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Histamine (HIST) and other biogenic amines found in fish and fishery products accumulated by the action of bacterial amino acid decarboxylase cannot be decomposed and eliminated by heating or other chemical methods. A simple method for HIST elimination is proposed by a coupling reaction of the fungal amine oxidase (FAO) and bacterial aldehyde oxidase (ALOX) of acetic acid bacteria. As a model reaction, FAO oxidized benzylamine to benzaldehyde, which in turn was oxidized spontaneously to benzoic acid with ALOX. Likely, in HIST elimination, FAO coupled well with ALOX to produce imidazole 4-acetic acid from HIST with an apparent yield of 100%. Imidazole 4-acetaldehyde was not detected in the reaction mixture. In the absence of ALOX, the coupling reaction was incomplete given a number of unidentified substances in the reaction mixture. The proposed coupling enzymatic method may be highly effective to eliminate toxic amines from fish and fishery products., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

D-Mannose isomerase (EC 5.3.1.7) catalyzing reversible conversion between D-mannose and D-fructose was found in acetic acid bacteria. Cell fractionation confirmed the enzyme to be a typical membrane-bound enzyme, while all sugar isomerases so far reported are cytoplasmic. The optimal enzyme activity was found at pH 5.5, which was clear contrast to the cytoplasmic enzymes having alkaline optimal pH. The enzyme was heat stable and the optimal reaction temperature was observed at around 40 to 60˚C. Purified enzyme after solubilization from membrane fraction showed the total molecular mass of 196 kDa composing of identical four subunits of 48 kDa. Washed cells or immobilized cells were well functional at nearly 80% of conversion ratio from D-mannose to D-fructose and reversely 20-25% of D-fructose to D-mannose. Catalytic properties of the enzyme were discussed with respect to the biotechnological applications to high fructose syrup production from konjac taro., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

We characterized the pyrroloquinoline quinone (PQQ)-dependent dehydrogenase 9 (PQQ-DH9) of Gluconobacter sp. strain CHM43, which is a homolog of PQQ-dependent glycerol dehydrogenase (GLDH). We used a plasmid construct to express PQQ-DH9. The expression host was a derivative strain of CHM43, which lacked the genes for GLDH and the membrane-bound alcohol dehydrogenase and consequently had minimal ability to oxidize primary and secondary alcohols. The membranes of the transformant exhibited considerable d-arabitol dehydrogenase activity, whereas the reference strain did not, even if it had PQQ-DH9-encoding genes in the chromosome and harbored the empty vector. This suggests that PQQ-DH9 is not expressed in the genome. The activities of the membranes containing PQQ-DH9 and GLDH suggested that similar to GLDH, PQQ-DH9 oxidized a wide variety of secondary alcohols but had higher Michaelis constants than GLDH with regard to linear substrates such as glycerol. Cyclic substrates such as cis-1,2-cyclohexanediol were readily oxidized by PQQ-DH9., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Due to the indigestibility, utilization of konjac taro, Amorphophallus konjac has been limited only to the Japanese traditional konjac food. Koji preparation with konjac taro was examined to utilize konjac taro as a source of utilizable carbohydrates. Aspergillus luchuensis AKU 3302 was selected as a favorable strain for koji preparation, while Aspergillus oryzae used extensively in sake brewing industry was not so effective. Asp. luchuensis grew well over steamed konjac taro by extending hyphae with least conidia formation. Koji preparation was completed after 3-day incubation at 30°C. D-Mannose and D-glucose were the major monosaccharides found in a hydrolyzate giving the total sugar yield of 50 g from 100 g of dried konjac taro. An apparent extent of konjac taro hydrolysis at 55°C for 24 h seemed to be completed. Since konjac taro is hydrolyzed into monosaccharides, utilization of konjac taro carbohydrates may become possible to various products of biotechnological interest.

Gluconobacter Frateurii: CHM 43 have D-mannitol dehydrogenase (quinoprotein glycerol dehydrogenase) and flavoprotein D-fructose dehydrogenase in the membranes. When the two enzymes are functional, D-mannitol is converted to 5-keto-D-fructose with 65% yield when cultivated on D-mannitol. 5-Keto-D-fructose production with almost 100% yield was realized with the resting cells. The method proposed here should give a smart strategy for 5-keto-D-fructose production.

