Identification of Enzymes from Pseudogluconobacter saccharoketogenes Producing d-Glucaric Acid from d-Glucose.

In 2004, the US Department of Energy listed d-glucaric acid as one of the top 12 bio-based chemicals and a potential biopolymer building block. In this study, we show that Pseudogluconobacter saccharoketogenes strains can produce d-glucaric acid from d-glucose, although in low yield because of the generation of the byproduct 2-keto-d-gluconic acid in large quantities. To improve d-glucaric acid yield, we generated Rh47-3, a P. saccharoketogenes IFO14464 mutant, which produced d-glucaric acid from d-gluconic acid and d-glucose with 81 and 53 mol% yields, respectively. Furthermore, the key enzymes involved in d-glucaric acid production, alcohol dehydrogenase (Ps-ADH), aldehyde dehydrogenase (Ps-ALDH), and gluconate 2-dehydrogenase (Ps-GADH), were purified and their roles in d-glucaric acid synthesis were evaluated. Ps-ADH and Ps-ALDH catalyzed d-glucaric acid production, which was mediated by d-gluconic acid and d-glucuronic acid pathways. In contrast, Ps-GADH inhibited d-glucaric acid production by promoting the formation of 2-keto-d-gluconic acid from d-glucose.

Oxidation of isomaltose, gentiobiose, and melibiose by membrane-bound quinoprotein glucose dehydrogenase from acetic acid bacteria.

Membrane-bound quinoprotein glucose dehydrogenase from acetic acid bacteria produces lactobionic acid by the oxidation of lactose. Its enzymatic activity on lactose and maltose is much lower than that on D-glucose. For that reason, the activity of the enzyme on disaccharides has been considered low. In this study, we show that the isomaltose-oxidizing activity of acetic acid bacteria is much higher than their lactose-oxidizing activity. In addition to isomaltose, the enzyme oxidized gentiobiose and melibiose to the same extent. According to the characteristics of the isomaltose-oxidizing activity and investigations using dehydrogenase-deficient mutant bacteria, we identified the responsible enzyme as membrane-bound quinoprotein glucose dehydrogenase.Abbreviations: AAB: acetic acid bacteria; m-GDH: membrane-bound quinoprotein glucose dehydrogenase; DCIP: 2,6-dichlorophenolindophenol; DP: degree of polymerization; HPAEC-PAD: high-performance anion-exchange chromatography with pulsed amperometric detection; NMR: nuclear magnetic resonance; TLC: thin layer chromatography; COSY: correlation spectroscopy.

Identifying membrane-bound quinoprotein glucose dehydrogenase from acetic acid bacteria that produce lactobionic and cellobionic acids.

Acetic acid bacteria are used in the commercial production of lactobionic acid (LacA). However, the lactose-oxidizing enzyme of these bacteria remains unidentified. Lactose-oxidizing activity has been detected in bacterial membrane fractions and is strongly inhibited by d-glucose, suggesting that the enzyme was a membrane-bound quinoprotein glucose dehydrogenase, but these dehydrogenases have been reported to be incapable of oxidizing lactose. Thus, we generated m-GDH-overexpressing and -deficient strains of Komagataeibacter medellinensis NBRC3288 and investigated their lactose-oxidizing activities. Whereas the overexpressing variants produced ~2-5-fold higher amounts of LacA than the wild-type strains, the deficient variant produced no LacA or d-gluconic acid. Our results indicate that the lactose-oxidizing enzyme from acetic acid bacteria is membrane-bound quinoprotein glucose dehydrogenase. Abbreviations: LacA: lactobionic acid; AAB: acetic acid bacterium; m-GDH: membrane-bound quinoprotein glucose dehydrogenase; DCIP: 2,6-dichlorophenolindophenol; HPAEC-PAD: high-performance anion-exchange chromatography with pulsed amperometric detection.

Lactobionic and cellobionic acid production profiles of the resting cells of acetic acid bacteria.

Lactobionic acid was produced by acetic acid bacteria to oxidize lactose. Gluconobacter spp. and Gluconacetobacter spp. showed higher lactose-oxidizing activities than Acetobacter spp. Gluconobacter frateurii NBRC3285 produced the highest amount of lactobionic acid per cell, among the strains tested. This bacterium assimilated neither lactose nor lactobionic acid. At high lactose concentration (30%), resting cells of the bacterium showed sufficient oxidizing activity for efficient production of lactobionic acid. These properties may contribute to industrial production of lactobionic acid by the bacterium. The bacterium showed higher oxidizing activity on cellobiose than that on lactose and produced cellobionic acid.

