title: Effect of N-linked glycosylation on the activity and other properties of recombinant endoglucanase IIa (Cel5A) from Penicillium verruculosum.
abstract: Endoglucanase IIa from Penicillium verruculosum (PvCel5A) has three potential N-glycosylation sites: Asn19, Asn42 and Asn194. In order to study the role of N-glycosylation, the wild type (wt) PvCel5A and its mutant forms, carrying Asn to Ala substitutions, were cloned into Penicillium canescens. All forms of the rPvCel5A were successfully expressed and purified for characterization. The MALDI-TOF mass spectrometry peptide fingerprinting showed that N-glycans linked to Asn42 and Asn194 represent variable oligosaccharides, according to the formula (Man)1-9(GlcNAc)2. No evidence for Asn19 glycosylation was found. Mutations had no notable effect on the enzyme thermostability; however, the N-linked glycans stabilized the enzyme against proteolytic attack. For N42A and N194A mutants, a slight shift of pH-optimum to pH 5.0 was observed (from pH-optimum of 4.5 for the native enzyme, rPvCel5A-wt and N19A mutant). The N19A mutation led to a notable decrease in the specific activity against carboxymethylcellulose and barley β-glucan (by 26% and 12% relative to the rPvCel5A-wt), while the N42A and N194A mutants displayed 12-13% and 32-35% increase in the activities. Similar effects of the mutations were observed in prolonged hydrolysis of β-glucan and milled aspen wood by rPvCel5A forms in the presence of purified β-glucosidase.
authors.affiliation: Department of Chemistry, M. V. Lomonosov Moscow State University, Vorobyovy Gory 1/11, Moscow 119991, Russia.|Federal Research Centre "Fundamentals of Biotechnology", Russian Academy of Sciences, Leninsky Pr. 33, Moscow 119071, Russia.
authors.forename: Anna S
authors.initials: AS
authors.lastname: Dotsenko
authors.affiliation: Department of Chemistry, M. V. Lomonosov Moscow State University, Vorobyovy Gory 1/11, Moscow 119991, Russia.|Federal Research Centre "Fundamentals of Biotechnology", Russian Academy of Sciences, Leninsky Pr. 33, Moscow 119071, Russia.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Gusakov
authors.affiliation: Department of Chemistry, M. V. Lomonosov Moscow State University, Vorobyovy Gory 1/11, Moscow 119991, Russia.|Federal Research Centre "Fundamentals of Biotechnology", Russian Academy of Sciences, Leninsky Pr. 33, Moscow 119071, Russia.
authors.forename: Aleksandra M
authors.initials: AM
authors.lastname: Rozhkova
authors.affiliation: Department of Chemistry, M. V. Lomonosov Moscow State University, Vorobyovy Gory 1/11, Moscow 119991, Russia.|Federal Research Centre "Fundamentals of Biotechnology", Russian Academy of Sciences, Leninsky Pr. 33, Moscow 119071, Russia.
authors.forename: Olga A
authors.initials: OA
authors.lastname: Sinitsyna
authors.affiliation: Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region 142292, Russia.
authors.forename: Vitaly A
authors.initials: VA
authors.lastname: Nemashkalov
authors.affiliation: Department of Chemistry, M. V. Lomonosov Moscow State University, Vorobyovy Gory 1/11, Moscow 119991, Russia.|Federal Research Centre "Fundamentals of Biotechnology", Russian Academy of Sciences, Leninsky Pr. 33, Moscow 119071, Russia.
authors.forename: Arkady P
authors.initials: AP
authors.lastname: Sinitsyn
keywords: 
            N-glycosylation; 
            Penicillium verruculosum
          ; cloning; endoglucanase (Cel5A); site-directed mutagenesis