A novel oxidation of D-pentonates to 4-keto-D-pentonates was analyzed with Gluconobacter thailandicus NBRC 3258. D-Pentonate 4-dehydrogenase activity in the membrane fraction was readily inactivated by EDTA and it was reactivated by the addition of PQQ and Ca 2+ . D-Pentonate 4-dehydrogenase was purified to two different subunits, 80 and 14 kDa. The absorption spectrum of the purified enzyme showed no typical absorbance over the visible regions. The enzyme oxidized D-pentonates to 4-keto-D-pentonates at the optimum pH of 4.0. In addition, the enzyme oxidized D-fructose to 5-keto-D-fructose, D-psicose to 5-keto-D-psicose, including the other polyols such as, glycerol, D-ribitol, D-arabitol, and D-sorbitol. Thus, D-pentonate 4-dehydrogenase was found to be identical with glycerol dehydrogenase (GLDH), a major polyol dehydrogenase in Gluconobacter species. The reaction versatility of quinoprotein GLDH was notified in this study.

D-Ribose and 2-deoxy-D-ribose were oxidized to 4-keto-D-ribonate and 2-deoxy-4-keto-D-ribonate respectively by oxidative fermentation, and the chemical structures of the oxidation products were confirmed to be as expected. Both pentoses are important sugar components of nucleic acids. When examined, purine nucleosidase activity predominated in the membrane fraction of acetic acid bacteria. This is perhaps the first finding of membrane-bound purine nucleosidase.

Further upstream of sldSLC, genes for FAD-dependent D-sorbitol dehydrogenase in Gluconobacter frateurii, three additional genes (sldR, xdhA, and perA) are found: for a transcriptional regulator, NAD(P)-dependent xylitol dehydrogenase, and a transporter protein, a member of major facilitator superfamily, respectively. xdhA and perA but not sldR were found to be in the same transcriptional unit. Disruption of sldR resulted in a dramatic decrease in sldSLC promoter activity, indicating that it is an activator for sldSLC expression. The recombinant protein of XdhA expressed in Escherichia coli showed NAD-dependent dehydrogenase activities with xylitol and D-sorbitol, but a mutant strain defective in this gene showed similar activities with both substrates as compared to the wild-type strain. Nonetheless, the growth of the xdhA mutant strain on D-sorbitol and xylitol was retarded, and so was that of a mutant strain defective in perA. These results indicate that xdhA and perA are involved in assimilation of D-sorbitol and xylitol.

Acetobacter tropicalis SKU1100 is a thermotolerant acetic acid bacterium that grows even at 42 °C, a much higher temperature than the limit for the growth of mesophilic strains. To elucidate the mechanism underlying the thermotolerance of this strain, we attempted to identify the genes essential for growth at high temperature by transposon (Tn10) mutagenesis followed by gene or genome analysis. Among the 4,000 Tn10-inserted mutants obtained, 32 exhibited a growth phenotype comparable to that of the parent strain at 30 °C but not at higher temperatures. We identified the insertion site of Tn10 on the chromosomes of all the mutant strains by TAIL (Thermal Asymmetric Interlaced)-PCR, and found 24 genes responsible for thermotolerance. The results also revealed a partial overlap between the genes required for thermotolerance and those required for acetic acid resistance. In addition, the origin and role of these thermotolerant genes are discussed.

In our previous study, a new microbial reaction yielding 4-keto-D-arabonate from 2,5-diketo-D-gluconate was identified with Gluconacetobacter liquefaciens RCTMR 10. It appeared that decarboxylation and dehydrogenation took place together in the reaction. To analyze the nature of the reaction, investigations were done with the membrane fraction of the organism, and 4-keto-D-arabinose was confirmed as the direct precursor of 4-keto-D-arabonate. Two novel membrane-bound enzymes, 2,5-diketo-D-gluconate decarboxylase and 4-keto-D-aldopentose 1-dehydrogenase, were involved in the reaction. Alternatively, D-arabonate was oxidized to 4-keto-D-arabonate by another membrane-bound enzyme, D-arabonate 4-dehydrogenase. More directly, D-arabinose oxidation was examined with growing cells and with the membrane fraction of G. suboxydans IFO 12528. 4-Keto-D-arabinose, the same intermediate as that from 2,5-diketo-D-gluconate, was detected, and it was oxidized to 4-keto-D-arabonate. Likewise, D-ribose was oxidized to 4-keto-D-ribose and then it was oxidized to 4-keto-D-ribonate. In addition to 4-keto-D-aldopentose 1-dehydrogenase, the presence of a novel membrane-bound enzyme, D-aldopentose 4-dehydrogenase, was confirmed in the membrane fraction. The formation of 4-keto-D-aldopentoses and 4-keto-D-pentonates (4-pentulosonates) was finally confirmed as reaction products of four different novel membrane-bound enzymes.

