Open AccessPurification and characterization of acidic endo‐polygalacturonase encoded by the PGL1‐1 gene from Saccharomyces cerevisiae
Author(s) -
Gainvors A,
Nedjaoum N,
Gognies S,
Muzart M,
Nedjma M,
Belarbi A
Publication year - 2000
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.1574-6968.2000.tb08946.x
Subject(s) - saccharomyces cerevisiae , pectinase , biochemistry , yeast , enzyme , open reading frame , recombinant dna , hydrolysis , peptide sequence , biology , chemistry , gene , microbiology and biotechnology
The PGL1 gene of the yeast Saccharomyces cerevisiae has been shown to encode polygalacturonase. Cloning of the PGL1 open reading frame behind the ADH1 promoter allowed overexpression of polygalacturonase activity in S. cerevisiae . This enzyme was purified to apparent homogeneity from cultures of recombinant S. cerevisiae on synthetic medium using one‐step purification by anionic exchange chromatography. The enzyme, named Pgl1P, had an apparent M r of 42 kDa as shown by SDS‐PAGE. Pgl1P was active from pH 3 to 5.5, with an optimum temperature at 25°C. This enzyme hydrolyzed polygalacturonic acid as an endo‐polygalacturonase as demonstrated by independent methods. The purified protein was N ‐glycosylated. However, the activity remained in the N ‐deglycosylated form. The N‐terminal amino acid sequence was also determined as D‐S‐C‐T‐L‐T‐G‐S‐S‐L.