Identification of Pathways for Production of D-Glucaric Acid by Pseudogluconobacter saccharoketogenes.

Pseudogluconobacter saccharoketogenes produces glucaric acid from D-glucose via two pathways, i.e., through D-glucuronic acid or D-gluconic acid. These pathways are catalyzed by alcohol dehydrogenase, aldehyde dehydrogenase, and gluconate dehydrogenase. Although D-glucaraldehyde and L-guluronic acid are also theorized to be produced in pathways throsugh D-glucuronic acid and D-gluconic acid, respectively, no direct data to identify these intermediates have been reported. In this study, the intermediates were purified and identified as D-glucaraldehyde and L-guluronic acid. The substrate specificities of the three enzymes on these intermediates and their oxidation products were studied, and the roles of alcohol, aldehyde, and gluconate dehydrogenases in D-glucaric acid-producing pathways were elucidated using the intermediates. Additionally, the substrate specificities of alcohol and aldehyde dehydrogenases on some alcohols, aldehydes, and aldoses were determined. Alcohol dehydrogenase showed wide substrate specificities, whereas the substrates oxidized by aldehyde dehydrogenase were limited. A 30-L scale reaction using the resting cells of Rh47-3 revealed that D-glucaric acid was produced from D-glucose and D-gluconic acid in 60.3 mol% (7.0 g/L) and 78.6 mol% (22.5 g/L) yields, respectively.

Optimization of lactobionic acid production by Acetobacter orientalis isolated from Caucasian fermented milk, "Caspian Sea yogurt".

We have reported that lactobionic acid is produced from lactose by Acetobacter orientalis in traditional Caucasian fermented milk. To maximize the application of lactobionic acid, we investigated favorable conditions for the preparation of resting A. orientalis cells and lactose oxidation. The resting cells, prepared under the most favorable conditions, effectively oxidized 2-10% lactose at 97.2 to 99.7 mol % yield.

Deracemization of 1-phenylethanols in a one-pot process combining Mn-driven oxidation with enzymatic reduction utilizing a compartmentalization technique.

Racemic 1-phenylethanols were converted into enantiopure (R)-1-phenylethanols via a chemoenzymatic process in which manganese oxide driven oxidation was coupled with enzymatic biotransformation by compartmentalization of the reactions, although the two reactions conducted under mixed conditions are not compatible due to enzyme deactivation by Mn ions. Achiral 1-phenylethanol is oxidized to produce acetophenone in the interior chamber of a polydimethylsiloxane thimble. The acetophenone passes through the membrane into the exterior chamber where enantioselective biotransformation takes place to produce (R)-1-phenylethanol with an enantioselectivity of >99% ee and with 96% yield. The developed sequential reaction could be applied to the deracemization of a wide range of methyl- and chloro-substituted 1-phenylethanols (up to 93%, >99% ee). In addition, this method was applied to the selective hydroxylation of ethylbenzene to afford chiral 1-phenylethanol.

Purification and characterization of a carbohydrate: acceptor oxidoreductase from Paraconiothyrium sp. that produces lactobionic acid efficiently.

A carbohydrate:acceptor oxidoreductase from Paraconiothyrium sp. was purified and characterized. The enzyme efficiently oxidized beta-(1-->4) linked sugars, such as lactose, xylobiose, and cellooligosaccharides. The enzyme also oxidized maltooligosaccharides, D-glucose, D-xylose, D-galactose, L-arabinose, and 6-deoxy-D-glucose. It specifically oxidized the beta-anomer of lactose. Molecular oxygen and 2,6-dichlorophenol indophenol were reduced by the enzyme as electron acceptors. The Paraconiothyrium enzyme was identified as a carbohydrate:acceptor oxidoreductase according to its specificity for electron donors and acceptors, and its molecular properties, as well as the N-terminal amino acid sequence. Further comparison of the amino acid sequences of lactose oxidizing enzymes indicated that carbohydrate:acceptor oxidoreductases belong to the same group as glucooligosaccharide oxidase, while they differ from cellobiose dehydrogenases and cellobiose:quinone oxidoreductases.

Preparation of branched cyclomaltoheptaose with 3-O-α-L-fucopyranosyl-α-D-mannopyranose and changes in fucosylation of HCT116 cells treated with the fucose-modified cyclomaltoheptaose.

