Asn64-glycosylation affects Hypocrea jecorina (syn. Trichoderma reesei) cellobiohydrolase Cel7A activity expressed in Pichia pastoris

Abstract

In this study, four N-glycosylation sites, Asn45, Asn64, Asn270 and Asn384 of Hypocrea jecorina (syn. Trichoderma reesei) Cel7A (family 7 cellobiohydrolase I) were replaced by serines using site-directed mutagenesis. These four mutants and wild type H. jecorina Cel7A gene were transformed into P. pastoris, and the recombinant enzymes were purified and analyzed. The enzymatic activities of recombinant Cel7A (rCel7A), and mutants N45S, N270S and N384S were very low while mutant N64S displayed about seven times higher activity than that of rCel7A, and about 10% of the wild-type Cel7A activity from H. jecorina. The results indicate that N-glycosylation of Asn64 had an effect on the activity of the Cel7A enzyme expressed in P. pastoris, and that glycosylation at this site would be only a subordinate reason for the low activity of the recombinant enzyme.

N-Glycoform Diversity of Cellobiohydrolase I from Penicillium decumbens and Synergism of Nonhydrolytic Glycoform in Cellulose Degradation

“…Recently, protein glycosylation has become widely accepted as an important factor affecting enzyme activity. CBHIs expressed in Saccharomyces cerevisiae and Pichia pastoris are hyperglycosylated, and neither exhibits any activity (9). By removing the Asn-384 glycosylation site and expression in Aspergillus niger, T. reesei CBHI shows 70% greater activity compared with the wild-type T. reesei enzyme (7).…”

Study of Phanerochaete chrysosporium Secretome Revealed Protein Glycosylation as a Substrate-Dependent Post-Translational Modification

“…reesei demonstrated wider pH-range activity and higher thermal stability compared to the native enzyme . Further, CBHIs expressed in Saccharomyces cerevisiae and Pichia pastoris were also hyperglycosylated, but neither exhibits any activities . Thus, degree of glycosylation can be important in enzyme activities.…”

“…The N-glycoproteins were classified by biological function. b The ions score cutoff was set at 30. exhibits any activities 51. Thus, degree of glycosylation can be important in enzyme activities.4.…”

Promotion of Extracellular Activity of Cellobiohydrolase I from Trichoderma reesei by Protein Glycosylation Engineering in Saccharomyces cerevisiae

“…Hyperglycosylation of S. cerevisiae inhibited the activity of recombiant Tr-Cel7A enzyme, a glycoprotein from T. reesei, significantly [11,23]. In order to investegate if the glycosylation in S. cerevisiae close somewhat to its native state could bring benefit to the extracellular Tr-Cel7A activity, the protein glycosylation engineering including disruption of the yeast mannosyltransferases Och1p, Mnn9p and Mnn1p, and heterogeneous expression of T. reesei mannosidase Tr-Mds1p, was performed.…”

“…Glycosylation might affect the protein secondary structure such as folding, disulfide bonds formation and so on [23,24,26]. For Cel7A and other enzymes with similar function, N-glycan in the catalytic domain might also affect its binding with the cellulose substrate and its stability [27][28][29].…”