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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. Plausible applications of konjac mannan into food, beverage, and chemical industries triggered by hydrolysis with konjac taro koji. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

l-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.25) from Rhodosporidium toruloides was utilized to remove l-phenylalanine ( l-Phe) from different commercial protein hydrolysates. A casein acid hydrolysate (CAH, l-Phe ~2.28 %) was employed as a model substrate. t-Cinnamic acid resulting from deamination of l-Phe was extracted, analyzed at λ = 290 nm, and used for PAL activity determination. Optimum reaction conditions, optimized using successive Doehlert design, were 35 mg mL of CAH and 800 mU mL of PAL, while temperature and pH were 42 °C and 8.7, respectively. Reaction kinetics of PAL with CAH was determined under optimized conditions. Then, removal of l-Phe from CAH was tested. Results showed that more than 92 % of initial l-Phe was eliminated. Similar results were obtained with other protein hydrolysates. These findings demonstrate that PAL is a useful biocatalyst for l-Phe removal from protein hydrolysates, which can be evaluated as potential ingredients in foodstuffs for PKU patients. [ABSTRACT FROM AUTHOR]

The article presents a study concerning the formation of 4-Pentulosonates (4-Keto-D-pentonates) and 4-Keto-D-aldopentoses having unidentified membrane-bound enzymes coming from acetic acid bacteria. Study uses various methods to examine the formation including thin layer chromatography (TLC), centrifugation, and ethylenediaminetetraacetic acid (EDTA) treatment. Findings reveal the unidentified membrane-bound enzymes which contribute in the formation of 4-Pentulosonates and 4-Keto-D-aldopentoses.

The article focuses on the production of 4-keto-D-arabonate (4KAB) by Gluconacetobacter liquefaciens strains. It states that the strains accumulated 2,5-diketo-d-gluconate (25DKA) and oxidized D-gluconate, D-glucose, and 2-keto-D-gluconate (2KGA). It says that after the accumulation, 25DKA was converted into 4KAB and 4KAB remained stable in Gluconacetobacter liquefaciens. Moreover, a study of M. Ameyama and K. Kondo on carbohydrate metabolism of acetic acid bacteria assumed the 4KAB occurrence.

The article presents a study regarding the productiuon of 3-dehydroshikimate (DSA) from quinate by immobilixing the membrane fraction of the acetic acid bacteria Gluconobacter oxydans IFO 3244. The study used several methods to realize and examine the immobilization process including encapsulation, ionic binding, and physical adsorption. Results show a conversion rate of almost 100% from quinate to 3-dehydroshikimate.

The article presents a study that focuses on the purification and characterization of the membrane-bound 3-dehydroshikimate dehydratase (mDSD) and enzyme solubilization from the membrane fraction of gluconobacter oxydans IFO 3244. The results revealed that the mDSD in the membrane fraction suggests the mDSD's significance of aromatization, solubilized and purified to high homogeneity. It says that dehydroshikimate (DSA) was confirmed as a direct precursor of phenylcyclohexylamine (PCA).

Chlorogenate hydrolase (EC 3.1.1.42, CHase) was highly induced in mycelia of Aspergillus sojae AKU 3312 grown in Czapek medium containing either instant coffee powder or coffee pulp as inducer. No CHase formation was observed in the mycelia when cultivated without the inducer. CHase was purified readily from CHase-induced mycelia to high homogeneity, and the purified CHase revealed the molecular weight of 180,000 consisting of two identical subunits of 88 kDa. Equimolar quinate (QA) and caffeate (CA) were confirmed on hydrolysis of chlorogenate (CGA). The purified CHase was only useful for a laboratory scale hydrolysis of CGA. For practical QA and CA production using scaled up hydrolysis of vegetable extracts of natural CGA resources, the enzyme activity of purified CHase decreased and denatured irreversibly. Preparation of coffee pulp koji and its application to QA and CA production were proposed instead of purified CHase. When coffee pulp koji was heated at 60°C for 30 min, CHase survived without any appreciable loss of enzyme activity while vegetative mycelial growth and spore germination were terminated. The heated coffee pulp koji thus prepared was effective itself as stable immobilized catalyst of CHase for QA and CA production from vegetable CGA resources such as coffee powders, coffee pulp, and others. [ABSTRACT FROM AUTHOR]

The article presents a study which examines the purification and properties of dihydroxyacetone (DHA) reductases in gluconobacter suboxydans grown in gycerol. It states that two DHA reductases were detected in the same cytoplasm gluconobacter suboxydans. It notes that NADH-dependent appears inducible and labile in nature while the NADPH-dependent appears constitutive and stable. It also notes that the two DHARS were specific only to DHA reduction to glycerol.

