose showed a substrate-binding mode compatible with oxidation at C3. To accommodate the sugar, a gating segment, 454FSY 456, in the substrate recognition loop partly unfolded to create a spacious and more polar active site that is distinct from the closed state of P2O. The crystal structure presented here shows that the preferred C2 oxidation where an ordered complex of P2O H167A with 3-deoxy-3-fluoro- d-glucose at 1.35 Å resolution was successfully trapped. In this semi-open C2-oxidation complex, the substrate recognition loop tightens to form an optimized substrate complex stabilized by interactions between Asp452 and glucose O4, as well as Tyr456 and the glucose O6 group, interactions that are not possible when glucose is positioned for oxidation at C3. The different conformations of the 454FSY 456 gating segment in the semi-open and closed states induce backbone and side-chain movements of Thr169 and Asp452 that add further differential stabilization to the individual states. We expect the semi-open state (C2-oxidation state) and closed state to be good approximations of the active-site structure during the reductive half-reaction (sugar oxidation) and oxidative half-reaction (O 2 reduction).

Enhanced Expression of Glucose 2-Oxidase in Phlebia tremellosa by Addition of Phthalates

Most fungi possess several hydrogen peroxide-generating enzymes, glucose oxidase and pyranose oxidase. Pyranose oxidase can use glucose as its substrate to generate hydrogen peroxide. White rot fungi, which degrade diverse recalcitrant compounds, contain lignin-degrading enzymes, and lignin peroxidase and manganese peroxidase require hydrogen peroxide for their enzymatic reactions. In this study, we isolated a cDNA fragment of pyranose oxidase from Phlebia tremellosa using PCR and examined its expression under the degradation conditions of diethylphthalate (DEP). Pyranose oxidase expression was enhanced up to 30% by the addition of DEP, and this result supports the possible involvement of pyranose oxidase in the degradation of recalcitrant compounds.

Offline glucose biomonitoring in yeast culture by polyamidoamine/ cysteamine‐modified gold electrodes

This article deals with the use of pyranose oxidase (PyOx) and glucose oxidase (GOx) enzymes in amperometric biosensor design and their application in monitoring fermentation processes with the combination of flow injection analysis (FIA). The amperometric studies were carried out at -0.7 V by following the oxygen consumption due to the enzymatic reactions for both batch and FIA modes. Optimization studies (enzyme amounts and pH) and analytical parameters such as linearity, repeatability, effect of interference, storage, and operational stabilities have been studied. Under optimized conditions, for the PyOx-based biosensor, linear graph was obtained from 0.025 to 0.5 mM glucose in phosphate buffer (50 mM) at pH 7.0 with the equation of y = 3.358x + 0.028 and R(2) = 0.998. Linearity was found to be 0.01-1.0 mM in citrate buffer (50 mM and pH 4.0) with the equation of y = 1.539x + 0.181 and R(2) = 0.992 for the GOx biosensor. Finally, these biosensor configurations were further evaluated in a conventional flow injection system. Results from batch experiments provide a guide to design sensitive, stable, and interference-free biosensors for FIA mode. Biosensor stability, dynamic range, and repeatability were also studied in FIA conditions, and the applicability for the determination of glucose in fermentation medium could be successfully demonstrated. The FIA-combined glucose biosensor was used for the offline monitoring of yeast fermentation. The obtained results correlated well with HPLC measurements.

Bioconversion of d-glucose into d-glucosone by Glucose 2-oxidase from Coriolus versicolor at Moderate Pressures