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.

Selective, high-yield production of 5-keto-D-gluconate (5KGA) from D-glucose by Gluconobacter was achieved without genetic modification. 5KGA production by Gluconobacter suffers byproduct formation of 2-keto-D-gluconate (2KGA). By controlling the medium pH strictly in a range of pH 3.5-4.0, 5KGA was accumulated with 87% conversion yield from D-glucose. The pH dependency of 5KGA formation appeared to be related to that of gluconate oxidizing activity.

Production of 4-keto-D-arabonate (4KAB) was confirmed in a culture medium of Gluconacetobacter liquefaciens strains, newly isolated from water kefir in Argentina. The strains rapidly oxidized D-glucose, D-gluconate (GA), and 2-keto-D-gluconate (2KGA), and accumulated 2,5-diketo-D-gluconate (25DKA) exclusively before reaching the stationary phase. 25DKA was in turn converted to 4KAB, and 4KAB remained stable in the culture medium. The occurrence of 4KAB was assumed by Ameyama and Kondo about 50 years ago in their study on the carbohydrate metabolism of acetic acid bacteria (Bull. Agr. Chem. Soc. Jpn., 22, 271-272, 380-386 (1958)). This is the first report confirming microbial production of 4KAB.

Acetobacter pasteurianus strains IFO3283, SKU1108, and MSU10 were grown under acetic acid fermentation conditions, and their growth behavior was examined together with their capacity for acetic acid resistance and pellicle formation. In the fermentation process, the cells became aggregated and covered by amorphous materials in the late-log and stationary phases, but dispersed again in the second growth phase (due to overoxidation). The morphological change in the cells was accompanied by changes in sugar contents, which might be related to pellicle polysaccharide formation. To determine the relationship between pellicle formation and acetic acid resistance, a pellicle-forming R strain and a non-forming S strain were isolated, and their fermentation ability and acetic acid diffusion activity were compared. The results suggest that pellicle formation is directly related to acetic acid resistance ability, and thus is important to acetic acid fermentation in these A. pasteurianus strains.

3-Dehydroshikimate dehydratase (DSD) is the first known enzyme catalyzing aromatization from 3-dehydroshikimate (DSA) to protocatechuate (PCA). Differently from cytosolic DSD (sDSD), a membrane-bound 3-dehydroshikimate dehydratase (mDSD) was found for the first time in the membrane fraction of Gluconobacter oxydans IFO 3244, and DSA was confirmed to be the direct precursor of PCA. In contrast to weak and instable sDSD, the abundance of mDSD in the membrane fraction suggested the metabolic significance of mDSD as the initial step in aromatization. mDSD was solubilized only by a detergent and was readily purified to high homogeneity. Its molecular weight was estimated to be 76,000. Purified mDSD showed a sole peak at 280 nm in the absorption spectrum and no critical cofactor requirements. The Km of DSA was measured at 0.5 mM, and the optimum pH was observed at pH 6-8. mDSD appeared to react only with DSA, and was inert to other compounds, such as 3-dehydroquinate, quinate, and shikimate.

The membrane fraction of Gluconobacter oxydans IFO 3244, involving membrane-bound quinoprotein quinate dehydrogenase and 3-dehydroquinate dehydratase, was immobilized into Ca-alginate beads. The Ca-alginate-immobilized bacterial membrane catalyzed a sequential reaction of quinate oxidation to 3-dehydroquinate and its spontaneous conversion to 3-dehydroshikimate under neutral pH. An almost 100% conversion rate from quinate to 3-dehydroshikimate was observed. NADP-Dependent cytoplasmic enzymes from the same organism, shikimate dehydrogenase and D-glucose dehydrogenase, were immobilized together with different carriers as an asymmetric reduction system forming shikimate from 3-dehydroshikimate. Blue Dextran 2000, Blue Dextran-Sepharose-4B, DEAE-Sephadex A-50, DEAE-cellulose, and hydroxyapatite were effective carriers of the two cytoplasmic enzymes, and the 3-dehydroshikimate initially added was converted to shikimate at 100% yield. The two cytoplasmic enzymes showed strong affinity to Blue Dextran 2000 and formed a soluble form of immobilized catalyst having the same catalytic efficiency as that of the free enzymes. This paper may be the first one on successful immobilization of NAD(P)-dependent dehydrogenases.

