KnowVolution of a GH5 Cellulase from <i>Penicillium verruculosum</i> to Improve Thermal Stability for Biomass Degradation

Abstract

Understanding the thermostability of cellulases is of high importance for their application in lignocellulosic biomass degradation, feedstock, and pulp and paper production. Cellulases have to withstand high temperatures and harsh conditions in various application areas, for instance, in bioethanol production. Engineering thermostable cellulases increases the cellulase lifetime in processes and contributes to more-sustainable production. Here we report the first KnowVolution campaign toward improving the thermostability of the endo-β-1,4-glucanase PvCel5A from Penicillium verruculosum. The C-terminal region of PvCel5A (eighth α-helix, amino acid residues 280–314) was identified as a key structural determinant to improve the thermostability of PvCel5A without affecting its specific activity. The most beneficial variant, PvCel5A-R17, harbors three substitutions (F16L/Y293F/Q289G); its half-life at 75 °C improved 5.5-fold (from 32 to 175 min) and the melting temperature was raised 7.7 °C (from 70.8 °C) when compared to those of wild-type PvCel5A. Exceptionally, the thermally improved PvCel5A-R17 variant retained its specific activity at low temperatures (40 °C). Computational analyses revealed that the stabilization of the C-terminal region of PvCel5A is responsible for the improved thermostability. This knowledge will facilitate shorter times in cellulase engineering and thereby enhance the performance and sustainability of processes.