Glucose 2-oxidase (pyranose oxidase, pyranose:oxygen-2-oxidoreductase, EC 1.1.3.10) from Coriolus versicolor catalyses the oxidation of D-glucose at carbon 2 in the presence of molecular O₂ producing D-glucosone (2-keto-glucose and D-arabino-2-hexosulose) and H₂O₂. It was used to convert D-glucose into D-glucosone at moderate pressures (i.e. up to 150 bar) with compressed air in a modified commercial batch reactor. Several parameters affecting biocatalysis at moderate pressures were investigated as follows: pressure, [enzyme], [glucose], pH, temperature, nature of fluid and the presence of catalase. Glucose 2-oxidase was purified by immobilized metal affinity chromatography on epoxy-activated Sepharose 6B-IDA-Cu(II) column at pH 6.0. The rate of bioconversion of D-glucose increased with the pressure since an increase in the pressure with compressed air resulted in higher rates of conversion. On the other hand, the presence of catalase increased the rate of reaction which strongly suggests that H₂O₂ acted as inhibitor for this reaction. The rate of bioconversion of D-glucose by glucose 2-oxidase in the presence of either nitrogen or supercritical CO₂ at 110 bar was very low compared with the use of compressed air at the same pressure. The optimum temperature (55 °C) and pH (5.0) of D-glucose bioconversion as well as kinetic parameters for this enzyme were determined under moderate pressure. The activation energy (E (a)) was 32.08 kJ mol⁻¹ and kinetic parameters (V(max), K(m), K(cat) and K(cat)/K(m)) for this bioconversion were 8.8 U mg⁻¹ protein, 2.95 mM, 30.81 s⁻¹ and 10,444.06 s⁻¹ M⁻¹, respectively. The biomass of C. versicolor as well as the cell-free extract containing glucose 2-oxidase activity were also useful for bioconversion of D-glucose at moderate pressures. The enzyme was apparently stable at moderate pressures since such pressures did not affect significantly the enzyme activity.

H-Bonding and Positive Charge at the N(5)/O(4) Locus Are Critical for Covalent Flavin Attachment in Trametes Pyranose 2-Oxidase

Flavoenzymes perform a wide range of redox reactions in nature, and a subclass of flavoenzymes carry covalently bound cofactor. The enzyme–flavin bond helps to increase the flavin's redox potential to facilitate substrate oxidation in several oxidases. The formation of the enzyme–flavin covalent bond—the flavinylation reaction—has been studied for the past 40 years. For the most advocated mechanism of autocatalytic flavinylation, the quinone methide mechanism, appropriate stabilization of developing negative charges at the flavin N(1) and N(5) loci is crucial. Whereas the structural basis for stabilization at N(1) is relatively well studied, the structural requisites for charge stabilization at N(5) remain less clear. Here, we show that flavinylation of histidine 167 of pyranose 2-oxidase from Trametes multicolor requires hydrogen bonding at the flavin N(5)/O(4) locus, which is offered by the side chain of Thr169 when the enzyme is in its closed, but not open, state. Moreover, our data show that additional stabilization at N(5) by histidine 548 is required to ensure high occupancy of the histidyl–flavin bond. The combination of structural and spectral data on pyranose 2-oxidase mutants supports the quinone methide mechanism. Our results demonstrate an elaborate structural fine-tuning of the active site to complete its own formation that couples efficient holoenzyme synthesis to conformational substates of the substrate-recognition loop and concerted movements of side chains near the flavinylation ligand.

Bioconversion of d-glucose into d-glucosone by immobilized glucose 2-oxidase from Coriolus versicolor at moderate pressures

The immobilized glucose 2-oxidase (pyranose oxidase, pyranose:oxygen-2-oxidoreductase, EC 1.1.3.10) from Coriolus versicolor was used to convert d-glucose into d-glucosone at moderate pressures, up to 150 bar, with compressed air in a modified commercial batch reactor. Several parameters affecting biocatalysis at moderate pressures were investigated as follows: pressure, different forms of immobilized biocatalysts, glucose concentration, pH, temperature and the presence of catalase. Glucose 2-oxidase (GOX2) was purified by immobilized metal affinity chromatography on epoxy-activated Sepharose 6B-IDA-Cu(II) column at pH 6.0. Purified enzyme and catalase were immobilized into a polyethersulfone (PES) membrane in the presence of glutaraldehyde and gelatin. Enhancement of the bioconversion of d-glucose was done by the pressure since an increase in the pressure with compressed air increases the conversion rates. The optimum temperature and pH for bioconversion of d-glucose were found to be 62 °C and pH 6.0, respectively and the activation energy ( E a) was 28.01 kJ mol −1. The apparent kinetic constants ( V ′ max , K ′ m , K ′ cat and K cat / K ′ m ) for this bioconversion were 2.27 U mg −1 protein, 11.15 mM, 8.33 s −1 and 747.38 s −1 M −1, respectively. The immobilized biomass of C. versicolor as well as crude extract containing GOX2 activity were also useful for bioconversion of d-glucose at 65 bar with a yield of 69.9 ± 3.8% and 91.3 ± 1.2%, respectively. The immobilized enzyme was apparently stable for several months without any significant loss of enzyme activity. On the other hand, this immobilized enzyme was also stable at moderate pressures, since such pressures did not affect significantly the enzyme activity.