The article reports on a study on the novel 3-dehydroquinate dehydrase (DQD) catalyzing extracellular formation of 3-dehydroshikimate by oxidative fermentation of Gluconobacter oxydans IFO 3244. The study showed that the same apparent molecular weights and same catalytic properties, but they were distinct from each other in subunit molecular mass.

The article reports on results of a study of physiological roles of two membrane-bound dehydrogenases responsible for d-sorbitol oxidation in Gluconobacter frateurii. A description of the study design and method is presented. The study concluded that pyroloquinoline quinone-dependent glycerol dehydrogenase (PQQ-GLDH) connects efficiently to cytochrome bo3 terminal oxidase.

The article examines the purification and properties of NADP-dependent shikimate dehydrogenase (SKDH) from Gluconobacter oxydans IFO 3244 and its application to enzymatic shikimate production. It showed a single protein band on native-polyacrylamide gel electrophoresis accompanying activity when SKDH was purified from Gluconobacter oxydans IFO 3244. It required NADP exclusively and catalyzed only the shuttle reaction between shikimate and 3-dehydroshikimate.

The article presents a study on the high shikimate (SKA) production from quinate with two enzymatic systems of acetic acid bacteria. 3-Dehydroshikimate was formed with a yield of 57 to 77 percent from quinate thru 3-dehydroquinate by two successive enzyme reactions, quinoprotein quinate dehydrogenase and 3-dehydroquinate dehydratase, in the cytoplasmic membranes of acetic acid bacteria. A strategy for high SKA production was proposed based on the data presented.

Discusses 3-dehydroquinate production from quinate by oxidative fermentation with Gluconobacter strains of acetic acid bacteria and the further conversion of 3-dehydroquinate to the intermediates in the shikimate pathway. Quinate oxidation by acetic acid bacteria; Quinate oxidation by immobilized cells of acetic acid bacteria; Chromatographic detection of oxidative products.

Discusses the purification and characterization of membrane-bound quinoprotein quinate dehydrogenase (QDH). Occurrence of QDH in aerobic bacteria; Titration of apo-QDH with PQQ; Properties of purified QDH; Identification of quinate oxidation product by the membrane-bound QDH.

Quinohemoprotein amine dehydrogenase (QH-AmDH) catalyzes the oxidative deamination of aliphatic and aromatic amines. The enzyme from Pseudomonas putida has an αβγ heterotrimeric structure with two heme c groups in the largest α subunit, and a novel quinone cofactor [cysteine tryptophylquinone (CTQ)] and hitherto unknown internal cross-bridges in the smallest γ subunit. The crystal structure of the enzyme in the complex with the inhibitor [p-nitrophenylhydrazine (pNPH)] has been determined at a 2.0 A˚ resolution.11Coordinates have been deposited in the Protein Data Bank (ID code 1JMZ). The hydrazone of the cofactor with the inhibitor was nicely modeled into the omit electron density map, identifying the C6 carbonyl group as the reactive site of the cofactor. The Asp33γ is unambiguously determined as the catalytic base to abstract the α-proton from a substrate, because Nβ atom of the inhibitor corresponding to the Cα atom of the substrate amine is neighbored to Asp33γ. The bound inhibitor is completely enclosed in the active site pocket formed by the residues from the β- and γ-subunits. The cofactor–inhibitor adduct may be predominantly in the hydrazone with the azo form as a minor component. The binding of the inhibitor causes minor but important conformational changes in the residues surrounding the active site. The inhibitor may have access to the active site pocket through the water-filled crevice between the β- and γ-subunits. [Copyright &y& Elsevier]

The article presents a study which examines the enzymatic synthesis of 4-Pentulosonate (4-Keto-D-pentonate) from D-Aldopentose and D-Pentonate using membrane enzymes of acetic acid bacteria. It states that the chemical structures of the 4-pentulosonates were confirmed by the analytical data. The study reveals a successful enzymatic synthesis of 4-pentulosonates.

