The functional role of N-link glycosylation in a novel cellobiohydrolase II (LsCel6A) from a white-rot fungus Lentinus sp. WR2.

Abstract

White-rot fungi produce a wide spectrum of lignocellulose-degradation enzymes, which can be used in bioenergy, bioremediation, and other industrial applications. This study identified a cellobiohydrolase II (Cel6A, GH6 cellobiohydrolase, EC 3.2.1.91) with high hydrolytic activity toward crystalline cellulose from a white-rot fungus Lentinus sp. WR2. Both native (nLsCel6A) and recombinant (rLsCel6A) enzymes expressed in Pichia pastoris were purified and characterized. Three N-glycosylation sites at Asn¹⁰², Asn¹⁴⁵, and Asn³⁹² containing high-mannose glycans, were confirmed by mass spectrometry. To elucidate the functional role of N-linked glycans, three deglycosylated mutants of rLsCel6A, i.e., N102A, N145A, and N392A, were created and characterized for their biochemical and kinetic properties. While no discernible changes in the secondary structure of the three mutants were determined by circular dichroism spectrometry, deterioration of thermostability was revealed in N392A but not in N102A and N145A. Structure modeling and molecular dynamics analyses revealed that the N-linked glycan on Asn³⁹² may restrict the flexibility of the C-terminal loop in LsCel6A, affecting the protein integrity and appropriate dynamics for the enzymatic function. In summary, this study identified a novel LsCel6A enzyme with high catalytic activity against insoluble forms of cellulose and demonstrated the role of N-linked glycosylation in the thermostability of the enzyme.