Characterisation of recombinant pyranose oxidase from the cultivated mycorrhizal basidiomycete Lyophyllum shimeji (hon-shimeji).

BackgroundThe flavin-dependent enzyme pyranose 2-oxidase (P2Ox) has gained increased attention during the last years because of a number of attractive applications for this enzyme. P2Ox is a unique biocatalyst with high potential for biotransformations of carbohydrates and in synthetic carbohydrate chemistry. Recently, it was shown that P2Ox is useful as bioelement in biofuel cells, replacing glucose oxidase (GOx), which traditionally is used in these applications. P2Ox offers several advantages over GOx for this application, e.g., its much broader substrate specificity. Because of this renewed interest in P2Ox, knowledge on novel pyranose oxidases isolated from organisms other than white-rot fungi, which represent the traditional source of this enzyme, is of importance, as these novel enzymes might differ in their biochemical and physical properties.ResultsWe isolated and over-expressed the p2ox gene encoding P2Ox from the ectomycorrhizal fungus Lyophyllum shimeji. The p2ox cDNA was inserted into the bacterial expression vector pET21a(+) and successfully expressed in E. coli Rosetta 2. We obtained active, flavinylated recombinant P2Ox in yields of approximately 130 mg per L of medium. The enzyme was purified by a two-step procedure based on anion exchange chromatography and preparative native PAGE, yielding an apparently homogenous enzyme preparation with a specific activity of 1.92 U/mg (using glucose and air oxygen as the substrates). Recombinant P2Ox from L. shimeji was characterized in some detail with respect to its physical and catalytic properties, and compared to the well-characterised enzymes from Phanerochaete chrysosporium and Trametes multicolor.ConclusionL. shimeji P2Ox shows properties that are comparable to those of P2Ox from white-rot fungal origin, and is in general characterised by lower Km and kcat values both for electron donor (sugar) as well as electron acceptor (ferrocenium ion, 1,4-benzoquinone, 2,6-dichloroindophenol). While L. shimeji P2Ox is the least thermostable of these three enzymes (melting temperature Tm of 54.9°C; half-life time of activity τ1/2 of 0.12 at 50°C and pH 6.5), P. chrysosporium P2Ox showed remarkable thermostability with Tm of 75.4°C and τ1/2 of 96 h under identical conditions.

Importance of the gating segment in the substrate‐recognition loop of pyranose 2‐oxidase