The article reports on the method of establishing enzymatic preparation of 3-dehydorquinate and 3-dehydroshikimate in the shikimate pathway by controlling the enzyme activity of 3-dehydroquinate dehydratase. 3-dehydroquinate was the predominant product formed when quinate was incubated with the membrane fraction of acetic acid bacteria at pH 5.0, while 3-dehydroshikimate was the sole product when incubated at pH 8.0. Moreover, the mutual separation of the metabolic intermediates was exemplified.

The article focuses on the study of growth promoting substance (GPS) in yeast extract for methylotrophic Candida boidinii. The study was conducted by purifying a GPS for methylotrophic yeasts from yeast extract. Reduction in the lag time of the growth of methylotrophic Candida boidinii indicated the growth promotion of purified GPS.

Acetic acid bacteria are gram-negative obligate aerobic bacteria assigned to the family Acetobacteraceae of Alphaproteobacteria . They are members of the genera Acetobacter , Gluconobacter , Gluconacetobacter , Acidomonas , Asaia , Kozakia , Swaminathania , Saccharibacter , Neoasaia , Granulibacter , Tanticharoenia , Ameyamaea , Neokomagataea , and Komagataeibacter . Many strains of Acetobacter and Komagataeibacter have been known to possess high acetic acid fermentation ability as well as the acetic acid and ethanol resistance, which are considered to be useful features for industrial production of acetic acid and vinegar, the commercial product. On the other hand, Gluconobacter strains have the ability to perform oxidative fermentation of various sugars, sugar alcohols, and sugar acids leading to the formation of several valuable products. Thermotolerant strains of acetic acid bacteria were isolated in order to serve as the new strains of choice for industrial fermentations, in which the cooling costs for maintaining optimum growth and production temperature in the fermentation vessels could be significantly reduced. Genetic modifications by adaptation and genetic engineering were also applied to improve their properties, such as productivity and heat resistance. [ABSTRACT FROM AUTHOR]

The article presents a study on the production of 5-keto-D-gluconate (5KGA) derived from D-glucose by Gluconobacter. The study indicates that the effect of potential of hydrogen (pH) is essential in the production of 5KGA is correlative with the pH dependency of 5KGA. It notes that the 5KGA compound holds potential as an intermediate in the food industry.

We isolated thermotolerant Gluconobacter strains that are able to produce 5-keto-D-gluconic acid (5KGA) at 37°C, a temperature at which regular mesophilic 5KGA-producing strains showed much less growth and 5KGA production. The thermotolerant strains produced 2KGA as the major product at both 30 and 37°C. The amount of ketogluconates produced at 37°C was slightly less than the amount produced at 30°C. To improve the yield of SKGA in these strains, we disrupted flavin adenine dinucleotide-gluconate dehydrogenase (FAD.GADH), which is responsible for 2KGA production. Genes for FAD-GADH were cloned by using inverse PCR and an in vitro cloning strategy. The sequences obtained for three thermotolerant strains were identical and showed high levels of identity to the FAD-GADH sequence reported for the genome of Gluconobacter oxydans 621 H. A kanamycin resistance gene cassette was used to disrupt the FAD-GADH genes in the thermotolerant strains. The mutant strains produced SKGA exclusively, and the final yields were over 90% at 30°C and 50% at 37°C. We found that the activity of pyrroloquinoline quinone (PQQ).dependent glycerol dehydrogenase, which is responsible for 5KGA production, increased in response to addition of PQQ and CaCI2 in vitro when cells were grown at 37°C. Addition of 5 mM CaCI2 to the culture medium of the mutant strains increased 5KGA production to the point where over 90% of the initial substrate was converted. The thermotolerant Glu- conobacter strains that we isolated in this study provide a promising new option for industrial 5KGA production. [ABSTRACT FROM AUTHOR]

The article discusses a study which investigated the energy metabolism and respiratory chain of Asaia bogorensis. A. bogorensis is a unique acetic acid bacterium with unusual properties including scanty acetic acid production and rapid growth. The found that A. bogorensis lacks quinoprotein alcohol dehydrogenase, but has sugar and sugar alcohol-oxidizing enzymes specific to the respiratory chain of Gluconobacter.

The article reports on the results of a study of the occurrence of a novel NADH dehydrogenase (NADH-dh), distinct from the old yellow enzyme, in gluconobacter strains A description of the experimental set-up and measurement methods is presented. The study concluded that the N-terminal amino acid sequence of NADPH-dh matched a putative oxidoreductase containing the old yellow enzyme-like FMN-binding domain.

