Semi-rational design in simultaneous improvement of thermostability and activity of β-1,3-glucanase from Alkalihalobacillus clausii KSMK16.

Abstract

Endo-β-1,3-glucanase (β-1,3-GA) is a key enzyme capable of acting on the β-1,3-glycosidic bond of β-1,3-glucan, resulting in the production of β-1,3-gluco-oligosaccharides with higher water solubility. Higher temperatures are beneficial for curdlan hydrolysis; however, low enzymatic activity and thermal stability limit their applicability. In this study, a mutant library of Endo-β-1,3-glucanase (AC-GA) derived from Alkalihalobacillus clausii KSM-K16 was constructed by a semi-rational design using amino-acid-based multiple sequence alignment and protein structure-based computer-aided engineering. The best combination mutant (S52T/M120L) was screened through ordered recombination mutations, which showed a 24.88 % increase in specific enzyme activity over the wild-type. The melting temperature (Tm) value, an enzyme protein denaturation temperature, was raised to 82.99 °C from 78.60 of the wild type. In comparison, the K_m for hydrolysis of curdlan by S52T/M120L was reduced by 12.1 %, while the k_cat was increased by 59.39 %, thus leading to a higher catalytic efficiency (k_cat/K_m, 227.73 vs 125.46 mL·s^-1·mg^-1). Molecular dynamics (MD) simulations showed that mutations resulted in a reduction in the overall flexibility of the enzyme, an increase in rigidity, and a more stable structure. An increase in the hydrophobic network at the entrance of the substrate increases the accessibility of the substrate to the enzyme, resulting in increased enzyme activity. High-efficiency mutants have potential industrial applications in the enzymatic preparation of β-1,3-gluco-oligosaccharides.