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With the crystalline preparation of alcohol oxidase of Kloeckera sp. No. 2201 grown on methanol as a sole carbon source, some properties of the enzyme were investigated. The visible absorption spectrum showed maxima at 373mƒÊ, 392mlƒÊ and 461 mƒÊ which were bleached by an addition of substrate and were restored by oxygenation. The pro sthetic group was identified to be FAD. The enzyme was unstable in acidic pH and higher temperature. The optima of pH and temperature for enzyme activity were pH 8.0 and 35•Ž, respectively. The Km values were 1.25•~l0-3M for methanol and 2.5 •10-3M for ethanol. The enzyme oxidized lower primary alcohols to the corresponding al dehydes and hydrogen peroxide.

Glutathione is synthesized from L-cysteine and glycine via the sequential enzymereactions catalyzed by y-glutamylcysteine synthetase (GSH-I) (EC 6.3.2.2) and glutathione synthetase (GSH-II) (EC.6.3.2.3) [Schemes (i) andAs shownpreviously,1* we have succeeded in the cloning of a gene (gsh I) for GSH-I and showed that the E. coli B strain having the hybrid plasmid with this gene was useful for the production of a large amount of glutathione. However, as a result of the increase in GSH-I activity* the reaction catalyzed by GSH-II seemed to be a rate limiting step in the glutathione production (Table I).To overcome this disadvantage in glutathione production and to construct a more potent glutathione producer, we isolated the gene (gsh II) for GSH-II and cloned it onto vector plasmid pBR322. This article deals with the cloning of the gene for GSH-II activity and its expression in E. coli B cells. Table I. Glutathione Content and GSH-I and -II Activities in Various E. coli B Cells Dosed with gsh I or gsh II Gene a The genotypic properties of these strains were described in our previous papers.2'3* b Glutathione content (mg/g wet wt. cells) was determined by the method previously described.4) c Activities of GSH-Iand GSH-II were determined by the method previously described.3) Activities are expressed as /zmol/h/mg-protein.