Open AccessMutagenesis of key amino acids alters activity of a Saccharomyces cerevisiae endo‐polygalacturonase expressed in Pichia pastoris
Author(s) -
Blanco Pilar,
Thow Graham,
Simpson Craig G.,
Villa Tomas G.,
Williamson Brian
Publication year - 2002
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2002.tb11179.x
Subject(s) - pichia pastoris , mutagenesis , saccharomyces cerevisiae , pectinase , pichia , biochemistry , site directed mutagenesis , biology , chemistry , yeast , enzyme , mutation , recombinant dna , gene , mutant
A polygalacturonase (PG)‐encoding gene from Saccharomyces cerevisiae ( PGU1 ) was successfully expressed in the methylotrophic yeast Pichia pastoris . PG secretion was efficiently directed by the S. cerevisiae α‐factor signal sequence, while the native ( PGU1 ) leader peptide was unable to direct protein export in P. pastoris . The level of PGU1 activity achieved in P. pastoris was significantly enhanced when compared to activity using the same gene in S. cerevisiae . Expression of PG proteins, engineered by site‐directed mutagenesis, in P. pastoris showed that aspartic acid residues at positions 179, 200, and 201, and histidine 222 were essential for enzyme activity. Mutation of the two potential glycosylation sites in PGU1 showed that the two residues individually (N318D, N330D) did not affect secreted enzyme activity, but the double mutant caused a 50% reduction in enzyme activity when compared to the wild‐type PGU1 transformant.