Membrane-bound glucono-delta-lactonase (MGL) was purified to homogeneity from the membrane fraction of Gluconobacter oxydans IFO 3244. After solubilization with 1 M CaCl2, MGL was purified in the presence of Ca2+ and detergent. A single band corresponding to 60 kDa appeared in SDS-PAGE. The molecular weight of MGL was judged to be 120 k. Differently from cytoplasmic lactonases, MGL showed optimum pH in an acidic range of 5-5.5. It was highly sensitive to metal-chelating agents such as EDTA, and the lost MGL activity was restored to the original level by the addition of divalent cations such as Ca2+ or Mg2+. The purified MGL was strictly dependent on Ca2+ and underwent rapid denaturing precipitation on Ca2+ depletion even in the presence of detergent. This communication can be the first one dealing with the solubilization, purification and properties of MGL.

Quinoprotein alcohol dehydrogenase (ADH) of acetic acid bacteria is a membrane-bound enzyme that functions as the primary dehydrogenase in the ethanol oxidase respiratory chain. It consists of three subunits and has a pyrroloquinoline quinone (PQQ) in the active site and four heme c moieties as electron transfer mediators. Of these, three heme c sites and a further site have been found to be involved in ubiquinone (Q) reduction and ubiquinol (QH2) oxidation respectively (Matsushita et al., Biochim. Biophys. Acta, 1409, 154-164 (1999)). In this study, it was found that ADH solubilized and purified with dodecyl maltoside, but not with Triton X-100, had a tightly bound Q, and thus two different ADHs, one having the tightly bound Q (Q-bound ADH) and Q-free ADH, could be obtained. The Q-binding sites of both the ADHs were characterized using specific inhibitors, a substituted phenol PC16 (a Q analog inhibitor) and antimycin A. Based on the inhibition kinetics of Q2 reductase and ubiquinol-2 (Q2H2) oxidase activities, it was suggested that there are one and two PC16-binding sites in Q-bound ADH and Q-free ADH respectively. On the other hand, with antimycin A, only one binding site was found for Q2 reductase and Q2H2 oxidase activities, irrespective of the presence of bound Q. These results suggest that ADH has a high-affinity Q binding site (QH) besides low-affinity Q reduction and QH2 oxidation sites, and that the bound Q in the QH site is involved in the electron transfer between heme c moieties and bulk Q or QH2 in the low-affinity sites.

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.

In addition to the cytoplasmic soluble form of 3-dehydroquinate dehydratase (sDQD) (EC 4.1.2.10), a novel form of DQD occurring in the periplasmic space was found in Gluconobacter oxydans IFO 3244. The novel DQD, tentatively designated as pDQD, appeared to have a practical function involved in oxidative fermentation extracellularly coupling with membrane-bound quinoprotein quinate dehydrogenase (QDH) yielding 3-dehydroshikimate from quinate via 3-dehydroquinate. pDQD was not detached from the membrane by mechanical disruption or extraction with high salt, but was solubilized only with detergent. pDQD and sDQD were purified to homogeneity and compared as to their enzymatic properties. They showed the same apparent molecular weights and same catalytic properties, but they were distinct each other in subunit molecular mass, 16 kDa for pDQD and 47 kDa for sDQD.

Acetic acid bacteria (AAB) are known as a vinegar producer on account of their ability to accumulate a high concentration of acetic acid due to oxidative fermentation linking the ethanol oxidation respiratory chain. Reactions in oxidative fermentation cause poor growth because a large amount of the carbon source is oxidized incompletely and the harmful oxidized products are accumulated almost stoichiometrically in the culture medium during growth, but a newly identified AAB, Asaia, has shown unusual properties, including scanty acetic acid production and rapid growth, as compared with known AAB as Acetobacter, Gluconobacter, and Gluconacetobacter. To understand these unique properties of Asaia in more detail, the respiratory chain and energetics of this strain were investigated. It was found that Asaia lacks quinoprotein alcohol dehydrogenase, but has other sugar and sugar alcohol-oxidizing enzymes specific to the respiratory chain of Gluconobacter, especially quinoprotein glycerol dehydrogenase. It was also found that Asaia has a cyanide-sensitive cytochrome bo(3)-type ubiquinol oxidase as sole terminal oxidase in the respiratory chain, and that it exhibits a higher H(+)/O ratio.