The article describes the preparation of two enzymes, 5-keto D-gluconate (5KGA) reductase (5KGR) and 2-keto D-gluconate (2KGA) reductase (2KGR) for quantification of 5KGA and 2KGA. According to the authors, the gene for 5KGR was found in the genome of Gluconobacter oxydans 621H and was identified as GOX2187, and the gene for 2KGR was identified as GOX0417 from the N-terminal amino acid sequence of the partially purified enzyme. The overexpression of 5KGR and 2KGR in Escherichia coli was noted.

Acetic acid bacteria are obligate aerobes able to oxidize ethanol, sugar alcohols, and sugars into their corresponding acids. Among them, Acetobacter and Gluconacetobacter species have very high ethanol oxidation capacity, leading to accumulation of vast amounts of acetic acid outside the cell. Since these bacteria are able to grow in media with high concentrations of acetic acid, they must possess a specific mechanism such as an efflux pump by which they can resist the toxic effects of acetic acid. In this study, the efflux pump of Acetobacter aceti IFO 3283 was examined using intact cells and membrane vesicles. The accumulation of acetic acid/acetate in intact cells was increased by the addition of a proton uncoupler and/or cyanide, suggesting the presence of an energy-dependent efflux system. To confirm this, right-side-out and inside-out membrane vesicles were prepared from A. aceti IFO 3283, and the accumulation of acetic acid/acetate in the vesicles was examined. Upon the addition of a respiratory substrate, the accumulation of acetic acid/acetate in the right-side-out vesicles was largely decreased, while its accumulation was very much increased in the inside-out vesicles. These respiration-dependent phenomena observed in both types of membrane vesicles were all sensitive to a proton uncoupler. Acetic acid/acetate uptake in the inside-out membrane vesicles was dependent not on ATP but on the proton motive force. Furthermore, uptake was shown to be rather specific for acetic acid and to be pH dependent, because higher uptake was observed at lower pH. Thus, A. aceti IFO 3283 possesses a proton motive force-dependent efflux pump for acetic acid. [ABSTRACT FROM AUTHOR]

Quino(hemo)protein alcohol dehydrogenases (ADH) that have pyrroloquinoline quinone (PQQ) as the prosthetic group are classified into 3 groups, types I, II, and III. Type I ADH is a simple quinoprotein having PQQ as the only prosthetic group, while type II and type III ADHs are quinohemoprotein having heme c as well as PQQ in the catalytic polypeptide. Type II ADH is a soluble periplasmic enzyme and is widely distributed in Proteobacteria such as Pseudomonas, Ralstonia, Comamonas, etc. In contrast, type III ADH is a membrane-bound enzyme working on the periplasmic surface solely in acetic acid bacteria. It consists of three subunits that comprise a quinohemoprotein catalytic subunit, a triheme cytochrome c subunit, and a third subunit of unknown function. The catalytic subunits of all the quino(hemo)protein ADHs have a common structural motif, a quinoprotein-specific superbarrel domain, where PQQ is deeply embedded in the center. In addition, in the type II and type III ADHs this subunit contains a unique heme c domain. Various type II ADHs each have a unique substrate specificity, accepting a wide variety of alcohols, as is discussed on the basis of recent X-ray crystallographic analyses. Electron transfer within both type II and III ADHs is discussed in terms of the intramolecular reaction from PQQ to heme c and also from heme to heme, and in terms of the intermolecular reaction with azurin and ubiquinone, respectively. Unique physiological functions of both types of quinohemoprotein ADHs are also discussed. [Copyright &y& Elsevier]

