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

Abstract

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 glycoslated 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 Ser⁸³, Ser¹⁴⁵, 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 Ser⁸³, Ser¹⁴⁵, and His²⁵⁹ in MDGL, are essential to enzyme activity. Asp¹⁹⁹ is also likely to be involved.