Two different membrane-bound enzymes oxidizing D-sorbitol are found in Gluconobacter frateurii THD32: pyroloquinoline quinone-dependent glycerol dehydrogenase (PQQ-GLDH) and FAD-dependent D-sorbitol dehydrogenase (FAD-SLDH). In this study, FAD-SLDH appeared to be induced by L-sorbose. A mutant defective in both enzymes grew as well as the wild-type strain did, indicating that both enzymes are dispensable for growth on D-sorbitol. The strain defective in PQQ-GLDH exhibited delayed L-sorbose production, and lower accumulation of it, corresponding to decreased oxidase activity for D-sorbitol in spite of high D-sorbitol dehydrogenase activity, was observed. In the mutant strain defective in PQQ-GLDH, oxidase activity with D-sorbitol was much more resistant to cyanide, and the H(+)/O ratio was lower than in either the wild-type strain or the mutant strain defective in FAD-SLDH. These results suggest that PQQ-GLDH connects efficiently to cytochrome bo(3) terminal oxidase and that it plays a major role in L-sorbose production. On the other hand, FAD-SLDH linked preferably to the cyanide-insensitive terminal oxidase, CIO.

A novel NADH dehydrogenase (NADH-dh) involving FAD as coenzyme, distinct from NADPH dehydrogenase (NADPH-dh, old yellow enzyme, EC 1.6.99.1), was found in the same cytoplasmic fraction of Gluconobacter strains. Conventional artificial electron acceptors were more effective than molecular oxygen in the NADH-dh reaction. NADH-dh did not appear to be identical with any previously described flavoproteins, although the N-terminal amino acid sequence showed 100% similarity with a non-heme chloroperoxidase. The N-terminal amino acid sequence of NADPH-dh matched 100% a putative oxidoreductase containing the old yellow enzyme-like FMN-binding domain. NADH-dh might function to regenerate NAD coupling with NAD-dependent dehydrogenases in the cytoplasm of Gluconobacter strains.

For easy measurement of 5-keto D-gluconate (5KGA) and 2-keto D-gluconate (2KGA), two enzymes, 5KGA reductase (5KGR) and 2KGA reductase (2KGR) are useful. The gene for 5KGR has been reported, and a corresponding gene was found in the genome of Gluconobacter oxydans 621H and was identified as GOX2187. On the other hand, the gene for 2KGR was identified in this study as GOX0417 from the N-terminal amino acid sequence of the partially purified enzyme. Several plasmids were constructed to express GOX2187 and GOX0417, and the final constructed plasmids showed good expression of 5KGR and 2KGR in Escherichia coli. From the two E. coli transformants, large amounts of each enzyme were easily prepared after one column chromatography, and the preparation was ready to use for quantification of 5KGA or 2KGA.

A method for enzymatic preparation of 3-dehydroquinate and 3-dehydroshikimate in the shikimate pathway was established by controlling the enzyme activity of 3-dehydroquinate dehydratase. When quinate was incubated with the membrane fraction of acetic acid bacteria at pH 5.0, 3-dehydroquinate was formed as the predominant product. 3-Dehydroshikimate was the sole product when incubated at pH 8.0. Mutual separation of the metabolic intermediates was also exemplified.

NADP-Dependent shikimate dehydrogenae (SKDH, EC 1.1.1.25) was purified from Gluconobacter oxydans IFO 3244. SKDH showed a single protein band on native-PAGE accompanying enzyme activity. It required NADP exclusively and catalyzed only the shuttle reaction between shikimate and 3-dehydroshikimate. The optimum pH for shikimate oxidation and 3-dehydroshikimate reduction was found at pH 10 and 7 respectively. SKDH proved to be a useful catalyst for shikimate production from 3-dehydroshikimate.

Acetobacter tropicalis SKU1100 produces a pellicle-forming capsular polysaccharide (CPS), consisting of galactose, glucose, and rhamnose. We cloned the galE gene, a UDP-galactose synthesis gene, from A. tropicalis SKU1100 by PCR. A galE-disruptant was prepared and found not to produce CPS and thus not to form a pellicle under the static condition. Instead, the DeltagalE mutant secreted an extracellular polysaccharide (EPS), which was purified and found to have a unique character, different from the original CPS.

