Gluconobacter Suboxydans IFO Research Articles

Enzymatic Synthesis of 4-Pentulosonate (4-Keto-D-pentonate) fromD-Aldopentose andD-Pentonate by Two Different Pathways Using Membrane Enzymes of Acetic Acid Bacteria

4-Keto-D-arabonate (D-threo-pent-4-ulosonate) and 4-keto-D-ribonate (D-erythro-pent-4-ulosonate) were prepared from D-arabinose and D-ribose by two successive reactions of membrane-bound enzymes, D-aldopentose 4-dehydrogenase and 4-keto-D-aldopentose 1-dehydrogenase of Gluconobacter suboxydans IFO 12528. Alternatively, they were prepared from D-arabonate and D-ribonate with another membrane-bound enzyme, D-pentonate 4-dehydrogenase. Analytical data confirmed the chemical structures of the 4-pentulosonates prepared. This is the first report of successful enzymatic synthesis of 4-pentulosonates.

Purification and Properties of Two Different Dihydroxyacetone Reductases inGluconobacter suboxydansGrown on Glycerol

It is well known that in oxidative fermentation microbial growth is improved by the addition of glycerol. In a wild strain, glycerol was converted rapidly to dihydroxyacetone (DHA) quantitatively in the early growth phase by the action of quinoprotein glycerol dehydrogenase (GLDH), and then DHA was incorporated into the cells by the early stationary phase. Two DHA reductases (DHARs), NADH-dependent (NADH-DHAR) (EC 1.1.1.6) and NADPH-dependent (NADPH-DHAR) (EC 1.1.1.156), were detected in the same cytoplasm of Gluconobacter suboxydans IFO 3255. The former appeared to be inducible and labile in nature while the latter was constitutive and stable. The two DHARs were separated each other and were finally purified to crystalline enzymes. This report might be the first one dealing with NADPH-DHAR that has been crystallized. The two DHARs were specific only to DHA reduction to glycerol and thus contributed to cytoplasmic DHA metabolism, resulting in an improved biomass yield with the addition of glycerol.

Formaldehyde elimination with formaldehyde and formate oxidase in membrane of acetic acid bacteria

Formaldehyde elimination was successfully carried out with Acetobacter sp. SKU 14, having strong formaldehyde-oxidizing activity in the cytoplasmic membrane. Formaldehyde was decomposed via formate to carbon dioxide by formaldehyde- and formate-oxidizing activities. A resting-cell suspension of the organism was more convenient for practical purposes than the isolated membrane fraction. In Gluconobacter suboxydans IFO 12528, formaldehyde elimination was not so prominent when compared with that in Acetobacter sp. SKU 14.

Cloning of a gluconate/polyol dehydrogenase gene from Gluconobacter suboxydans IFO 12528, characterisation of the enzyme and its use for the production of 5-ketogluconate in a recombinant Escherichia coli strain.

A 5-ketogluconate (5-KGA)-forming membrane quinoprotein, gluconate dehydrogenase, was isolated from Gluconobacter suboxydans strain IFO 12528 and partially sequenced. Partial sequences of five internal tryptic peptides were elucidated by mass spectrometry and used to isolate the two adjacent genes encoding the enzyme (EBI accession no. AJ577472). These genes share close homology with sorbitol dehydrogenase from another strain of G. suboxydans (IFO 3255). Substrate specificity of gluconate 5-dehydrogenase (GA 5-DH) turned out to be quite broad, covering many polyols, amino derivatives of carbohydrates, and simple secondary alcohols. There is a broad correlation between the substrate specificity of GA 5-DH and the empirical Bertrand-Hudson rule that predicts the specificity of oxidation of polyols by acetic acid bacteria. Escherichia coli transformed with the genes encoding gluconate dehydrogenase were able to convert gluconic acid into 5-KGA at 75% yield. Furthermore, it was found that 5-KGA can be converted into tartaric acid semialdehyde by a transketolase. These results provide a basis for designing a direct fermentation-based process for conversion of glucose into tartaric acid.

