KnowVolution of a GH5 Cellulase from Penicillium verruculosum to Improve Thermal Stability for Biomass Degradation

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.