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Effect of N-linked glycosylation on the activity and other properties of recombinant endoglucanase IIa (Cel5A) from Penicillium verruculosumстатья
Информация о цитировании статьи получена из
Web of Science,
Scopus
Статья опубликована в журнале из списка Web of Science и/или Scopus
Дата последнего поиска статьи во внешних источниках: 8 января 2017 г.
- Авторы: Dotsenko A.S., Gusakov A.V., Rozhkova A.M., Sinitsyna O.A., Nemashkalov V.A., Sinitsyn A.P.
- Журнал: Protein Engineering, Design and Selection
- Том: 29
- Номер: 11
- Год издания: 2016
- Издательство: Oxford University Press
- Местоположение издательства: United Kingdom
- Первая страница: 495
- Последняя страница: 502
- DOI: 10.1093/protein/gzw030
- Аннотация: 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.
- Добавил в систему: Гусаков Александр Васильевич