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Uracil-thymine dehydrogenase (UTDH), which catalyzes the irreversible oxidation of uracil to barbituric acid in oxidative pyrimidine metabolism, was purified from Rhodococcus erythropolis JCM 3132. The finding of unusual stabilizing conditions (pH 11, in the presence of NADP+ or NADPH) enabled the enzyme purification. The purified enzyme was a heteromer consisting of three different subunits. The enzyme catalyzed oxidation of uracil to barbituric acid with artificial electron acceptors such as methylene blue, phenazine methosulfate, benzoquinone, and α-naphthoquinone; however, NAD+, NADP+, flavin adenine dinucleotide, and flavin mononucleotide did not serve as electron acceptors. The enzyme acted not only on uracil and thymine but also on 5-halogen-substituted uracil and hydroxypyrimidine (pyrimidone), while dihydropyrimidine, which is an intermediate in reductive pyrimidine metabolism, and purine did not serve as substrates. The activity of UTDH was enhanced by cerium ions, and this activation was observed with all combinations of substrates and electron acceptors. • Uracil-thymine dehydrogenase was found to be a heteromer consisting of three different subunits. • Uracil-thymine dehydrogenase catalyzed oxidation of uracil with artificial electron acceptors. • The activity of uracil-thymine dehydrogenase was enhanced by cerium ions. [ABSTRACT FROM AUTHOR]

Mead acid (MA; 20:3ω9) is one of the ω9 series of polyunsaturated fatty acids (PUFAs). MA is used to inhibit the inflammation of joints and is applied to the medicinal or health food field. We aimed to construct MA-producing strains with disruption of the Δ12-desaturase gene (Δ1 2ds) via an efficient gene-targeting system using the lig4 -disrupted strain of Mortierella alpina 1S-4 as the host. The transformants showed a unique fatty acid composition that only comprised ω9-PUFAs and saturated fatty acids, while ω6-and ω3-PUFAs were not detected, and the total composition of ω9-PUFAs, including oleic acid (18:1ω9), 18:2ω9, 20:1ω9, 20:2ω9, and MA, was up to 68.4% of the total fatty acids. The MA production in the Δ1 2ds -disruptant reached 0.10 g/L (8.5%), which exceeded 0.050 g/L (4.6%) in the conventional Δ1 2ds -defective mutant JT-180. [ABSTRACT FROM AUTHOR]

To produce tomato juice with health-promoting functions, lactic acid bacteria (LAB) capable of converting l -glutamic acid in tomatoes into γ-aminobutyric acid (GABA) was screened from LAB stocks isolated from Japanese pickles. Lactiplantibacillus plantarum KB1253 was selected as the highest GABA producer among 74 strains of LAB stocks. gad gene expression and glutamic acid decarboxylation activity increased at low pH (3.0–3.5), whereas the growth decreased. Under optimal reaction conditions using resting cells as catalysts, this strain produced 245.8 ± 3.4 mM GABA. Furthermore, this strain produced 41.0 ± 1.1 mM GABA from l -glutamic acid in tomato juice under optimal fermentation conditions (pH 4.0, 20°Bx). This study may provide the basis for developing health-promoting functional foods rich in GABA from tomatoes and other agricultural products. [ABSTRACT FROM AUTHOR]

Enzymatic glycosylation is an industrially useful technique for improving the properties of compounds with hydroxy groups, and the biological activities of the resulting glycosides differ depending on the glycosylation position. Therefore, regioselective glycosyltransferases are required for precise synthesis of glycosides. We found that Rhizobium pusense JCM 16209T could catalyze the regioselective glycosylation of resveratrol. To identify the regioselective glycosyltransferase, two α-glucosidases of R. pusense JCM 16209T (RpG I and RpG II) were cloned and expressed in Escherichia coli. The molecular mass of purified recombinant RpG I and II was estimated to be 60 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). RpG I showed strong glycosylation activity toward resveratrol with 4′-selectivity of 98.3%. The enzyme activity was maximized at pH 8.0 and 50 °C, and enhanced in the presence of Cs+ and Li+ ions. The maximum molar yield of resveratrol 4′- O -α-glucoside from resveratrol reached 41.6% at 30 min, and the concentration of the product was 2.08 mmol L−1. Glycosylation activity was observed toward resveratrol as well as toward caffeic acid, ferulic acid, 6-gingerol, flavonoid, and isoflavonoid compounds with high regioselectivity, indicating that RpG I could glycosylate a wide range of substrates. To the best of our knowledge, there are few reports on microbial glycosyltransferases that are useful for regioselective glycosylation. This research could be the first step toward developing technologies for the precise synthesis of glycosides. [ABSTRACT FROM AUTHOR]