From a mixture of 4-nitrophenyl α-L-fucopyranoside and D-mannopyranose, 3-O-α-L-fucopyranosyl-D-mannopyranose was synthesised through the transferring action of α-fucosidase (Sumizyme PHY). 6(I),6(IV)-Di-O-(3-O-α-L-fucopyranosyl-α-D-mannopyranosyl)-cyclomaltoheptaose {8, 6(I),6(IV)-di-O-[α-L-Fuc-(1→3)-α-D-Man]-ßCD} was chemically synthesised using the trichloroacetimidate method. The structures were confirmed by MS and NMR spectroscopy. A cell-based assay using the fucosyl ßCD derivatives, including the newly synthesised 8, showed that derivatives with two branches of the α-L-Fuc or α-L-Fuc-(1→3)-α-D-Man residues possessed slight growth-promoting effects and lower toxicity in HCT116 cells compared to those with one branch. These compounds may be useful as drug carriers in targeted drug delivery systems.

Cyclic GMP acts as a common regulator for the transcriptional activation of the flavonoid biosynthetic pathway in soybean.

Cyclic GMP (cGMP) is an important signaling molecule that controls a range of cellular functions. So far, however, only a few genes have been found to be regulated by cGMP in higher plants. We investigated the cGMP-responsiveness of several genes encoding flavonoid-biosynthetic enzymes in soybean (Glycine max L.) involved in legume-specific isoflavone, phytoalexin and anthocyanin biosynthesis, such as phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, 4-coumarate:CoA ligase, chalcone synthase, chalcone reductase, chalcone isomerase, 2-hydroxyisoflavanone synthase, 2-hydroxyisoflavanone dehydratase, anthocyanidin synthase, UDP-glucose:isoflavone 7-O-glucosyltransferase, and isoflavone reductase, and found that the majority of these genes were induced by cGMP but not by cAMP. All cGMP-induced genes were also stimulated by sodium nitroprusside (SNP), a nitric oxide (NO) donor, and illumination of cultured cells with white light. The NO-dependent induction of these genes was blocked by 6-anilino-5,8-quinolinedione, an inhibitor of guanylyl cyclase. Moreover, cGMP levels in cultured cells were transiently increased by SNP. Consistent with the increases of these transcripts, the accumulation of anthocyanin in response to cGMP, NO, and white light was observed. The treatment of soybean cotyledons with SNP resulted in a high accumulation of isoflavones such as daidzein and genistein. Loss- and gain-of-function experiments with the promoter of chalcone reductase gene indicated the Unit I-independent activation of gene expression by cGMP. Together, these results suggest that cGMP acts as a second messenger to activate the expression of genes for enzymes involved in the flavonoid biosynthetic pathway in soybean.

Characterization of dimers of hydroquinone glucosides produced by peroxidase-catalyzed polymerization.

4'-Hydroxyphenyl alpha-glucoside and 4'-hydroxyphenyl beta-glucoside were polymerized with horseradish peroxidase. The isolated dimers were found to have linkages at C3' of the hydroxyphenyl moieties and proved to be fluorescent. Low accumulation of oligomers was attributed to increasing electrochemical reactivity with polymerization degrees, which were expected from the levels of highest occupied molecular orbital.

Purification and characterization of a novel alpha-glucuronidase from Aspergillus niger specific for O-alpha-D-glucosyluronic acid alpha-D-glucosiduronic acid.

A new alpha-glucuronidase that specifically hydrolyzed O-alpha-D-glucosyluronic acid alpha-D-glucosiduronic acid (trehalose dicarboxylate, TreDC) was purified from a commercial enzyme preparation from Aspergillus niger, and its properties were examined. The enzyme did not degrade O-alpha-D-glucosyluronic acid alpha-D-glucoside, O-alpha-D-glucosyluronic acid beta-D-glucosiduronic acid, O-alpha-D-glucosyluronic acid-(1-->2)-beta-D-fructosiduronic acid, p-nitrophenyl-O-alpha-D-glucosiduronic acid, methyl-O-alpha-D-glucosiduronic acid, or 6-O-alpha-(4-O-alpha-D-glucosyluronic acid)-D-glucosyl-beta-cyclodextrine. Furthermore, it showed no activity on alpha-glucuronyl linkages of 4-O-methyl-D-glucosyluronic acid-alpha-(1-->2)-xylooligosaccharides, derived from xylan, a supposed substrate of alpha-glucuronidases.The molecular mass of the enzyme was estimated to be 120 kDa by gel filtration and 58 kDa by SDS-PAGE suggesting, the enzyme is composed of two identical subunits. It was most active at pH 3.0-3.5 and at 40 degrees C. It was stable in pH 2.0-4.5 and below 30 degrees C. It hydrolyzed O-alpha-D-glucosyluronic acid alpha-D-glucosiduronic acid to produce alpha- and beta-anomers of D-glucuronic acid in an equimolar ratio. This result suggests that inversion of the anomeric configuration of the substrate is involved in the hydrolysis mechanism.