3-Dehydroshikimate (3-DHS) is a key intermediate for the synthesis of various compounds, including the antiviral drug oseltamivir. The Gluconobacter oxydans strain NBRC3244 intrinsically oxidizes quinate to produce 3-dehydroquinate (3-DHQ) in the periplasmic space. Even though a considerable activity is detected in the recombinant G. oxydans homologously overexpressing type II dehydroquinate dehydratase (DHQase) encoded in the aroQ gene at a pH where it grows, an alkaline shift of the culture medium is required for 3-DHS production in the middle of cultivation. Here, we attempted to adopt type I DHQase encoded in the aroD gene of Gluconacetobacter diazotrophicus strain PAL5 because the type I DHQase works optimally at weak acid, which is preferable for growth conditions of G. oxydans. In addition, we anticipated that subcellular localization of DHQase is the cytoplasm, and therefore, transports of 3-DHQ and 3-DHS across the cytoplasmic membrane are rate-limiting steps in the biotransformation. The Sec- and TAT-dependent signal sequences for secretion were attached to the N terminus of AroD to change the subcellular localization. G. oxydans that expresses the TAT-AroD derivative achieved 3-DHS production at a tenfold higher rate than the reference strain that expresses wild-type AroD even devoid of alkaline shift. Enzyme activity with the intact cell suspension and signal sequence cleavage supported the relocation of AroD to the periplasmic space. The present study suggests that the relocation of DHQase improves 3-DHS production in G. oxydans and represents a proof of concept for the potential of enzyme relocation in metabolic engineering. Key points: • Type-I dehydroquinate dehydratase (DHQase) was expressed in Gluconobacter oxydans. • Cytoplasmic DHQase was relocated to the periplasmic space in G. oxydans. • Relocation of DHQase in G. oxydans improved 3-dehydroshikimate production. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Dihydroxyacetone (DHA), a chemical suntan agent, is produced by the regiospecific oxidation of glycerol with Gluconobacter thailandicus NBRC3255. However, this microorganism consumes DHA produced in the culture medium. Here, we attempted to understand the pathway for DHA metabolism in NBRC3255 to minimize DHA degradation. The two gene products, NBRC3255_2003 (DhaK) and NBRC3255_3084 (DerK), have been annotated as DHA kinases in the NBRC 3255 draft genome. Because the double deletion derivative for dhaK and derK showed ATP-dependent DHA kinase activity similar to that of the wild type, we attempted to purify DHA kinase from ∆dhaK ∆derK cells to identify the gene for DHA kinase. The identified gene was NBRC3255_0651, of which the product was annotated as glycerol kinase (GlpK). Mutant strains with several combinations of deletions for the dhaK, derK, and glpK genes were constructed. The single deletion strain ∆glpK showed approximately 10% of wild-type activity and grew slower on glycerol than the wild type. The double deletion strain ∆derK ∆glpK and the triple deletion strain ∆dhaK ∆derK ∆glpK showed DHA kinase activity less than a detection limit and did not grow on glycerol. In addition, although ΔderK ΔglpK consumed a small amount of DHA in the late phase of growth, ∆dhaK ΔderK ΔglpK did not show DHA consumption on glucose–glycerol medium. The transformants of the ∆dhaK ΔderK ΔglpK strain that expresses one of the genes from plasmids showed DHA kinase activity. We concluded that all three DHA kinases, DhaK, DerK, and GlpK, are involved in DHA metabolism of G. thailandicus. Key points: • Dihydroxyacetone (DHA) is produced but degraded by Gluconobacter thailandicus. • Phosphorylation rather than reduction is the first committed step in DHA metabolism. • Three kinases are involved in DHA metabolism with the different properties. [ABSTRACT FROM AUTHOR]

A novel oxidation of D-pentonates to 4-keto-D-pentonates was analyzed withGluconobacter thailandicusNBRC 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 Ca2+. 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 inGluconobacterspecies. The reaction versatility of quinoprotein GLDH was notified in this study. [ABSTRACT FROM AUTHOR]

The bacterial aerobic respiratory chain has a terminal oxidase of the heme-copper oxidase superfamily, comprised of cytochrome c oxidase (COX) and ubiquinol oxidase (UOX); UOX evolved from COX. Acetobacter pasteurianus , an α-Proteobacterial acetic acid bacterium (AAB), produces UOX but not COX, although it has a partial COX gene cluster, ctaBD and ctaA , in addition to the UOX operon cyaBACD . We expressed ctaB and ctaA genes of A. pasteurianus in Escherichia coli and demonstrated their function as heme O and heme A synthases. We also found that the absence of ctaD function is likely due to accumulated mutations. These COX genes are closely related to other α-Proteobacterial COX proteins. However, the UOX operons of AAB are closely related to those of the β/γ-Proteobacteria (γ-type UOX), distinct from the α/β-Proteobacterial proteins (α-type UOX), but different from the other γ-type UOX proteins by the absence of the cyoE heme O synthase. Thus, we suggest that A. pasteurianus has a functional γ-type UOX but has lost the COX genes, with the exception of ctaB and ctaA , which supply the heme O and A moieties for UOX. Our results suggest that, in AAB, COX was replaced by β/γ-Proteobacterial UOX via horizontal gene transfer, while the COX genes, except for the heme O/A synthase genes, were lost. [ABSTRACT FROM AUTHOR]

