Tatsurokuro Tochikura

Purification and properties of glutaminase from Aspergillus oryzae

Abstract Glutaminase activity was found in a water extract of a wheat bran koji (extracellular fraction) of Aspergillus oryzae strains Lee-1, H-16 and MA-27-IM isolated from a commercial koji ssed for soy sauce fermentation, as well as in thier mycelia (intracellular fraction). Both the intracellular and the extracellular glutaminases were purified from strain MA-27-IM. Polyacrylamide gel electrophoresis of each purified preparation gave a single band with identical electrophoretic mobility. The molecular weight of the intracellular and the extracellular glutaminases were estimated to be approximately 113, 000. Both preparations hydrolyzed various γ-glutamyl compounds besides l -glutamine but did not exhibit asparaginase activity. Further investigation of these preparations inidicated that these glutaminases possessed almost the same properties, suggesting their similarity.

Molecular cloning of Escherichia coli K-12 ggt and rapid isolation of γ-glutamyltranspeptidase

Abstract Based on the results of mapping of ggt , eight strains were selected from a gene library of E. coli . One of the strains harboring pLC9-12 was found to show 14 times higher γ-glutamyltranspeptidase activity per cell than the wild type strain. The ggt was subcloned to the Bam HI site of pUC18 and the recombinant plasmid sSH101 was obtained. Ggt− phenotype of γ-glutamyltranspeptidase-deficient mutants was complemented by pSH101. The specific activity of the enzyme in cells harboring pSH101 was 37-fold higher than that in the wild type cells. γ-Glutamyltranspeptidase was isolated from the periplasmic fraction of the cells by simple two steps and crystallized.

Elucidation of the role of sugar chains in glucoamylase using endo-β-N-acetylglucosaminidase from Flavobacterium sp.

Abstract Endo-β-N-acetylglucosaminidase (glycopeptide- d -mannosyl-N4-(N-acetyl- d -glucosaminyl)2-asparagine 1,4-N-acetyl-β-glucosaminohydrolase, EC 3.2.1.96), homogenized from the culture filtrate of Flavobacterium sp., could liberate about 50% of the sugar chains from the glucoamylase of Rhizopus niveus. The native and carbohydrate-depleted glucoamylases were compared in their various enzymatic properties. It was found that they were identical in their catalytic activities. However, the carbohydrate-depleted glucoamylase was less thermally stable than the native glucoamylase. Moreover, the carbohydrate-depleted glucoamylase was more sensitive to proteinases such as pronase, subtilisin and trypsin. These results suggest that the sugar chains of the glucoamylase contribute to the high stability of the enzyme. However, circular dichroism spectra of the native and carbohydrate-depleted glucoamylase were found to be identical.

Formation of γ-glutamylglycyglycine by extracellular glutaminase of Aspergillus oryzae

Abstract γ-Glutamylglycylglycine (γ-GluGlyGly) was formed through the γ-glutamyltranspeptidase (GGT) reaction catalyzed by glutaminase in a water extract of wheat bran koji obtained with Aspergillus oryzae MA-27-IM. The yield of γ-GluGlyGly was about 18% from l -glutamine in a reaction mixture containing 50 mM l -glutamine, 50 mM glycylglycine, and the extract (0.1 unit ml as GGT activity) in a 100 mM Tris-HCl buffer solution (pH 7.2), which was incubated for 7 h at 30°C. The γ-GluGlyGly formed was purified by ion exchange chromatographies, and the identified by chemical and enzymatic methods as well as by infrared and PMR spectroscopic analyses.

Mortality of bifidobacteria in boiled yogurt

Abstract Bifidobacterial strains showed various mortalities during storage at 30°C in boiled yogurt prepared with Streptococcus sp. and Lactobacillus sp. Bifidobacterium breve 203 was most stable, maintaining its initial cell number for more than 5 days. It was also stable at 4 or 10°C in fresh yogurt. B. longum 401 rapidly lost viability in the boiled yogurt and was unstable in the fresh yogurt at any temperature. Lactobacillus sp. remained fully viable for one week or more at 4–30°C while Streptococcus sp. lost viability at 20°C or above. The difference in mortality between B. breve 203 and B. longum 401 was mainly due to their sensitivity to the acidic environment, with temperature during storage having a secondary effect. Effects of lysozyme, pepsin and bile acids on the two strains were also investigated.