Membrane-bound d-sorbitol dehydrogenase of Gluconobacter suboxydans IFO 3255—enzymatic and genetic characterization

Gluconobacter strains effectively produce l-sorbose from d-sorbitol because of strong activity of the d-sorbitol dehydrogenase (SLDH). l-sorbose is one of the important intermediates in the industrial vitamin C production process. Two kinds of membrane-bound SLDHs, which consist of three subunits, were reportedly found in Gluconobacter strains [Agric. Biol. Chem. 46 (1982) 135,FEMS Microbiol. Lett. 125 (1995) 45]. We purified a one-subunit-type SLDH (80 kDa) from the membrane fraction of Gluconobacter suboxydans IFO 3255 solubilized with Triton X-100 in the presence of d-sorbitol, but the cofactor could not be identified from the purified enzyme. The SLDH was active on mannitol, glycerol and other sugar alcohols as well as on d-sorbitol to produce respective keto-aldoses. Then, the SLDH gene ( sldA) was cloned and sequenced. It encodes the polypeptide of 740 residues, which contains a signal sequence of 24 residues. SLDH had 35–37% identity to those of membrane-bound quinoprotein glucose dehydrogenases (GDHs) from Escherichia coli, Gluconobacter oxydans and Acinetobacter calcoaceticus except the N-terminal hydrophobic region of GDH. Additionally, the sldB gene located just upstream of sldA was found to encode the polypeptide consisting of 126 very hydrophobic residues that is similar to the one-sixth N-terminal region of the GDH. Development of the SLDH activity in E. coli required co-expression of the sldA and sldB genes and the presence of PQQ. The sldA gene disruptant showed undetectable oxidation activities on d-sorbitol in growing culture, and resting-cell reaction (pH 4.5 and 7); in addition, they showed undetectable activities on d-mannitol and glycerol. The disruption of the sldB gene by a gene cassette with a downward promoter to express the sldA gene resulted in formation of a larger size of the SLDH protein and in undetectable oxidation of the polyols. In conclusion, the SLDH of the strain 3255 functions as the main polyol dehydrogenase in vivo. The sldB polypeptide possibly has a chaperone-like function to process the SLDH polypeptide into a mature and active form.

Molecular cloning and functional expression of D-sorbitol dehydrogenase from Gluconobacter suboxydans IF03255, which requires pyrroloquinoline quinone and hydrophobic protein SldB for activity development in E. coli.

The sldA gene that encodes the D-sorbitol dehydrogenase (SLDH) from Gluconobacter suboxydans IFO 3255 was cloned and sequenced. It encodes a polypeptide of 740 residues, which contains a signal sequence of 24 residues. SLDH had 35-37% identity to the membrane-bound quinoprotein glucose dehydrogenases (GDHs) from E. coli, Gluconobacter oxydans, and Acinetobacter calcoaceticus except the N-terminal hydrophobic region of GDH. Additionally, the sldB gene located just upstream of sldA was found to encode a polypeptide consisting of 126 very hydrophobic residues that is similar in sequence to the one-sixth N-terminal region of the GDH. For the development of the SLDH activity in E. coli, co-expression of the sldA and sldB genes and the presence of pyrrloquinolone quinone as a co-factor were required.

Main Polyol Dehydrogenase ofGluconobacter suboxydansIFO 3255, Membrane-boundD-Sorbitol Dehydrogenase, That Needs Product of Upstream Gene,sldB, for Activity

The D-sorbitol dehydrogenase gene, sldA, and an upstream gene, sldB, encoding a hydrophobic polypeptide, SldB, of Gluconobacter suboxydans IFO 3255 were disrupted in a check of their biological functions. The bacterial cells with the sldA gene disrupted did not produce L-sorbose by oxidation of D-sorbitol in resting-cell reactions at pHs 4.5 and 7.0, indicating that the dehydrogenase was the main D-sorbitol-oxidizing enzyme in this bacterium. The cells did not produce D-fructose from D-mannitol or dihydroxyacetone from glycerol. The disruption of the sldB gene resulted in undetectable oxidation of D-sorbitol, D-mannitol, or glycerol, although the cells produced the dehydrogenase. The cells with the sldB gene disrupted produced more of what might be signal-unprocessed SldA than the wild-type cells did. SldB may be a chaperone-like component that assists signal processing and folding of the SldA polypeptide to form active D-sorbitol dehydrogenase.

Purification and properties of membrane-bound D-sorbitol dehydrogenase from Gluconobacter suboxydans IFO 3255.