3-Dehydroshikimate was formed with a yield of 57-77% from quinate via 3-dehydroquinate by two successive enzyme reactions, quinoprotein quinate dehydrogenase (QDH) and 3-dehydroquinate dehydratase, in the cytoplasmic membranes of acetic acid bacteria. 3-Dehydroshikimate was then reduced to shikimate (SKA) with NADP-dependent SKA dehydrogenase (SKDH) from the same organism. When SKDH was coupled with NADP-dependent D-glucose dehydrogenase (GDH) in the presence of excess D-glucose as an NADPH re-generating system, SKDH continued to produce SKA until 3-dehydroshikimate added initially in the reaction mixture was completely converted to SKA. Based on the data presented, a strategy for high SKA production was proposed.

A growth-promoting substance (GPS) for methylotrophic yeasts was purified from yeast extract. The purified GPS showed growth promotion by reducing the lag time of the growth of methylotrophic Candida boidinii, while the growth rate at the exponential phase was the same as that of growth without GPS. GPS was finally identified to be L-proline by the criteria of instrumental analysis and chemical shift assignments.

Membrane-bound NADP-independent formaldehyde-oxidizing enzyme was purified to homogeneity from the membrane of Acetobacter sp. SKU 14 isolated in Thailand. The enzyme was solubilized from the membrane fraction of glycerol-grown cells with 1% Tween 20 at pH 2.85, and purified to homogeneity through the steps of column chromatographies on DEAE-Sephadex A-50 and Q-Sepharose in the presence of 0.1% Tween 20 and 0.1% Triton X-100. The enzyme purified together with a cytochrome c showed a single protein band on native-PAGE, and was dissociated into three different subunits upon SDS-PAGE with molecular masses of 78 kDa, 55 kDa, and 18 kDa. The purified enzyme was finally characterized as a quinoprotein alcohol dehydrogenase (QADH), and this is the first indication that QADH highly oxidizes formaldehyde. The substrate specificity of the enzyme was found to be broad toward aldehydes and alcohols, and alcohols, especially n-butanol, n-propanol, and ethanol, were oxidized more rapidly than formaldehyde.

There are two types of membrane-bound D-sorbitol dehydrogenase (SLDH) reported: PQQ-SLDH, having pyrroloquinoline quinone (PQQ), and FAD-SLDH, containing FAD and heme c as the prosthetic groups. FAD-SLDH was purified and characterized from the PQQ-SLDH mutant strain of a thermotolerant Gluconobacter frateurii, having molecular mass of 61.5 kDa, 52 kDa, and 22 kDa. The enzyme properties were quite similar to those of the enzyme from mesophilic G. oxydans IFO 3254. This enzyme was shown to be inducible by D-sorbitol, but not PQQ-SLDH. The oxidation product of FAD-SLDH from D-sorbitol was identified as L-sorbose. The cloned gene of FAD-SLDH had three open reading frames (sldSLC) corresponding to the small, the large, and cytochrome c subunits of FAD-SLDH respectively. The deduced amino acid sequences showed high identity to those from G. oxydans IFO 3254: SldL showed to other FAD-enzymes, and SldC having three heme c binding motives to cytochrome c subunits of other membrane-bound dehydrogenases.

Type II NADH dehydrogenase of Corynebacterium glutamicum (NDH-2) was purified from an ndh overexpressing strain. Purification conferred 6-fold higher specific activity of NADH:ubiquinone-1 oxidoreductase with a 3.5-fold higher recovery than that previously reported (K. Matsushita et al., 2000). UV-visible and fluorescence analyses of the purified enzyme showed that NDH-2 of C. glutamicum contained non-covalently bound FAD but not covalently bound FMN. This enzyme had an ability to catalyze electron transfer from NADH and NADPH to oxygen as well as various artificial quinone analogs at neutral and acidic pHs respectively. The reduction of native quinone of C. glutamicum, menaquinone-2, with this enzyme was observed only with NADH, whereas electron transfer to oxygen was observed more intensively with NADPH. This study provides evidence that C. glutamicum NDH-2 is a source of the reactive oxygen species, superoxide and hydrogen peroxide, concomitant with NADH and NADPH oxidation, but especially with NADPH oxidation. Together with this unique character of NADPH oxidation, phylogenetic analysis of NDH-2 from various organisms suggests that NDH-2 of C. glutamicum is more closely related to yeast or fungal enzymes than to other prokaryotic enzymes.

