Bioscience, Biotechnology, and Biochemistry

ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20

Secretion of Mono- and Diacylglycerol Lipase from Penicillium camembertii U-150 by Saccharomyces cerevisiae and Site-directed Mutagenesis of the Putative Catalytic Sites of the Lipase Shotaro Yamaguchi, Tamio Mase & Kazuyuki Takeuchi To cite this article: Shotaro Yamaguchi, Tamio Mase & Kazuyuki Takeuchi (1992) Secretion of Mono- and Diacylglycerol Lipase from Penicillium camembertii U-150 by Saccharomyces cerevisiae and Site-directed Mutagenesis of the Putative Catalytic Sites of the Lipase, Bioscience, Biotechnology, and Biochemistry, 56:2, 315-319, DOI: 10.1271/bbb.56.315 To link to this article: http://dx.doi.org/10.1271/bbb.56.315

Published online: 12 Jun 2014.

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Date: 08 June 2016, At: 20:12

Biosci. Biotech. Biochem., 56 (2), 315-319, 1992

Secretion of Mono- and Diacylglycerol Lipase from Penicillium camembert;; U-I50 by Saccharomyces cerevisiae and Site-directed Mutagenesis of the Putative Catalytic Sites of the Lipase Shotaro YAMAGUCHI, Tamio MASE, * and Kazuyuki TAKEUCHI Tsukuba Research Laboratories, Amano Pharmaceutical Co., Ltd., 22 Miyukigaoka, Tsukuba, Ibaraki 305, Japan Amano Pharmaceutical Co. Ltd., Nishiharu, Nishikasugai, Aichi 481, Japan Received September 12, 1991

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*Research and Development Division,

Yeast cells carrying intronless mono- and diacylglycerol lipase (MDGL) genes, constructed by recombination of the genomic gene and cDNA, secreted MDGL into the culture supernatant. Most of the yeast MDGL were extensively glycosylated while they had a similar glyceride specificity to that of native MDGL. Site-directed mutagenesis was used to directly confirm the involvements in enzyme activity of the presumptive amino acid residues to form the catalytic center of MDGL. These residues were conserved in the primary structure alignment of a lipase family from filamentous fungi. Mutant lipase proteins in which Ser 83 , Ser 145 , or His 259 was replaced with glycine were secreted by yeast transformants as inactive proteins. Mutant proteins replacing ASp199 with glycine or asparagine were not detected in the culture supernatant. Replacing other two highly conserved aspartic acids (at positions 232 and 243) with glycine did not render the enzyme inactive. These results indicate that Ser 83 , Ser l4 S, and His 259 in MDGL, are essential to enzyme activity. ASp199 is also likely to be involved.

Lipase (triacylglycerol lipase [Ee 3.1.1.3J) acts on In this paper, we report on the expression of MDGL in water-insoluble substrates such as triacylglycerol at an the yeast Saccharomyces cerevisiae and on site-directed oil-water interface. The finding of lipase reactions· in or- mutagenesis of the putative catalytic sites of MDGL to ganic solvents as well as the broad substrate specificity investigate the relationships between MDGL structure and variety in reaction modes of lipase has created many and triacylglycerollipase structure. applications using lipases in various fields of industry. 1) To clarify the molecular mechanism in the catalytic action Materials and Methods of lipase, the three-dimensional structures of some triacylStrains and plasmid. Eschericia coli DH51X (BRL, Gaithersburg, MD, glycerol lipases (Mucor miehei,2) human pancreas,3) and F-

Secretion of mono- and diacylglycerol lipase from Penicillium camembertii U-150 by Saccharomyces cerevisiae and site-directed mutagenesis of the putative catalytic sites of the lipase.

Yeast cells carrying intronless mono- and diacylglycerol lipase (MDGL) genes, constructed by recombination of the genomic gene and cDNA, secreted MDGL...
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