Pyranose 2-oxidase from Trametes multicolor is a 270 kDa homotetrameric enzyme that participates in lignocellulose degradation by wood-rotting fungi and oxidizes a variety of aldopyranoses present in lignocellulose to 2-ketoaldoses. The active site in pyranose 2-oxidase is gated by a highly conserved, conformationally degenerate loop (residues 450-461), with a conformer ensemble that can accommodate efficient binding of both electron-donor substrate (sugar) and electron-acceptor substrate (oxygen or quinone compounds) relevant to the sequential reductive and oxidative half-reactions, respectively. To investigate the importance of individual residues in this loop, a systematic mutagenesis approach was used, including alanine-scanning, site-saturation and deletion mutagenesis, and selected variants were characterized by biochemical and crystal-structure analyses. We show that the gating segment ((454)FSY(456)) of this loop is particularly important for substrate specificity, discrimination of sugar substrates, turnover half-life and resistance to thermal unfolding, and that three conserved residues (Asp(452), Phe(454) and Tyr(456)) are essentially intolerant to substitution. We furthermore propose that the gating segment is of specific importance for the oxidative half-reaction of pyranose 2-oxidase when oxygen is the electron acceptor. Although the position and orientation of the slow substrate 2-deoxy-2-fluoro-glucose when bound in the active site of pyranose 2-oxidase variants is identical to that observed earlier, the substrate-recognition loop in F454N and Y456W displays a high degree of conformational disorder. The present study also lends support to the hypothesis that 1,4-benzoquinone is a physiologically relevant alternative electron acceptor in the oxidative half-reaction.

ChemInform Abstract: Enzymatic Formation of Dicarbonyl Sugars: C-2 Oxidation of 1→6 Disaccharides Gentiobiose, Isomaltose and Melibiose by Pyranose 2-Oxidase from Trametes multicolor.

ABSTRACT Pyranose 2-oxidase, a homotetrameric FAD-flavoprotein from the fungus Trametes multicolor, catalyzes the oxidation of 1→6 disaccharides gentiobiose (β-D-Glcp-(1→6)-D-Glc), melibiose (α-D-Galp-(1→6)-D-Glc), and isomaltose (α-D-Glcp-(1→6)-D-Glc) at C-2 of their reducing moiety. The products were identified as their hydrazone derivatives.

Kinetic Isotope Effects on the Noncovalent Flavin Mutant Protein of Pyranose 2-Oxidase Reveal Insights into the Flavin Reduction Mechanism

Pyranose 2-oxidase (P2O) from Trametes multicolor contains a flavin adenine dinucleotide (FAD) cofactor covalently linked to the N(3) atom of His167. The enzyme catalyzes the oxidation of aldopyranoses by molecular oxygen to generate 2-keto-aldoses and H(2)O(2) as products. In this study, the transient kinetics and primary and solvent kinetic isotope effects of the mutant in which His167 has been replaced with Ala (H167A) were investigated, to elucidate the functional role of the 8a-N(3)-histidyl FAD linkage and to gain insights into the reaction mechanism of P2O. The results indicate that the covalent linkage is mainly important for a reductive half-reaction in which the FAD cofactor is reduced by d-glucose, while it is not important for an oxidative half-reaction in which oxygen reacts with the reduced FAD to generate H(2)O(2). d-Glucose binds to H167A via multiple binding modes before the formation of the active Michaelis complex, and the rate constant of flavin reduction decreases approximately 22-fold compared to that of the wild-type enzyme. The reduction of H167A using d-glucose isotopes (2-d-d-glucose, 3-d-d-glucose, and 1,2,3,4,5,6,6-d(7)-d-glucose) as substrates indicates that the primary isotope effect results only from substitution at the C2 position, implying that H167A catalyzes the oxidation of d-glucose regiospecifically at this position. No solvent kinetic isotope effect was detected during the reductive half-reaction of the wild-type or H167A enzyme, implying that the deprotonation of the d-glucose C2-OH group may occur readily upon the binding to P2O and is not synchronized with the cleavage of the d-glucose C2-H bond. The mutation has no drastic effect on the oxidative half-reaction of P2O, as H167A is very similar to the wild-type enzyme with respect to the kinetic constants and the formation of the C4a-hydroperoxyflavin intermediate. Kinetic mechanisms for both half-reactions of H167A were proposed on the basis of transient kinetic data and were verified by kinetic simulations and steady-state kinetic parameters.