3-Dehydroquinate production from quinate by oxidative fermentation with Gluconobacter strains of acetic acid bacteria was analyzed for the first time. In the bacterial membrane, quinate dehydrogenase, a typical quinoprotein containing pyrroloquinoline quinone (PQQ) as the coenzyme, functions as the primary enzyme in quinate oxidation. Quinate was oxidized to 3-dehydroquinate with the final yield of almost 100% in earlier growth phase. Resting cells, dried cells, and immobilized cells or an immobilized membrane fraction of Gluconobacter strains were found to be useful biocatalysts for quinate oxidation. 3-Dehydroquinate was further converted to 3-dehydroshikimate with a reasonable yield by growing cells and also immobilized cells. Strong enzyme activities of 3-dehydroquinate dehydratase and NADP-dependent shikimate dehydrogenase were detected in the soluble fraction of the same organism and partially fractionated from each other. Since the shikimate pathway is remote from glucose in the metabolic pathway, the entrance into the shikimate pathway from quinate to 3-dehydroquinate looks advantageous to produce metabolic intermediates in the shikimate pathway.

Several bacterial strains carrying quinoprotein quinate dehydrogenase (QDH) were screened through acetic acid bacteria and other bacteria. Strong enzyme activity was found in the membrane fraction of Gluconobacter melanogenus IFO 3294, G. oxydans IFO 3292, G. oxydans IFO 3244, and some strains of Acinetobacter calcoaceticus. Interestingly, in the membrane fraction of A. calcoaceticus AC3, which is unable to produce pyrroloquinoline quinone (PQQ), fairly large amounts of apo-QDH were formed, and were converted to holo-QDH only by the addition of PQQ. It was difficult to detach PQQ from the holo-QDH by EDTA treatment, and EDTA treatment with apo-QDH prior to PQQ addition gave no significant holo-QDH. For QDH purification, Gluconobacter strains were not suitable due to the presence of huge amounts of quinohemoprotein alcohol dehydrogenase (ADH) in the same membrane, which was co-solubilized with QDH and disturbed purification of QDH. Purification of holo-QDH was done with Acinetobacter sp. SA1 instead, which contained no ADH. Apo-QDH was purified from A. aclcoaceticus AC3. This is the first report dealing with QDH purification, and two different criteria of QDH purification were given. A combination of two steps using butyl-Toyopearl and hydroxyapatite columns gave a highly purified holo-QDH which was monodispersed and showed enough purity, though the specific activity did not increase as much as expected. When QDH purification was done with A. calcoaceticus AC3 in the absence of PQQ, purified apo-QDH appeared to be a dimer, which was converted to the monomer on addition of PQQ. Since QDH was highly hydrophobic, one-step chromatography on a DEAE-Sepharose column was tried. Purified holo-QDH of higher specific activity was obtained with a higher yield. The molecular mass of QDH was estimated to be 88 kDa. There was no characteristic absorption spectrum with the purified QDH except for a small bump around 420 nm. QDH oxidized only quinate and shikimate so far examined. The optimal QDH activity was found at pH 6-7 when assayed with artificial electron acceptors. QDH was formed in the presence or absence of quinate in the culture medium, although stronger induction was usually observed in the presence of quinate.

Molecular cloning of the gene of quinohemoprotein alcohol dehydrogenase (ADH IIB) from Pseudomonas putida HK5 was done. The gene (qbdA) was 690 amino acids in length, containing a 22-amino acid signal sequence. Another gene (qbdB) upstream of qbdA, probably in the same transcriptional unit, was found. Further upstream, a gene divergently transcribed against qbdBA had identity with NAD-dependent aldehyde dehydrogenase.

High NAD-dependent alcohol dehydrogenase (ADH) activity was found in the cytoplasm when a membrane-bound, quinoprotein, ADH-deficient mutant strain of Acetobacter pasteurianus SKU1108 was grown on ethanol. Two NAD-dependent ADHs were separated and purified from the supernatant fraction of the cells. One (ADH I) is a trimer, consisting of an identical subunit of 42 kDa, while the other (ADH II) is a homodimer, having a subunit of 31 kDa. One of the two ADHs, ADH II, easily lost the activity during the column chromatographies, which could be stabilized by the addition of DTT and MgCl2 in the column buffer. ADH I but not ADH II contained approximately one zinc atom per subunit. The N-terminal amino acid analysis indicated that ADH I and ADH II have homology to the long-chain and short-chain ADH families, respectively. ADH I showed a preference for primary alcohols, while ADH II had a preference for secondary alcohols. The two ADHs showed clear difference in their kinetics on ethanol, acetaldehyde, NAD, and NADH. The physiological function of both ADH I and ADH II are also discussed.