D-Sorbitol dehydrogenase was solubilized from the membrane fraction of Gluconobacter suboxydans IFO 3255 with Triton X-100 in the presence of D-sorbitol. Purification of the enzyme was done by fractionation with column chromatographies of DEAE-Cellulose, DEAE-Sepharose, hydroxylapatite, and Sephacryl HR300 in the presence of Triton X-100. The molecular mass of the enzyme was 800 kDa, consisting of homologous subunits of 80 kDa. The optimum pH of the enzyme activity was 6.0, and the optimum temperature was 30 degrees C. Western blot analysis suggested the occurrence of the enzyme in all the Gluconobacter strains tested.

Membrane-bound quinoprotein D-arabitol dehydrogenase of Gluconobacter suboxydans IFO 3257: a versatile enzyme for the oxidative fermentation of various ketoses.

Solubilization of membrane-bound quinoprotein D-arabitol dehydrogenase (ARDH) was done successfully with the membrane fraction of Gluconobacter suboxydans IFO 3257. In enzyme solubilization and subsequent enzyme purification steps, special care was taken to purify ARDH as active as it was in the native membrane, after many disappointing trials. Selection of the best detergent, keeping ARDH as the holoenzyme by the addition of PQQ and Ca2+, and of a buffer system involving acetate buffer supplemented with Ca2+, were essential to treat the highly hydrophobic and thus labile enzyme. Purification of the enzyme was done by two steps of column chromatography on DEAE-Toyopearl and CM-Toyopearl in the presence of detergent and Ca2+. ARDH was homogenous and showed a single sedimentation peak in analytical ultracentrifugation. ARDH was dissociated into two different subunits upon SDS-PAGE with molecular masses of 82 kDa (subunit I) and 14 kDa (subunit II), forming a heterodimeric structure. ARDH was proven to be a quinoprotein by detecting a liberated PQQ from SDS-treated ARDH in HPLC chromatography. More preliminarily, an EDTA-treated membrane fraction lost the enzyme activity and ARDH activity was restored to the original level by the addition of PQQ and Ca2+. The most predominant unique character of ARDH, the substrate specificity, was highly versatile and many kinds of substrates were oxidized irreversibly by ARDH, not only pentitols but also other polyhydroxy alcohols including D-sorbitol, D-mannitol, glycerol, meso-erythritol, and 2,3-butanediol. ARDH may have its primary function in the oxidative fermentation of ketose production by acetic acid bacteria. ARDH contained no heme component, unlike the type II or type III quinoprotein alcohol dehydrogenase (ADH) and did not react with primary alcohols.

Membrane-bound sugar alcohol dehydrogenase in acetic acid bacteria catalyzes L-ribulose formation and NAD-dependent ribitol dehydrogenase is independent of the oxidative fermentation.

To identify the enzyme responsible for pentitol oxidation by acetic acid bacteria, two different ribitol oxidizing enzymes, one in the cytosolic fraction of NAD(P)-dependent and the other in the membrane fraction of NAD(P)-independent enzymes, were examined with respect to oxidative fermentation. The cytoplasmic NAD-dependent ribitol dehydrogenase (EC 1.1.1.56) was crystallized from Gluconobacter suboxydans IFO 12528 and found to be an enzyme having 100 kDa of molecular mass and 5 s as the sedimentation constant, composed of four identical subunits of 25 kDa. The enzyme catalyzed a shuttle reversible oxidoreduction between ribitol and D-ribulose in the presence of NAD and NADH, respectively. Xylitol and L-arabitol were well oxidized by the enzyme with reaction rates comparable to ribitol oxidation. D-Ribulose, L-ribulose, and L-xylulose were well reduced by the enzyme in the presence of NADH as cosubstrates. The optimum pH of pentitol oxidation was found at alkaline pH such as 9.5-10.5 and ketopentose reduction was found at pH 6.0. NAD-Dependent ribitol dehydrogenase seemed to be specific to oxidoreduction between pentitols and ketopentoses and D-sorbitol and D-mannitol were not oxidized by this enzyme. However, no D-ribulose accumulation was observed outside the cells during the growth of the organism on ribitol. L-Ribulose was accumulated in the culture medium instead, as the direct oxidation product catalyzed by a membrane-bound NAD(P)-independent ribitol dehydrogenase. Thus, the physiological role of NAD-dependent ribitol dehydrogenase was accounted to catalyze ribitol oxidation to D-ribulose in cytoplasm, taking D-ribulose to the pentose phosphate pathway after being phosphorylated. L-Ribulose outside the cells would be incorporated into the cytoplasm in several ways when need for carbon and energy sources made it necessary to use L-ribulose for their survival. From a series of simple experiments, membrane-bound sugar alcohol dehydrogenase was concluded to be the enzyme responsible for L-ribulose production in oxidative fermentation by acetic acid bacteria.

