Enhancement of Thermostability and Catalytic Activity of β-1,4-Xylanase via Rational and Semi-rational Collaborative Engineering

β-1,4-Xylanase is a key enzyme that hydrolyzes β-1,4 glycosidic bonds between xylose units. It is widely used in the degradation and conversion of lignocellulose. However, the thermal stability and catalytic activity of xylanase limit their industrial application. Therefore, in this study, we rationally and semirationally engineered the active site and flexible noncatalytic regions of a xylanase from Bacillus amyloliquefaciens BH072 and expressed it in Lactococcus lactis NZ9000. This approach successfully constructed the mutant Mut-1, which exhibited significantly enhanced catalytic activity and thermostability, improving its performance in industrial applications. The specific activity and optimal temperature of Mut-1 were 1929.30 U/mg and 65 °C, respectively, representing increases of 174.84% and 15 °C compared to the wild-type. Additionally, Mut-1 showed significantly enhanced catalytic activity at medium-to-high temperatures (70–90 °C).This study demonstrates that multistrategy collaborative engineering is an effective approach to optimize the thermal stability and catalytic activity of BaXynA.