Shikimate and 3-dehydroshikimate are useful chemical intermediates for the synthesis of various compounds, including the antiviral drug oseltamivir. Here, we show an almost stoichiometric biotransformation of quinate to 3-dehydroshikimate by an engineered Gluconobacter oxydans strain. Even under pH control, 3-dehydroshikimate was barely detected during the growth of the wild-type G. oxydans strain NBRC3244 on the medium containing quinate, suggesting that the activity of 3-dehydroquinate dehydratase (DHQase) is the rate-limiting step. To identify the gene encoding G. oxydans DHQase, we overexpressed the gox0437 gene from the G. oxydans strain ATCC621H, which is homologous to the aroQ gene for type II DHQase, in Escherichia coli and detected high DHQase activity in cell-free extracts. We identified the aroQ gene in a draft genome sequence of G. oxydans NBRC3244 and constructed G. oxydans NBRC3244 strains harboring plasmids containing aroQ and different types of promoters. All recombinant G. oxydans strains produced a significant amount of 3-dehydroshikimate from quinate, and differences between promoters affected 3-dehydroshikimate production levels with little statistical significance. By using the recombinant NBRC3244 strain harboring aroQ driven by the lac promoter, a sequential pH adjustment for each step of the biotransformation was determined to be crucial because 3-dehydroshikimate production was enhanced. Under optimal conditions with a shift in pH, the strain could efficiently produce a nearly equimolar amount of 3-dehydroshikimate from quinate. In the present study, one of the important steps to convert quinate to shikimate by fermenting G. oxydans cells was investigated. [ABSTRACT FROM AUTHOR]

We succeeded in obtaining a strain adapted to higher temperature from a thermotolerant strain, Gluconobacter frateurii CHM43, for sorbose fermentation. The adapted strain showed higher growth and l-sorbose production than original CHM43 strain at higher temperature around 38.5-40 °C. It was also shown to be useful even with the fermentation without temperature control. To understand the sorbose fermentation ability of the adapted strain at higher temperature, d-sorbitol-oxidizing respiratory chain was compared with the CHM43 strain and the adapted strain. We found that the activity of pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase (GLDH), which is a primary dehydrogenase of the respiratory chain and responsible for l-sorbose production, was decreased when the temperature increased, but the decreased activity of GLDH was recovered by the addition of PQQ. Since the adapted strain was found to produce more PQQ than the CHM43 strain, it was suggested that the adapted strain keeps GLDH as holoenzyme with the increased PQQ production, and thus produces more l-sorbose and grows better under higher temperature. [ABSTRACT FROM AUTHOR]

The article discusses the findings of a study which determined the role of genes in oxidative fermentation of D-sorbitol in Gluconobacter frateurii bacteria. The researchers described the oxidizing characteristic of the Gluconobacter in producing L-sorbose, 2- and 5-keto-D-gluconates, and the Miglitol drug. The experiment confirmed the identification and characterization of D-sorbitol dehydrogenase genes including sldR, xdhA, and perA.

The article presents a study that attempts to identify the genes essential for growth at high temperature by transposon (Tn10) mutagenesis to elucidate the mechanism underlying the thermotolerance of mesophilic strains. It states that Acetobacter tropicalis SKU1100 is a thermotolerant acetic acid bacterium that dwells even at high temperatures. Results have further supported the partial overlap between the genes required for acetic acid resistance and those needed for thermotolerance.

The article presents a study concerning acetic acid fermentation of Acetobacter pasteurianus. The study involves the examination of the relationship between pellicle polysaccharide formation and acetic acid resistance. It shows that acetic acid resistance ability pellicle formation is primarily related to pellicle formation, thus is essential to acetic acid fermentation in A. pasteurianus strains.