Production of 5-keto- d-gluconate by acetic acid bacteria is catalyzed by pyrroloquinoline quinone (PQQ)-dependent membrane-bound d-gluconate dehydrogenase

Gluconobacter suboxydans IFO 12528 was selected as the best strain for 5-keto- d-gluconate (5KGA) production by oxidative fermentation. 5KGA was markedly accumulated by the strain during cultivation in a medium containing d-glucose and/or d-gluconate. The resting cells and the membrane fraction also catalyzed 5KGA formation with a minimal formation of 2-keto- d-gluconate (2KGA), an alternative keto- d-gluconate from d-gluconate. The membrane fraction of the organism was confirmed to contain a membrane-bound d-gluconate dehydrogenase (GADH) catalyzing d-gluconate oxidation to 5KGA of which optimum pH and temperature were found at pH 4 and 15°C, respectively. After treating the membrane fraction with EDTA allowing conversion from holo-GADH to the apoenzyme, 5KGA-forming GADH was confirmed to be a pyrroloquinoline quinone (PQQ)-dependent enzyme by the fact that the enzyme activity was restored by the addition of CaCl 2 and PQQ. The 5KGA-forming GADH was totally distinct from 2KGA-forming GADH in which a covalently bound FAD functions as coenzyme. 5KGA-forming GADH was well solubilized from the membrane fraction with n-octyl- β- d-thioglucoside and 5KGA formation was favourably catalyzed at relatively lower temperature, while 2KGA-forming enzyme was solubilized with Triton X-100 and relatively higher temperatures was optimum for 2KGA formation. These results are completely discrepant from the conclusion proposed by Klasen et al. [R. Klasen, S. Bringer-Mayer, H. Sahm, J. Bacteriol., 177, 1995, 2637] claiming that 5KGA was produced by d-gluconate oxidation catalyzed by NADP-dependent cytoplasmic 5KGA reductase from Gluconobacter species at fairly alkaline pH such as 10.

Crystalline NADP-Dependent D-Mannitol Dehydrogenase from Gluconobacter suboxydans.

D-Mannitol dehydrogenase (EC 1.1.1.138) was purified and crystallized for the first time from the cell-free extract of Gluconobacter suboxydans IFO 12528. The enzyme was purified about 100-fold by a procedure involving ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography, and gel filtration by a Sephadex G-75 column. The enzyme was completely separated from a similar enzyme, NAD-dependent D-mannitol dehydrogenase (EC 1.1.1.67), during enzyme purification. There being sufficient purity of the enzyme at this stage, the enzyme was crystallized, by the addition of ammonium sulfate, to fine needles. The crystalline enzyme showed a single sedimentation peak in analytical ultracentrifugation, giving an apparent sedimentation constant of 3.6 s. The molecular mass of the enzyme was estimated to be 50 kDa by SDS-PAGE and gel filtration chromatography. Oxidation of D-mannitol to D-fructose and reduction of D-fructose to D-mannitol were specifically catalyzed with NADP and NADPH, respectively. NAD and NADH were inert for the enzyme. Since the reaction equilibrium declined to D-fructose reduction over a wide pH range, the enzyme showed several advantages for direct enzymatic measurement of D-fructose. Even in the presence of a large excess of D-glucose and other substances, oxidation of NADPH to NADP was highly specific and stoichiometric to the D-fructose reduced.

Crystallization and Properties of NAD-Dependent D-Sorbitol Dehydrogenase from Gluconobacter suboxydans IFO 3257.