Engineered Pyranose 2‐Oxidase: Efficiently Turning Sugars into Electrical Energy

AbstractDue to the recent interest in enzymatic biofuel cells (BFCs), sugar oxidizing enzymes other than the commonly used glucose oxidase are gaining more importance as possible bioelements of implantable microscale‐devices, which can, for example, be used in biosensors and pacemakers. In this study we used rational and semi‐rational protein design to improve the catalytic activity of the enzyme pyranose 2‐oxidase (P2Ox) with its alternative soluble electron acceptors 1,4‐benzoquinone and ferricenium ion, which can serve as electron mediators, to possibly boost the power output of enzymatic BFCs. Using a screening assay based on 96‐well plates, we identified the variant H450G, which showed lower KM and higher kcat values for both 1,4‐benzoquinone and ferricenium ion compared to the wild‐type enzyme, when either D‐glucose or D‐galactose were used as saturating electron donors. Besides this variant, we analyzed the variants V546C and T169G/V546C for their possible application in enzymatic BFCs. The results obtained in homogeneous solution were compared with those obtained when P2Ox was immobilized on the surface of graphite electrodes and either “wired” to an osmium redox polymer or using soluble 1,4‐benzoquinone as mediator. According to the spectrophotometrically determined kinetic constants, the possible energy output, measured in flow injection analysis experiments with these variants, increased up to 4‐fold compared to systems employing the wild‐type enzyme.

Evaluation of different expression systems for the heterologous expression of pyranose 2-oxidase from Trametes multicolor in E. coli

The heterologous production of the industrially relevant fungal enzyme pyranose 2-oxidase in the prokaryotic host E. coli was investigated using 3 different expression systems, i.e. the well-studied T7 RNA polymerase based pET21d+, the L-arabinose inducible pBAD and the pCOLD system. Preliminary experiments were done in shaking flasks at 25°C and optimized induction conditions to compare the productivity levels of the different expression systems. The pET21d+ and the pCOLD system gave 29 U/L·h and 14 U/L·h of active pyranose 2-oxidase, respectively, whereas the pBAD system only produced 6 U/L·h. Process conditions for batch fermentations were optimized for the pET21d+ and the pCOLD systems in order to reduce the formation of inactive inclusion bodies. The highest productivity rate with the pET21d+ expression system in batch fermentations was determined at 25°C with 32 U/L·h. The pCOLD system showed the highest productivity rate (19 U/L·h) at 25°C and induction from the start of the cultivation. Using the pCOLD system in a fed batch fermentation at 25°C with a specific growth rate of μ = 0.15 h-1resulted in the highest productivity rate of active pyranose oxidase with 206 U/L·h.

A Conserved Active-site Threonine Is Important for Both Sugar and Flavin Oxidations of Pyranose 2-Oxidase

Pyranose 2-oxidase (P2O) catalyzes the oxidation by O(2) of d-glucose and several aldopyranoses to yield the 2-ketoaldoses and H(2)O(2). Based on crystal structures, in one rotamer conformation, the threonine hydroxyl of Thr(169) forms H-bonds to the flavin-N5/O4 locus, whereas, in a different rotamer, it may interact with either sugar or other parts of the P2O.sugar complex. Transient kinetics of wild-type (WT) and Thr(169) --> S/N/G/A replacement variants show that D-Glc binds to T169S, T169N, and WT with the same K(d) (45-47 mm), and the hydride transfer rate constants (k(red)) are similar (15.3-9.7 s(-1) at 4 degrees C). k(red) of T169G with D-glucose (0.7 s(-1), 4 degrees C) is significantly less than that of WT but not as severely affected as in T169A (k(red) of 0.03 s(-1) at 25 degrees C). Transient kinetics of WT and mutants using d-galactose show that P2O binds d-galactose with a one-step binding process, different from binding of d-glucose. In T169S, T169N, and T169G, the overall turnover with d-Gal is faster than that of WT due to an increase of k(red). In the crystal structure of T169S, Ser(169) O gamma assumes a position identical to that of O gamma 1 in Thr(169); in T169G, solvent molecules may be able to rescue H-bonding. Our data suggest that a competent reductive half-reaction requires a side chain at position 169 that is able to form an H-bond within the ES complex. During the oxidative half-reaction, all mutants failed to stabilize a C4a-hydroperoxyflavin intermediate, thus suggesting that the precise position and geometry of the Thr(169) side chain are required for intermediate stabilization.