A wide variety of S-substituted cysteine derivatives occur in plant metabolites. For example, S -allyl- l -cysteine (SAC), mainly contained in garlic, gathers huge interest because of its favorable bioactivities for human health. However, conventional methods for preparing SAC suffer from several drawbacks with regard to efficiency and toxicity, which highlights the need for improved processes for SAC synthesis. This study aims to develop a novel bioprocess to produce SAC by microbial enzymes from easily available substrates. We found that Escherichia coli had the ability to synthesize SAC from allyl mercaptan, pyruvic acid, and ammonium sulfate. An enzyme purification through 3-step column chromatography, followed by determination of the N-terminal amino acid sequence revealed that tryptophanase (TnaA) was the enzyme responsible for SAC formation. Although the enzyme catalyzed the reversible reaction for synthesizing and degrading SAC, the degradation proceeded significantly faster than the synthesis. Interestingly, TnaA catalyzed the synthesis of a wide range of S-substituted cysteines with alkyl chains or aromatic rings, some of which are present in Allium and Petiveria plants. Our results showed a novel substrate specificity of TnaA toward various S-substituted cysteine. TnaA is a promising biocatalyst for developing a new process to supply various valuable S-substituted cysteine derivatives for medicinal and health-promoting applications. [ABSTRACT FROM AUTHOR]

Quantification of leuco-indigo is most important for Aizome , Japanese indigo-dyeing; however, there has been no convenient quantitative method. This study demonstrated that normal pulse voltammetry under quiescent conditions can be used to detect leuco-indigo. As a result of quantification of leuco-indigo in the depth direction in fermenting suspensions, the steady-state concentrations of leuco-indigo showed sigmoidal profiles in the depth direction. The steady state is caused by competitive reactions of microbial reduction of indigo and autoxidation of leuco-indigo by O 2 dissolved from the air interface of the suspension. In addition, we investigated the effects of stirring the suspension and adding some nutrients to the concentration profile. The weakened activity was partially recovered by the addition of ethanol and remarkably recovered by the addition of hipolypepton or glucose. Knowledge is essential for the proper management of indigo-dye-fermenting suspensions. [ABSTRACT FROM AUTHOR]

ω3-Docosapentaenoic acid (ω3-DPA), an ω3-polyunsaturated fatty acid (ω3-PUFA), is expected to have beneficial physiological functions to humans; however, because of its rarity in nature, it has not been fully analyzed. We isolated an ω3-DPA producing microorganism strain T7 from brackish areas in Japan. Although most oleaginous microorganisms rarely accumulate ω3-DPA (<5% of total lipid), strain T7 accumulated ω3-DPA with more than 20% of total fatty acids. The strain T7 was identified as a related species of Aurantiochytrium. In Aurantiochytrium sp. T7, ω3-DPA production reached 164 mg/L culture broth, and the ω3-DPA content reached 23.5% of the total fatty acids when cultivated in a medium containing 2% glucose as the carbon source and 1% yeast extract as the nitrogen source, with a salinity equivalent to 50% of that of seawater and a pH in the acidic range (pH < 5.5). Aurantiochytrium sp. T7 is a promising producer of high-purity ω3-DPA containing-lipid for the functional analysis of ω3-DPA whose physiological function has hardly been elucidated, and a useful strain for investigating the novel metabolic pathway of fatty acids. [ABSTRACT FROM AUTHOR]

Cyclic voltammetry was successfully applied to in-vivo monitoring of leuco-indigo in indigo-fermenting suspensions under quiescent conditions without deoxygenation; the working and counter electrodes were kept on the surface of each suspension by a polyethylene vinyl alcohol tube holder. The anodic peak current was used as a measure of the leuco-indigo concentration. The voltammetric wave shape suggested partial solubilization of the indigo with some macromolecules in the fermenting suspensions, which lead to an in-situ method without any electrode surface pretreatment. The anodic peak current well reflected the dyeing activity of a suspensions. The results obtained for laboratory-level fermentation systems clarified the number of days required for dye fermentation, the effectiveness of addition of old suspension as an additive for preparing fresh fermenting suspensions, and the importance of addition of a nitrogen-based nutrient as well as a glucose-based one to recover the indigo-reducing activity. The method can also be applied to determine the amounts of indigo in used dye suspensions and extracts of fermented indigo leaves (sukumo) by adding a chemical reduction pretreatment. [Display omitted] [ABSTRACT FROM AUTHOR]