We isolated several thermotolerant Acetobacter species of which MSU10 strain, identified as Acetobacter pasteurianus, could grow well on agar plates at 41°C, tolerate to 1.5% acetic acid or 4% ethanol at 39°C, similarly seen with A. pasteurianus SKU1108 previously isolated. The MSU10 strain showed higher acetic acid productivity in a medium containing 6% ethanol at 37°C than SKU1108 while SKU1108 strain could accumulate more acetic acid in a medium supplemented with 4–5% ethanol at the same temperature. The fermentation ability at 37°C of these thermotolerant strains was superior to that of mesophilic A. pasteurianus IFO3191 strain having weak growth and very delayed acetic acid production at 37°C even at 4% ethanol. Alcohol dehydrogenases (ADHs) were purified from MSU10, SKU1108, and IFO3191 strains, and their properties were compared related to the thermotolerance. ADH of the thermotolerant strains had a little higher optimal temperature and heat stability than that of mesophilic IFO3191. More critically, ADHs from MSU10 and SKU1108 strains exhibited a higher resistance to ethanol and acetic acid than IFO3191 enzyme at elevated temperature. Furthermore, in this study, the ADH genes were cloned, and the amino acid sequences of ADH subunit I, subunit II, and subunit III were compared. The difference in the amino acid residues could be seen, seemingly related to the thermotolerance, between MSU10 or SKU1108 ADH and IFO 3191 ADH. [ABSTRACT FROM AUTHOR]

The article discusses membrane-bound glucono-δ-lactonase (MGL) properties, solubility, and enzyme purification. It presents the process wherein MGL is purified from Gluconobacter oxydans. It also discusses that the involved acetic acid bacteria and aerobic bacteria in the oxidative fermentation contains a well-developed periplasmic oxydase system which yields bioenergy formation.

The article studies tightly-bound quinone functions in the ubiquinone reaction sites of the quinoprotein alcohol dehydrogenase (ADH) of the Gluconobacter suboxydans bacterium in acetic acid. It includes an in-depth analysis of the solubilization and purification of ADH with dodecyl maltoside, as well as a discussion on the issues' implications for biological chemistry.

Pseudomonas putida HK5 produces three different quinoprotein alcohol dehydrogenases: ADH-I, ADH-IIB and ADH-IIG. Gene organization of qedA, the gene for ADH-I, and other 10 genes in the cluster was related to the genome sequences of five other Pseudomonas strains. Insertion mutations in either qedA, exaE or agmR eliminated ADH-I activity, although the mutants were still able to grow on ethanol but more slowly than the wild-type strain. Mutant analysis demonstrated the requirement of agmR and exaE in ADH-I expression, and the tentative involvement of agmR, but not exaE, in the induction of ADH-IIB and ADH-IIG activities. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

Most Gluconobacter species produce and accumulate 2-keto-D-gluconate (2KGA) and SKGA simultaneously from D-glucose via GA in culture medium. 2KGA is produced by membrane-bound flavin adenine dinucleotide-containing GA 2-dehydrogenase (FAD-GADH). FAD-GADH was purified from ‘Gluconobacter dioxyacetonicus’ IFO 3271, and N-terminal sequences of the three subunits were analyzed. PCR primers were designed from the N-terminal sequences, and part of the FAD-GADH genes was cloned as a PCR product. Using this PCR product, gene fragments containing whole FAD-GADH genes were obtained, and finally the nucleotide sequence of 9,696 bp was determined. The cloned sequence had three open reading frames (ORFs), gndS, gndL, and gndC, corresponding to small, large, and cytochrome c subunits of FAD-GADH, respectively. Seven other OREs were also found, one of which showed identity to glucono-δ-lactonase, which might be involved directly in 2KGA production. Three mutant strains defective in either gndL or sldA (the gene responsible for 5KGA production) or both were constructed. Ferricyanide-reductase activity with GA in the membrane fraction of the gndL-defective strain decreased by about 60% of that of the wild-type strain, while in the sldA-defective strain, activity with GA did not decrease and activities with glycerol, D-arabitol, and D-sorbitol disappeared. Unexpectedly, the strain defective in both gndL and sldA (double mutant) still showed activity with GA. Moreover, 2KGA production was still observed in gndL and double mutant strains. 5KGA production was not observed at all in sldA and double mutant strains. Thus, it seems that ‘G. dioxyacetonicus’ IFO 3271 has another membrane-bound enzyme that reacts with GA, producing 2KGA. [ABSTRACT FROM AUTHOR]

