UDP-N-Acetylglucosamine 4-epimerase (EC 5.1.3.7) was purified from a cell extract of Bacillus subtilis by protamine sulfate treatment, ammonium sulfate fractionation, and column chromatographies on DEAE-Sephadex, hydroxylapatite, and Sephadex G-150. The purified enzyme was homogeneous on disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 62, 000 by gel filtration. The enzyme had an optimal pH in the range of 7.0 to 9.0, and was active at 30-35°C in the absence of added NAD and more active at 40-45°C in the presence of NAD. The enzyme activity was highly stimulated by small amounts of NAD. The estimated Km values were 4.4mM for UDP-N-acetylglucosamine and 0.13mM for NAD. No UDP-glucose 4-epimerase activity was found in the purified enzyme. The enzyme was inhibited by high concentrations of glucosamine and inhibition by glucosamine was competitive with UDP-N-acetylglucosamine. The inhibition constant was 37.0mM. N-Acetylglucosamine did not inhibit the enzyme.
Metabolites from salicylic acid by microorganisms were investigated. About eighty strains of bacteria which were able to utilize salicylic acid as a sole source of carbon were isolated from soil and other natural sources. Among these bacteria, several strains produced a large amount of keto acids in the culture fluid during the cultivation. The acid was isolated from the culture fluid of strain K 102 in crystalline form. The crystal was identified as α-ketoglutaric acid by physicochemical methods. From the taxonomical studies, the isolated bacterial strains K 102 and K 362 were assumed to be Pseudomonas sp.
Journal Article Elucidation of the Role of Sugar Chains in Glycosylated-enzymes Using Endo-γ-N-acetylglu-cosaminidase from Flavo-bacterium sp. Get access Kenji Yamamoto, Kenji Yamamoto Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan Search for other works by this author on: Oxford Academic Google Scholar Kaoru Takegawa, Kaoru Takegawa Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan Search for other works by this author on: Oxford Academic Google Scholar Hidehiko Kumagai, Hidehiko Kumagai Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan Search for other works by this author on: Oxford Academic Google Scholar Tatsurokuro Tochikura Tatsurokuro Tochikura Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan Search for other works by this author on: Oxford Academic Google Scholar Agricultural and Biological Chemistry, Volume 50, Issue 8, 1 August 1986, Pages 2167–2169, https://doi.org/10.1080/00021369.1986.10867721 Published: 01 August 1986 Article history Received: 06 May 1986 Published: 01 August 1986
1) The production of α-ketoglutaric acid by the washed cells of coli-aerogenes from glucose was observed to be inhibited in the presence of αα'-dipyridyl. Moreover, it was found that both αα'-dipyridyl and O-phenanthroline extremely inhibited the bacterial synthesis of α-ketoglutaric acid from pyruvate, while the consumption of pyruvic acid was not greatly affected and acetic acid or acetoine could be produced even in the presence of these inhibitors. It was thus concluded that inorganic iron would be a kind of cofactor of the enzyme system relating to the synthesis of α-ketoglutaric acid from pyruvate, since these inhibiting actions could be reduced by the addition of inorganic iron such as FeSO4. 2) It was demonstrated that the enzyme preparations such as dried cells and cell-free extracts revealed the ability of synthesizing α-ketoglutaric acid from pyruvate. 3) Lactic acid was found to be a potent precursor in the production of α-ketoglutaric acid by either the washed cells or the growing culture, and the same inhibiting action was again observed by the reagents forming Fe-complex compound. 4) The most presumable mechanism for the synthesis of α-ketoglutaric acid from C3-compound such as pyruvic and lactic acids was discussed.
Two types of α-L-fucosidase (F-I and F-II), that differ in substrate specificity, were produced in the culture fluid by Bacillus circulans isolated from soil when the bacterium was cultivated on medium containing porcine gastric mucin. F-I was able to cleave the α-(1→2), α-(1→3) and α-(1→4)-L-fucosidic linkages in various oligosaccharides and glyco proteins, but not p-nitrophenyl α-L-fucoside, as previously reported [Y. Tsuji et al. (1990) J. Biochem. 107, 324-330]. F-II was purified from the culture fluid obtained with glucose medium by ammonium sulfate fractionation and various subsequent column chromatogra phies. The purified enzyme was found to be homogeneous on PAGE and its molecular weight was estimated to be approximately 250,000. The maximal activity was observed between pH 6.0 to 7.0, the stable pH range being 6.0 to 8.5. The enzyme specifically cleaved a L-fucosidic bonds in low molecular weight substrates. The enzyme cleaved not only p-nitrophenyl α-L-fucOSide, but also 2′-fucosyllactose and 3-fucosyllactose. The enzyme was also able to act on the α-(1→6)-L-fucosidase linkages to N-acetyiglucosamine in 6-O-α L-fucopyranosyl-N-acetylglucosamlne, and bi- and tetrα-antennary oligosaceharides derived from porcine pancreatic lipase, which were not hydrolyzed by F-I.
The optimum condition for the formation of CDP-choline was studied: (1) the reaction proceeded more effectively at 35°C than at 28 or 40°C. (2) the maximum formation of CDP-choline was obtained at pH 7.5, when pH levels were kept constant throughout the reaction. (3) twenty #x03BC;moles per ml of 5′-CMP was the optimum concentration for the formation of CDP-choline. When higher concentration of 5′-CMP was employed, the substrate was decomposed to uridine, uracil, etc., and the yield of CDP-choline decreased. By the application of feeding method, 5′-CMP was utilized to the effective formation of CDP-choline without further formation of side-products.
