Construction and Properties of β-1,3-1,4-Glucanase Thermostable Mutants

• 1、School of Biotechnology,Jiangnan University,Wuxi,Jiangsu 214122

Abstract：β-1,3-1,4-glucanase (EC 3.2.1.73, β-glu) is an important β-glucanohydrolase that can specifically hydrolyze β-glucan and β-glucan. The β-1,4 glycosidic bond adjacent to the 1,3 glycosidic bond is an important enzyme preparation in industrial applications such as beer brewing and feed industry. However, the β-glu currently on the market has the problem of poor thermal stability. To solve this problem, this study selected β-glu (Aaglu) derived from Aspergillus awamori, used the prediction software FoldX to calculate the potential thermostable mutation sites, and designed 6 single-point mutants using Pichia pastoris (Pichia pastoris). ) for heterologous secretory expression. The differences between the mutant and the wild enzyme were analyzed through enzymatic properties, thermal stability and enzymatic reaction kinetics, and finally three mutants VR, AP and TP with significantly improved thermal stability were obtained. The enzymatic properties showed that the optimal reaction temperature of the three mutants and wild enzymes was 50℃, and the optimal reaction pH was 5.0. The thermal stability experiments showed that the relative enzyme activities of the three mutants were significantly improved compared with the wild enzyme after incubation at 60℃-90℃ for 20 min. Among them, the relative enzyme activities of mutant VR, AP and TP were 3.6, 3.0 and 1.6 times higher than those of wild enzyme after high temperature treatment at 90℃. In addition, the kinetic analysis of the enzymatic reaction showed that the Km values of the three mutants VR, AP and TP were 73.5%, 64.9% and 37% lower than those of the wild enzyme, respectively, and the kcat/Km The values were 4.53, 3.41 and 2.31 times that of the wild enzyme, respectively. It indicated that the catalytic efficiency of the three mutants was improved. The analysis showed that new hydrogen bonds, hydrophobic interactions and insertion of proline in the loop region were formed in the mutants, which played an important role in improving the thermostability of the enzyme. In this study, the stability of Aaglu was modified by software calculation and site-directed mutagenesis, and mutants with improved catalytic efficiency and enhanced thermal stability were obtained, which laid a good foundation for the wide application of β-glu in the food and feed industries.

Keywords： β-1,3-1,4-glucanase Pichia pastoris mutation thermostability