REVIEW ARTICLE
Impact of Glycosylation on Saccharomyces cerevisiae Endopolygalacturonase PGU1 Activity and Stability
V. Houle1, 3, M.C. Gagnon1, 3, E. Dubé2, 3, Y. Hurtubise1, M. Beauregard1, 3, *
Article Information
Identifiers and Pagination:
Year: 2008Volume: 2
First Page: 36
Last Page: 42
Publisher ID: TOBIOTJ-2-36
DOI: 10.2174/1874070700802010036
Article History:
Received Date: 06/12/2007Revision Received Date: 17/01/2008
Acceptance Date: 17/01/2008
Electronic publication date: 6/2/2008
Collection year: 2008
open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Endopolygalacturonases are among the best selling enzymes for a number of commercial applications such as food processing. For such enzymes, a potentially important component of production cost is glycosylation. This important modification of endopolygalacturonase has been detected for a number of species, but its real importance has not been thoroughly studied. Here we investigated endopolygalacturonase PGU1 from Saccharomyces cerevisiae CECT 1389 produced in S. cerevisiae INVSc 1. Combinatorial mutagenesis of recombinant S. cerevesiae PGU1 putative glycosylation sites was performed, where asparagines 318 and 330 were replaced with either aspartic acid or glutamine. Electrophoretic analysis of the different recombinant enzymes studied here demonstrates that the putative sites 318 and 330 are indeed glycosylated when produced in S. cerevesiae INVSc 1. The optimal activity of these enzymes was detected at pH 4.5 and 55-60 ºC. As for stability, all enzymes studied were less than 50% active after an incubation of two hours at 50 ºC and at pH between 4.5 and 6.0. Glycosylation did not provide any significant stabilisation of PGU1, but the replacement of Asn 330 with Gln had a deleterious effect on stability. The secondary structure spectra are characteristics of proteins mostly composed of beta sheets. The Tm values measured for PGU1, PGU1 deglycosylated with endo H, and three mutants ranged from 53 to 55.4ºC, indicating that glycosylation had no impact on PGU1 conformation.
