tailieunhanh - Báo cáo khóa học: Heterogeneity of homologously expressed Hypocrea jecorina (Trichoderma reesei ) Cel7B catalytic module

The catalytic module ofHypocrea jecorina(previously Trichoderma reesei) Cel7B was homologously expressed by transformation of strain QM9414. Post-translational modi-fications in purified Cel7B preparations were analysed by enzymatic digestions, high performance chromatography, mass spectrometry and site-directed mutagenesis. Of the five potential sites found in the wild-type enzyme, only Asn56 and Asn182 were found to be N-glycosylated. GlcNAc2Man5was identified as the predominant N-glycan, although lesser amounts of GlcNAc2Man7 and glycans carrying a mannophosphodiester bond were also detected. Repartition of neutral and charged glycan structures over the two glycosylation sites mainly accounts for the observed microheterogeneity of the protein. . | Eur. J. Biochem. 271 1266-1276 2004 FEBS 2004 doi Heterogeneity of homologously expressed Hypocrea jecorina Trichoderma reesei Cel7B catalytic module Torny Eriksson1 I Ingeborg Stals2 I Anna Collen1 Folke Tjerneld1 Marc Claeyssens2 Henrik Stalbrand1 and Harry Brumer3 1 Department of Biochemistry Center for Chemistry and Chemical Engineering Lund University Sweden 2Laboratory for Biochemistry Department of Biochemistry Physiology and Microbiology Ghent University Belgium 3 Department of Biotechnology Royal Institute of Technology KTH AlbaNova University Centre Stockholm Sweden The catalytic module of Hypocrea jecorina previously Trichoderma reesei Cel7B was homologously expressed by transformation of strain QM9414. Post-translational modifications in purified Cel7B preparations were analysed by enzymatic digestions high performance chromatography mass spectrometry and site-directed mutagenesis. Of the five potential sites found in the wild-type enzyme only Asn56 and Asn182 were found to be N-glycosylated. GlcNAc2Man5 was identified as the predominant N-glycan although lesser amounts of GlcNAc2Man7 and glycans carrying a mannophosphodiester bond were also detected. Repartition of neutral and charged glycan structures over the two glycosylation sites mainly accounts for the observed microheterogeneity of the protein. However partial deamidation of Asn259 and a partially occupied O-glycosylation site give rise to further complexity in enzyme preparations. Keywords protein glycosylation O-glycan N-glycan Tricho-derma reesei cellulase. The filamentous fungus Hypocrea jecorina previously Trichoderma reesei 1 produces several extracellular cellulases which cooperate in the degradation of paracrystalline cellulose. The five known endoglucanases including Cel7B generally act by hydrolysing the b 1fi4 glucan chains internally 2 3 whereas the two cellobiohydrolases Cel6A and Cel7A release cellobiose from the nonreducing and reducing chain ends