Abstract: PqqC/D converts the biosynthetic intermediate purified from a pqqC mutant to pyrroloquinoline quinone (PQQ), and both NAD(P)H and cytosolic fraction, named as activating factor (ActF), are required to show its higher production. Dithiothreitol alone, as well as ActF plus NAD(P)H, enhanced the PQQ production by PqqC/D. Thioredoxin–thioredoxin reductase system with NADPH showed similar effect. PqqC/D made a tight complex with PQQ, however, in the presence of dithiothreitol, PQQ was dissociated from the protein. ActF showed NADPH oxidase activity which was enhanced by the addition of PQQ. These data suggest that PqqC/D produces the reduced PQQ from the intermediate in vivo, but in vitro, it is further oxidized by molecular oxygen and then the oxidized PQQ is trapped in PqqC/D to show product inhibition. [Copyright &y& Elsevier]

The article discusses the study about the conversion of capsular polysaccharide (CPS) to extracellular polysaccharide (EPS) by galE deletion in acetobacter tropicalis. A clone of the galE gene from Acetobacter tropicalis was performed. Results show that the cloned gene does not produce CPS, but secreted an EPS, which is purified and having a unique characteristic from the original CPS.

The article discusses the results of a study which examined membrane-bound NAD(P)-independent formaldehyde-oxidizing enzyme from Acetobacter sp. SKU 14. The enzyme was solubilized from the membrane fraction of glycerol-grown cells and was purified to homogeneity through column chromatographies. The purified enzyme was characterized as a quinoprotein alcohol dehydrogenase.

The article discusses the findings of a study of the characterization and spontaneous mutation of a novel gene, polE, involved in pellicle formation in Acetobacter tropicalis SKU1 100. The study established that Acetobacter tropicalis SKU1100 produces a pellicle polysaccharide which consists of galactose, glucose and rhamnose. The strain forms two types of colony on agar plates, a rough-surfaced colony and a mucoid smooth-surfaced colony.

Depending on the alcohols used as growth substrates, Pseudomonas putida HK5 produces two distinct quinohemoprotein alcohol dehydrogenases, ADH-IIB and ADH-IIG, both of which contain pyrroloquinoline quinone (PQQ) and heme c as the prosthetic groups but show different substrate specificities, especially for diol substrates. Molecular cloning of the gene of ADH-IIB and its crystal structure are already reported. Here, molecular cloning of the gene, qgdA, and solution of the three-dimensional structure of ADH-IIG are reported. The enzyme consists of 718 amino acid residues including a signal sequence of 29 amino acid residues. The PQQ domain is highly homologous to other quinoproteins, especially to quinohemoproteins. The crystal structure of ADH-IIG, determined at 2.2Å resolution, shows that the overall structure and the amino acid residues involved in PQQ binding are quite similar to ADH-IIB and to another quinohemoprotein ADH, qhEDH from Comamonas testosteroni. However, the lengths of the linker regions connecting the PQQ and the cytochrome domains are different from each other, leading to a significant difference in orientation of the cytochrome domain with respect to the PQQ domain. Apart from ADH-IIB and qhEDH, ADH-IIG has an extra 12-residue helix within loop 3 in the PQQ domain and an extra 310 helix in the C terminus of the cytochrome domain, and both helices appear parallel and linked by a hydrogen bond. The amino acid residues contacting substrate/product in the crystal structures are also different among them. In the crystal structure of ADH-IIG with 1,2-propanediol, one of the hydroxyl groups of the substrate forms a hydrogen bond with O5 of PQQ and OD1 of Asp300, and the other interacts with a water molecule and with NE2 of Trp386, the corresponding residue of which is not found in ADH-IIB and qhEDH, and might be the residue responsible for making ADH-IIG prefer diol substrates. [Copyright &y& Elsevier]

Examines the molecular properties of membrane-bound FAD-containing D-sorbitol dehydrogenase (SLDH) from thermotolerant Gluconobacter frateurii, an acetic acid bacteria. Industrial application of Gluconobacter strains; Role of SLDH in L-sorbose fermentation; Method used for the purification of FAD-SLDH from Gluconobacter frateurii.

Cites a study presenting the purification of type II NADH dehydrogenase of Corynebacterium glutamicum (NDH-2) from an ndh over-producing strain. Enrichment of NDH-2 by 6-fold higher specific activity of NADH after purification; Ability of the purified enzyme to catalyze electron transfer from NADH and NADPH to oxygen as well as various artificial quinone analogs; Presentation of C. glutamicum NDH-2 as a source of the reactive oxygen species, superoxide and hydrogen peroxide, concominant with NADH and NADPH oxidation.