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Cloning and Expression of Two Chitin Deacetylase Genes of Saccharomyces cerevisiae
Author(s) -
MISHRA CHITRA,
SEMINO CARLOS E.,
McCREATH KENNETH J.,
DE LA VEGA HUMBERTO,
JONES BEVERLY J.,
SPECHT CHARLES A.,
ROBBINS PHILLIPS W.
Publication year - 1997
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/(sici)1097-0061(19970330)13:4<327::aid-yea96>3.0.co;2-t
Subject(s) - biology , orfs , saccharomyces cerevisiae , chitin , microbiology and biotechnology , mutant , open reading frame , biochemistry , histone deacetylase , chitin synthase , acetylation , yeast , gene , peptide sequence , chitosan , histone
Chitin deacetylase (EC 3.5.1.41), which hydrolyses the N‐acetamido groups of N‐acetyl‐ d ‐glucosamine residues in chitin, has been demonstrated in crude extracts from sporulating Saccharomyces cerevisiae . Two S. cerevisiae open reading frames (ORFs), identified by the Yeast Genome Project, have protein sequence homology to a chitin deacetylase from Mucor rouxii . Northern blot hybridizations show each ORF was transcribed in diploid cells after transfer to sporulation medium and prior to formation of asci. Each ORF was cloned in a vector under transcriptional control of the GAL 1, 10 promoter and introduced back into haploid strains of S. cerevisiae . Chitin deacetylase activity was detected by in vitro assays from vegetative cells grown in galactose. Chemical analysis of these cells also demonstrated the synthesis of chitosam in vivo . Both recombinant chitin deacetylases showed similar qualitative and quantitative activities toward chitooligosaccharides in vitro . A diploid strain deleted of both ORFs, when sporulated, did not show deacetylase activity. The mutant spores were hypersensitive to lytic enzymes (Glusulase or Zymolyase). © 1997 John Wiley & Sons, Ltd.