Purification and characterization of acidic endo-polygalacturonase encodedby the PGL1-1 gene from Saccharomyces cerevisiae

The PGL1 gene of the yeast Saccharomyces cerevisiae has been shown to encod e polygalacturonase. Cloning of the PGL1 open reading frame behind the,ADH1 promoter allowed overexpression of polygalacturonase activity in S. cerevi siae. This enzyme was purified to apparent homogeneity from cultures of rec ombinant S. cerevisiae on synthetic medium using one-step purification by a nionic 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 a n optimum temperature at 25 degrees C. This enzyme hydrolyzed polygalacturo nic acid as an endo-polygalacturonase as demonstrated by independent method s. The purified protein was M-glycosylated. However, the activity remained in the N-deglycosylated form. The N-terminal amino acid sequence was also d etermined as D-S-C-T-L-T-G-S-S-L. (C) 2000 Federation of European Microbiol ogical Societies. Published by Elsevier Science B.V. All rights reserved.