NAD-dependent D-sorbitol dehydrogenase (EC 1.1.1.14) was crystallized from the cytosolic fraction of Gluconobacter suboxydans IFO 3257. This is the first example of the enzyme crystallized from acetic acid bacteria. The enzyme catalyzed oxidoreduction between D-sorbitol and D-fructose in the presence of NAD or NADH. The crystalline enzyme showed a single sedimentation peak in analytical ultracentrifugation, giving an apparent sedimentation constant of 5.1s. Gel filtration on a Sephadex G-200 column gave the molecular mass of 98 kDa for the enzyme, which dissociated into 26-kDa subunits on SDS-PAGE, indicating that the enzyme is composed of four identical subunits. Oxidation of D-sorbitol to D-fructose and xylitol to D-xylulose predominated in the presence of NAD at the optimum pH of 9.5-10.0. Reductions of D-fructose to D-sorbitol and D-xylulose to xylitol were also observed in the presence of NADH with the optimum pH around 6.0. The relative rate of D-fructose reduction was about one-fourth of that of D-sorbitol oxidation. NADP and NADPH were inert for the both reactions. Since the reation rate in D-sorbitol oxidation predominated over D-fructose reduction at some alkaline pH, the enzyme could be available for direct enzymatic measurement of D-sorbitol. Even in the presence of a large excess of D-glucose and other substances, reduction of NAD to NADH was highly specific and stoichiometric to the D-sorbitol oxidized.

Continous production of free gluconic acid by Gluconobacter suboxydans IFO 3290 immobilized by adsorption on ceramic honeycomb monolith: effect of reactor configuration on further oxidation of gluconic acid to keto-gluconic acid

Gluconobacter suboxydans IFO 3290 was immobilized by adsorption on ceramic honeycomb monolith, and continuous production of free gluconic acid from 100 g/l glucose was carried out in one- and three-stage monolith reactors. Further oxidation of gluconic acid to keto-gluconic acid by the immobilized cells has been found to be more suppressed in the three-stage monolith reactor. This finding can be explained by the fact that, with the three-stage reactor, the opportunity to oxidize gluconic acid further was decreased because the residence time of the reaction mixture at glucose conversion above the threshold value was shorter.

Characterization of production of free gluconic acid by Gluconobacter suboxydans adsorbed on ceramic honeycomb monolith

Gluconobacter suboxydans IFO 3290 was immobilized by adsorption on ceramic honeycomb monolith and continuous production of free gluconic acid from glucose was performed in an aerated reactor. The effects of reactor residence time, aeration rate, and glucose concentration were investigated on the gluconic acid yield. Observation of SEM photographs revealed that the cells were adsorbed with a high density not only on the outer surface of the support but also on the inner surface of large pores. From measurement of the number of the adsorbed cells, it was elucidated that the biofilm comprised a monolayer or bilayer of the cells. Maximum specific rate of growth was estimated for the free and adsorbed cells, and the adsorbed cells were found to grow at a fast rate compared with the free cells. In the continuous fermentation performed for one month at the glucose concentration of 100 kg/m(3), reactor residence time of 3.5 h and aeration rate of 900 cm(3)/min, the activity of the adsorbed cells was appreciably stable. The high productivity of 26.3 kg/(m(3)-reactor . h) was attained with the gluconic acid yield of 84.6% and glucose conversion of 94%.

NADPH production from NADP+ with a glucose dehydrogenase system involving whole cells and immobilized cells of Gluconobacter suboxydans

A convenient and efficient method of NADPH production from NADP+ has been established using a glucose dehydrogenase system involving whole cells and immobilized cells of Gluconobacter suboxydans IFO 3172. Using airdried cells of the bacterium, the optimum conditions for NADPH production were examined, including the cell and glucose concentrations, NADP+ concentration, pH, buffer and reaction temperature. Under suitable conditions, the conversion ratio and the amount of NADPH accumulated reached about 100% and 73 mg/ml of the reaction mixture, respectively, after 1-h reaction. Intact cells of the bacterium also showed high NADPH production even in the reaction mixture without a surfactant. The addition of Triton X-100 to the reaction mixture and freeze-thawing treatment of intact cells enhanced the production. The NADPH production method was further improved by using cells of the bacterium immobilized by entrapment in a κ-carrageenan gel lattice. The immobilized cells had almost the same enzymatic properties as the air-dried cells. The conditions for the continuous production of NADPH with an immobilized cell column were also investigated. NADPH was produced in a good yield (about 95%) with this continuous process.