A rapid assay method for ammonia using glutamine synthetase from glutamate-producing bacteria

A rapid enzymatic assay method for ammonia was developed by using glutamine synthetase from glutamate-producing bacteria together with pyruvate kinase, lactate dehydrogenase, and NADH. The time required for determination of 25 nmol of ammonia was 5 min with 1 unit of glutamine synthetase, as opposed to 14-30 min with 1 unit of glutamate dehydrogenases from various sources. The present method was used to determine ammonia in serum, microbiol-culture broth, and waste water. The method can be modified for spectrophotometry in the visible region by substituting pyruvate oxidase, peroxidase, and appropriate chromogens for lactate dehydrogenase and NADH. With 4-aminoantipyrine (4AA) and phenol, and with 4AA and N-ethyl-N-2-hydroxyethyl-m-toluidine as chromogens, the sensitivity of ammonia determination was 0.65 and 1.7 times that with glutamate dehydrogenase, respectively. The present method was also applicable to the continuous detection of the activity of some ammonia-forming enzymes such as guanase, adenosine deaminase, and urease and to the determination of 0.5-30 microM ATP-ADP after some modification of the mixture.

Nucleotide sequence of the yeast glutathione S-transferase cDNA

The nucleotide sequence (658 bp) of the cDNA coding for glutathione S-transferase Y-2 of yeast Issatchenkia orientalis was obtained. The cDNA clone contains an open reading frame of 570 nucleotides encoding a polypeptide comprising 190 amino acids with a molecular weight of 21,520. The primary amino acid sequence of the enzyme exhibits only 25.0% and 21.1% identity with 177 and 151 amino acid residues of maize glutathione S-transferase I and rat glutathione S-transferase Yb2, respectively.

Utilization of the γ-glutamyltranspeptidase reaction for glutathione synthesis

Abstract This paper describes a new method for the enzymatic production of glutathione. The transpeptidase activity of γ-glutamyltranspeptidase (EC 2.3.2.2) from Escherichia coli K-12 was utilized for the enzymatic production of glutathione analogues ( S -benzyl-glutathione monomethyl ester and S -benzyl-glutathione) from L-glutamine and S -benzyl-L-cysteinylglycine methyl ester. The optimum conditions for the production of each glutathione analogue were determined, and the products were isolated and identified.

Excretion and rapid purification of γ-glutamyltranspeptidase from Escherichia coli K-12

tolA strains of Escherichia coli harboring plasmids carrying the ggt gene excreted about 70% of their γ-glutamyltranspeptidase (GGT) (EC 2.3.2.2) into the medium. The excreted GGT was purified to electrophoretic homogeneity by a simple two-step method with a yield of 51%.

Glutathione S-transferase in yeast: Induction of mRNA, cDNA cloning and expression in Escherichia coli

Glutathione S-transferase Y-2 mRNA synthesis was induced in yeast Issatchenkia orientalis approximately 37-fold by cultivation with o-dinitrobenzene. A cDNA library complementary to poly (A)+RNA of I. orientalis grown with o-dinitrobenzene was screened by colony hybridization. Twenty positive clones were obtained from 6,000 clones and seven of twenty positive clones expressed glutathione S-transferase activity in E. coli. One of the expressing clones harboring plasmid pHT108 had 28 times more glutathione S-transferase activity induced by Isopropyl-beta-D-thio-galactopyranoside than a strain harboring plasmid pUC118. Expressed glutathione S-transferase Y-2 protein comigrated with yeast glutathione S-transferase Y-2 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as detected by immunoblot analysis.