Improving the thermostability of a thermostable endoglucanase from Chaetomium thermophilum by engineering the conserved noncatalytic residue and N-glycosylation site

Endoglucanases provide an attractive avenue for the bioconversion of lignocellulosic materials into fermentable sugars to supply cellulosic feedstock for biofuels and other value-added chemicals. Thermostable endoglucanases with high catalytic activity are preferred in practical processes. To improve the thermostability and activity of the thermostable beta-1,4-endoglucanase CTendo45 isolated from the thermophilic fungus Chaetomium thermophilum, structure-based rational design was performed by using site-directed mutagenesis. When inactivated mutation of the unique N-glycosylation sequon (N88-E89-T90) was implemented and the conserved Y173 residue was substituted with phenylalanine, a double mutant T90A/Y173F demonstrated enzymatic activity that dramatically increased 2.12- and 1.82-fold towards CMC-Na and beta-D-glucan, respectively. Additionally, T90A/Y173F exhibited extraordinary heat endurance after 300 min of incubation at elevated temperatures. This study provides a valid approach to the improvement of enzyme redesign protocols and the properties of this endoglucanase mutant distinguish it as an excellent candidate enzyme for industrial biomass conversion. (C) 2020 Elsevier B.V. All rights reserved.