Alcohol-FET sensor based on a complex cell membrane enzyme system

An alcohol -FET sensor was developed by use of a complex enzyme system in a cell membrane and an ion-sensitive field effect transistor (ISFET). The cell membrane of Gluconobacter suboxydans IFO 12528, which converts ethanol to acetic acid, was immobilized on the gate of an ISFET with calcium alginate gel coated with nitrocellulose. This ISFET (1), a reference ISFET without the cell membrane (ISFET 2) and an Ag/AgCl reference electrode were placed in 5 mM Trismalate buffer (pH 5.5, 25°C), and the differential output between ISFETS 1 and 2 was measured. The output of the sensor was stabilized by adding pyrroloquinoline quinone. The response time was ca. 10 min., and there was a linear relationship between the differential output voltage and the ethanol concentration up to 20 mg l −1. The output of the sensor was stable for 40 h below 30°C. The sensor responded to ethanol, propan- 1-ol and butan- 1-ol, but not to methanol, propan-2-ol and butan-2-ol. The sensor was used to determine blood ethanol.

Amperometric alcohol sensor based on an immobilised bacteria cell membrane.

A simple amperometric alcohol sensor was developed using a cell membrane of acetic acid bacteria, a gas-permeable membrane and an oxygen electrode. The cell membrane of Gluconobacter suboxydans IFO 12528 was adsorbed on a nitrocellulose filter and attached to the Teflon membrane of the oxygen electrode and these membranes were then covered with the gas-permeable membrane (porous Teflon). When a sample solution containing ethanol was injected into the alcohol sensor system, the sensor current decreased markedly with time and reached a steady state after approximately 3 min. A linear relationship was observed between the decrease in current and the ethanol concentration up to 25 mg l–1. The sensor was found to have a good selectivity, which was better than that of an enzyme electrode with alcohol oxidase. The response of the sensor decreased at temperatures greater than 45 °C, and good responses were obtained over a period of 15 d. The sensor was applied to the determination of serum ethanol. There was a good correlation between the results obtained by the sensor and those obtained by gas chromatography (correlation coefficient r= 0.99). The current was reproducible with a relative error of ±6.2% when the ethanol concentration of the serum was 2.3 mg ml–1. The standard deviation was 0.07 mg ml–1 in nine experiments.

Purification, crystallization and properties of 6-phospho-D-gluconate dehydrogenase from Gluconobacter suboxydans.

6-Phospho-D-gluconate dehydrogenase (EC 1.1.1.44) was purified and crystallized from a cell free extract of Gluconobacter suboxydans IFO 12528. Affinity chromatography on a blue-dextran Sepharose 4B column was effective through the purification steps, and a 280-fold purification was achieved with a 56% yield. The crystalline enzyme was homogeneous, as judged by polyacrylamide gel electrophoresis and analytical ultracentrifugation. Although the enzyme from G. suboxydans catalyzed the oxidative decarboxylation of 6-phospho-D-gluconate to D-ribulose-5-phosphate, it differed from enzymes derived from other sources in respect to substrate specificity and molecular properties. Unlike the others, this Gluconobacter enzyme has a requirement for similar amounts of NAD and NADP as coenzymes and the pH optimum of this protein with two coenzymes was samely 6.5, while other enzyme preparations have the optimum pH at 8.0. The molecular weight of this enzyme was 175, 000, and the enzyme was composed of four identical subunits of molecular weight 43, 000. The enzyme activity was inhibited competitively by ATP, D-fructose-1, 6-diphosphate and NAD(P)H. It was shown that the NADPH formed by the action of the enzyme was reoxidized by the old yellow enzyme of the organism.

D-glucose dehydrogenase of Gluconobacter suboxydans: Solubilization,purification and characterization.

D-Glucose dehydrogenase was purified to homogeneity from the membrane of Gluconobacter suboxydans IFO 12528. The enzyme was solubilized from the membrane with Triton X-100 and fractionated with SE-cellulose and hydroxyapatite columns. Other species of membrane-bound dehydrogenases which were cosolubilized with D-glucose dehydrogenase were eliminated by treating the enzyme at fairly acidic pH in the initial step of enzyme purification. The purified enzyme was homogeneous in analytical ultracentrifugation and sucrose density gradient centrifugation. The enzyme had an apparent sedimentation constant of 4.2 S and showed a molecular weight of 87,000 by urea-sodium dodecylsulfate gel electrophoresis. The spectral studies with the purified enzyme preparation showed the participation of pyrroloquinoline quinone, a new prosthetic group, in the enzyme. Phenazine methosulfate was found to be the best electron acceptor in D-glucose oxidation with the enzyme. The optimum pH of D-glucose oxidation was at pH 3.0 with po...