Thermostable Variants of Pyranose 2-Oxidase Showing Altered Substrate Selectivity for Glucose and Galactose

The homotetrameric flavoprotein pyranose 2-oxidase (P2Ox) has several proposed biotechnological applications, among others as a biocatalyst for carbohydrate transformations toward higher-value products. To improve some of the catalytic properties of P2Ox from Trametes multicolor, we selected a semirational enzyme engineering approach, namely, saturation mutagenesis of the amino acid His450 located at a pivotal point of the active site loop and subsequent screening of the libraries thus obtained for improved activity with the sugar substrate d-galactose. A variant with improved catalytic characteristics identified was H450G, which showed a significant, 3.6-fold decrease in K(M) together with a 1.4-fold increase in k(cat) for its substrate D-galactose and an overall improvement in the catalytic efficiency by a factor of 5. By combining H450G with other amino acid replacements, we obtained the P2Ox variants H450G/V546C and H450G/E542K/V546C, which can be of interest for applications in food industry due to their increased activity with D-galactose, high activity with D-glucose, and considerably increased stability for the latter variant. While the His-tagged recombinant wild-type enzyme strongly prefers D-glucose to D-galactose as its substrate, H450G/E542K/V546C converts both sugars, which are found in lactose hydrolysates, concomitantly, as was shown by laboratory-scale biotransformation experiments. The 2-keto sugars thus obtained can conveniently be reduced to the corresponding ketoses D-fructose and D-tagatose.

Rheological properties of keto-sugars with high-density carbonyl groups

Keto-dextran with high density of carbonyl groups was synthesized by the reactions of three enzymes (pyranose 2-oxidase, catalase, and dextransucrase). To evaluate the structure and interaction of keto-dextran with carbonyl groups in water, the viscosity of keto-dextran was compared with that of dextran. At a dilute concentration, the viscosity of keto-dextran is lower than that of dextran, because keto-dextran contracted due to intra-molecular interactions via hydrogen bonding with the carbonyl groups. Dynamic light scattering results showed that the hydrodynamic radii of dextran and keto-dextran were 24.1 and 16.7 nm, respectively. To evaluate only the inter-molecular interactions of the carbonyl groups, viscosities of glucose and keto-glucose were determined. The difference in the viscosity between keto-glucose and glucose was apparent within the concentrated region due to the inter-molecular interactions of the carbonyl groups in keto-glucose. The coefficients estimated by the extended Jones–Dole equation confirmed that keto-sugars have stronger hydration and association characteristics.

Maltose biosensing based on co-immobilization of α-glucosidase and pyranose oxidase

A new bi-enzymatic system was designed by co-immobilization of alpha-glucosidase (AG) and pyranose oxidase (PyOx) for maltose analysis. The immobilization was carried out by cross-linking enzyme mixture, chitosan (CHIT) and carbon nanotube (CNT) via glutaraldehyde. The structure of biosensor including enzyme, CHIT, glutaraldehyde and CNT amount together with operational conditions like pH, temperature and applied potential were optimized. Then analytical characterization was performed. A fast linear response of the biosensor was observed for maltose in the concentration range from 0.25 to 2.0 mM at 35 degrees C and pH 6.0. The effect of CNT addition into the immobilization matrix was also investigated. The linear relationships between sensor response (y; microA/cm(2)) and substrate concentration (x; mM) were defined by the equations of y=0.844x+0.029 (R(2)=0.999) and y=0.882x+0.0625 (R(2)=0.996) for AG/PyOx/CHIT and AG/PyOx/CHIT-CNT biosensors, respectively. All other data were also given as comparison of two systems one with CNT-modified and CNT-free. Finally, for the sample application, maltose was analyzed in beer samples. As a result, it has been found that; complex matrix of natural beer samples had no influence on the biosensing response. Also the results were in good agreement with those obtained by spectrophotometric measurements.