Artificial trimerization of β-glucosidase for enhanced thermostability and activity via computational redesign

Abstract

The improvement of enzyme thermostability is of great significance in the biotechnology industry. Herein, we modified the C-terminus of β-glucosidase via the computer-aided rational design and successfully obtained six variants that exhibit improved optimal temperatures and thermal inactivation half-lives at 65 °C. Among these variants, the thermal inactivation half-life and hydrolytic activity of TrCel1b-H13 at 65 °C were increased by 416 and 3223 folds, respectively, compared to those of the wild type. The optimal catalytic temperature and melting temperature (Tm) of TrCel1b-H13 were elevated by 55 °C and 20 °C respectively, compared to those of the wild type. The Kcat/Km value of TrCel1b-H13 was improved by 13.3-fold, compared to that of the wild type. Furthermore, we found that the modified β-glucosidase, TrCel1b-H13, self-assembles into a trimeric structure, which was confirmed by X-ray crystallography and blue native polyacrylamide gel electrophoresis. The amino acid residues, including E28, H60, E99, K106, and K471, located at the trimeric interface of the adjacent subunits, were found to play an important role in maintaining the crystal structure of the protein trimer. Thus, protein multimerization via the computer-aided rational design method provides a cost-effective tool for simultaneously improving the thermostability and activity of enzymes.