Lignocellulosic biomass is a most promising feedstock in the production of second-generation biofuels. Efficient degradation of lignocellulosic biomass requires a synergistic action of several cellulases and hemicellulases. Cellulases depolymerize cellulose, the main polymer of the lignocellulosic biomass, to its building blocks. The production of cellulase cocktails has been widely explored, however, there are still some main challenges that enzymes need to overcome in order to develop a sustainable production of bioethanol. The main challenges include low activity, product inhibition, and the need to perform fine-tuning of a cellulase cocktail for each type of biomass. Protein engineering and directed evolution are powerful technologies to improve enzyme properties such as increased activity, decreased product inhibition, increased thermal stability, improved performance in non-conventional media, and pH stability, which will lead to a production of more efficient cocktails. In this review, we focus on recent advances in cellulase cocktail production, its current challenges, protein engineering as an efficient strategy to engineer cellulases, and our view on future prospects in the generation of tailored cellulases for biofuel production.

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.

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