The gut bacterium Gordonibacter urolithinfaciens DSM 27213 metabolizes ellagic acid into three polyphenol compounds, namely, urolithin M5, urolithin M6, and urolithin C, which are collectively called urolithin. The key reactions of this metabolic pathway are the dehydroxylation of the phenolic hydroxy group, i.e., conversion of urolithin M5 to urolithin M6, and successive conversion of urolithin M6 to urolithin C. By testing the effects of various electron-transferring compounds on the dehydroxylation reactions, methylviologen was found to effectively support the dehydroxylation catalyzed by the cell free extracts. The urolithin dehydroxylating enzymes were found in the soluble fraction of the cell free extracts. The urolithin dehydroxylation was found to be coupled with reduction of dicationic methylviologen to a cation radical form catalyzed by enzymes with hydrogen as an electron donor, which was also found with the soluble fraction. Further investigation of the reaction in the presence of natural cofactors with or without methylviologen and hydrogen revealed the involvement of NADPH and FAD in the electron transportation systems of the urolithin dehydroxylation. [ABSTRACT FROM AUTHOR]

Nitrobacter winogradskyi is an abundant, intensively studied autotrophic nitrite-oxidizing bacterium, which is frequently used as a model strain in the two-step nitrification of ammonia (NH 3) to nitrate (NO 3 −) via nitrite (NO 2 −), either in activated sludge, agricultural field studies or more recently in artificial microbial consortia for organic hydroponics. We observed a hitherto unknown cobalt ion-dependent inhibition of cell growth and NO 2 − oxidation activity of N. winogradskyi in a mineral medium, which strongly depended on accompanying Ca2+ and Mg2+ concentrations. This inhibition was bacteriostatic, but susceptible to natural chelators. l- Histidine effectively restored cell growth and NO 2 − oxidation activity of N. winogradskyi in mineral media containing Co2+ with >90% recovery. Our results suggest that Co2+ competed with alkaline earth metals during uptake and that its toxicity was significantly reduced by complexation. Image 1 [ABSTRACT FROM AUTHOR]

Microorganisms with medium-chain alkane-producing activity are promising for the bio-production of drop-in fuel. In this study, we screened for microorganisms producing tridecane from tetradecanal. The activity of aldehyde decarbonylation was found in a wide range of microbes. In particular, the genus Klebsiella in the Enterobacteriaceae family was found to have a high ability to produce alkanes from aldehydes via enzyme catalyzed reaction. [ABSTRACT FROM AUTHOR]

Microorganisms were screened for transribosylation activity between 2′- O -methyluridine (2′-OMe-UR) and nucleobases, for the purpose of developing a biotransformation process to synthesize 2′- O -methylribonucleosides (2′-OMe-NRs), which are raw materials for nucleic acid drugs. An actinomycete, Agromyces sp. MM-1 was found to produce 2′- O -methyladenosine (2′-OMe-AR) when whole cells were used in a reaction mixture containing 2′-OMe-UR and adenine. The enzyme responsible for the transribosylation was partially purified from Agromyces sp. MM-1 cells through a six-step separation procedure, and identified as a nucleoside hydrolase family enzyme termed Ag NH. Ag NH was a bi-functional enzyme catalyzing both hydrolysis towards 2′-OMe-NRs and transribosylation between 2′-OMe-UR and various nucleobases as well as adenine. In the hydrolysis reaction, Ag NH preferred guanosine analogues as its substrates. In the transribosylation reaction, Ag NH showed strong activity towards 6-chloroguanine, with 25-fold relative activity when adenine was used as the acceptor substrate. The transribosylation reaction product from 2′-OMe-UR and 6-chloroguanine was determined to 2′- O -methyl-6-chloroguanosine (2′-OMe-6ClGR). Under the optimal conditions, the maximum molar yield of 2′-OMe-6ClGR reached 2.3% in a 293-h reaction, corresponding to 440 mg/L. [ABSTRACT FROM AUTHOR]

2′- O -Methylribonucleosides (2′-OMe-NRs) are promising raw materials for the production of nucleic acid drugs. We previously reported that Lb NH, a nucleoside hydrolase from Lactobacillus buchneri LBK78 (NITE P-01581), was the first enzyme found to act on 2′-OMe-NRs. In the present study, we determined that Lb NH also has the transribosylation activity between 2′-OMe-NRs and nucleobases, in addition to the hydrolyzing activity towards 2′-OMe-NRs. When 2′- O -methyluridine (2′-OMe-UR) and adenine were reacted with Lb NH, 2′- O -methyladenosine (2′-OMe-AR) was produced. Lb NH preferred purine nucleobases as its acceptor substrates for the transribosylation with 2′-OMe-UR as a donor substrate. Kinetic analysis of Lb NH revealed that adenine behaved as a mixed inhibitor of the hydrolysis of 2′-OMe-UR. Under the optimal reaction conditions, the maximum molar yield of enzymatic 2′-OMe-AR produced reached 0.97% towards 2′-OMe-UR, corresponding to 0.16 g/L. [ABSTRACT FROM AUTHOR]