Acetobacter strains able to produce a thick pellicle at 37 degrees C were screened among many thermotolerant strains isolated from fruits in Thailand. As a result, Acetobacter sp. SKU 1100 was selected as the producer of a relatively thick pellicle even when cultured at higher temperatures such as 37 degrees C or 40 degrees C. This strain could produce a pellicle polysaccharide in a shaking submerged culture as well as under static culture conditions. The polysaccharide was found to be attached to the bacterial cells. Although the polysaccharide production was higher at 30 degrees C than at 37 degrees C in shaking submerged culture, the productivity in static culture was not decreased even at higher temperatures. The membrane-attached polysaccharide was purified from the SKU 1100 strain by cell disruptions using either ultrasonic treatment or lysozyme treatment, followed by ultracentrifugation, enzyme treatments, dialysis against SDS, DEAE-cellulose column chromatography, alcohol precipitation, and gel filtration chromatography. The polysaccharide purified by the sonic treatment and also by the mild conditions using lysozyme treatment had the same average molecular mass of 120 kDa. The purified polysaccharide was composed of three different monosaccharides; glucose, galactose, and rhamnose, in an approximately equimolar ratio of 1:1:1.

Membrane-bound NAD(P)-independent malate dehydrogenase (EC 1.1.99.16) was purified to homogeneity from the membrane of thermotolerant Acetobacter sp. SKU 14, an isolate from Thailand. The enzyme was solubilized from the membrane fraction of glycerol-grown cells with 1% Triton X-100 in the presence of 0.1 M KCl, and purified to homogeneity through steps of column chromatographies on DEAE-Sephadex A-50 and DEAE-Toyopearl in the presence of 0.1% Triton X-100. The purified enzyme showed a single protein band in both native-PAGE and SDS-PAGE. The enzyme was a homodimer with a molecular mass of 60 kDa subunit and had noncovalently bound FAD as the cofactor. The enzyme was stable over pH 5 and had its maximum activity at pH 11.0 when ferricyanide was used as an electron acceptor. The enzyme activity was elevated by the addition of ammonium ions. The substrate specificity was very strict to only L-malate, of which the apparent Km was 10 mM and over 20 compounds involving D-malate were not oxidized by the enzyme.

Thermotolerant Gluconobacter frateurii CHM 43 was selected for L-erythrulose production from mesoerythritol at higher temperatures. Growing cells and the membrane fraction of the strain rapidly oxidized mesoerythritol to L-erythrulose irreversibly with almost 100% of recovery at 37 degrees C. L-Erythrulose was also produced efficiently by the resting cells at 37 degrees C with 85% recovery. The enzyme responsible for mesoerythritol oxidation was found to be located in the cytoplasmic membrane of the organism. The EDTA-resolved enzyme required PQQ and Ca2+ for L-erythrulose formation, suggesting that the enzyme catalyzing meso-erythritol oxidation was a quinoprotein. Quinoprotein membrane-bound mesoerythritol dehydrogenase (QMEDH) was solubilized and purified to homogeneity. The purified enzyme showed a single band in SDS-PAGE of which the molecular mass corresponded to 80 kDa. The optimum pH of QMEDH was found at pH 5.0. The Michaelis constant of the enzyme was found to be 25 mM for meso-erythritol as the substrate. QMEDH showed a broad substrate specificity toward C3-C6 sugar alcohols in which the erythro form of two hydroxy groups existed adjacent to a primary alcohol group. On the other hand, the cytosolic NAD-denpendent meso-erythritol dehydrogenase (CMEDH) of the same organism was purified to a crystalline state. CMEDH showed a molecular mass of 60 kDa composed of two identical subunits, and an apparent sedimentation constant was 3.6 s. CMEDH catalyzed oxidoreduction between mesoerythritol and L-erythrulose. The oxidation reaction was observed to be reversible in the presence of NAD at alkaline pHs such as 9.0-10.5. L-Erythrulose reduction was found at pH 6.0 with NADH as coenzyme. Judging from the catalytic properties, the NAD-dependent enzyme in the cytosolic fraction was regarded as a typical pentitol dehydrogenase of NAD-dependent and the enzyme was independent of the oxidative fermentation of L-erythrulose production.