Rational design and structure insights for thermostability improvement of Penicillium verruculosum Cel7A cellobiohydrolase

Abstract

Thermostability is a fundamental characteristic of enzymes that is of high importance for industrial implementation of enzymatic catalysis. Cellobiohydrolases are enzymes capable to hydrolyze the most abundant natural polysaccharide – cellulose. These enzymes are widely applied in industry for processing of cellulose containing materials. However, structural and functional engineering of cellobiohydrolases for improving their properties is a challenging task. In this study, the thermostability of Penicillium verruculosum Cel7A cellobiohydrolase was increased through rational design of substitutions with proline. The stabilizing substitution G415P resulted in 3.4-fold increase in half-life time at 60 °C compared to wild-type enzyme. Molecular dynamics simulations indicated a clear effect of the stabilizing substitution G415P and the destabilizing substitutions D62P, S191P, and S273P on the stability of the enzyme tertiary structure. The stabilizing substitution G415P decreased flexibility of the lateral sides of the enzyme active site tunnel, while the considered destabilizing substitutions increased their flexibility.