We constructed dihomo-γ-linolenic acid (DGLA)-producing strains with disruption of the Δ5-desaturase ( Δ5ds ) gene, which encodes a key enzyme catalyzing the bioconversion of DGLA to arachidonic acid (ARA), by efficient gene-targeting, using Δlig4 strain of Mortierella alpina 1S-4 as the host. In previous study, we had already identified and disrupted the lig4 gene encoding DNA ligase 4, which involves in non-homologous end joining, in M. alpina 1S-4, and the Δlig4 strain had showed efficient gene-targeting. In this study, the uracil auxotroph of Δlig4 strain was constructed, and then transformed for disruption of Δ5ds . The isolation of nine Δ5ds -disruptants out of 18 isolates indicated that the disruption efficiency was 50%. The ratio of DGLA among the total fatty acids of the Δ5ds -disruptants reached 40.1%; however, no ARA was detected. To our knowledge, this is the first study to report the construction of DGLA-producing transformants by using the efficient gene-targeting system in M. alpina 1S-4. [ABSTRACT FROM AUTHOR]

We investigated the omega-3 eicosatetraenoic acid (ETA) production by molecular breeding of the oleaginous fungus Mortierella alpina , which can slightly accumulate ETA only when cultivated at a low temperature. The endogenous ω3-desaturase gene or the heterologous Saprolegnia diclina Δ17 ( sdd17m ) desaturase gene were overexpressed in M. alpina S14, a Δ5-desaturation activity-defective mutant derived from M. alpina 1S-4. M. alpina S14 transformants introduced with the endogenous ω3-desaturase gene showed ETA at 42.1% content in the total lipids that was 84.2-fold and 3.2-fold higher than that of the wild-type strain 1S-4 and host strain S14, respectively, when cultivated at 12°C. No accumulation of ETA was observed at 28°C. In contrast, transformants with the heterologous sdd17m gene showed 24.9% of the content of total lipids at 28°C. These results indicated that these M. alpina S14 transformants are promising strains for the production of ETA, which is hard to obtain from natural sources. [ABSTRACT FROM AUTHOR]

Linoleic acid Δ9 hydratase, which is involved in linoleic acid saturation metabolism of Lactobacillus plantarum AKU 1009a, was cloned, expressed as a his-tagged recombinant enzyme, purified with an affinity column, and characterized. The enzyme required FAD as a cofactor and its activity was enhanced by NADH. The maximal activities for the hydration of linoleic acid and for the dehydration of 10-hydroxy- cis -12-octadecenoic acid (HYA) were observed at 37 °C in buffer at pH 5.5 containing 0.5 M NaCl. Free C16 and C18 fatty acids with cis -9 double bonds and 10-hydroxy fatty acids served as substrates for the hydration and dehydration reactions, respectively. The apparent K m value for linoleic acid was estimated to be 92 μM, with a k cat of 2.6∙10 −2 s −1 and a Hill factor of 3.3. The apparent K m value for HYA was estimated to be 98 μM, with a k cat of 1.2∙10 −3 s −1 . [ABSTRACT FROM AUTHOR]

We found a new aldehyde oxidase (ALOD), which catalyzes the conversion of glycolaldehyde to glycolate, from Burkholderia sp. AIU 129. The enzyme further oxidized aliphatic aldehydes, an aromatic aldehyde, and glyoxal, but not glycolate or alcohols. The molecular mass of this enzyme was 130 kDa, and it was composed of three different subunits (αβγ structure), in which the α, β, and γ subunits were 76 kDa, 36 kDa, and 14 kDa, respectively. The N-terminal amino acid sequences of each subunit showed high similarity to those of putative subunits of xanthine dehydrogenase. Metals (copper, iron and molybdenum) and chelating reagents (α,α′-dipyridyl and 8-hydroxyquinoline) inhibited the ALOD activity. The ALOD showed highest activity at pH 6.0 and 50°C. Twenty mM glycolaldehyde was completely converted to glycolate by incubation at 30°C for 3 h, suggesting that the ALOD found in this study would be useful for enzymatic production of glycolate. [ABSTRACT FROM AUTHOR]

Trehalose confers protection against various environmental stresses on yeast cells. In this study, trehalase gene deletion mutants that accumulate trehalose at high levels showed significant stress tolerance to acetic acid. The enhancement of trehalose accumulation can thus be considered a target in the breeding of acetic acid-tolerant yeast strains. [ABSTRACT FROM AUTHOR]