木聚糖酶在催化木聚糖生成木糖部分中起着最重要的作用。GH11木聚糖酶已广泛应用于许多领域,但是大多数GH11木聚糖酶是嗜温酶。为了提高黑曲霉木聚糖酶(Xyn-WT)的催化活性和热稳定性,我们通过多种计算机辅助酶工程策略预测了Xyn-WT潜在的关键突变位点。我们介绍了一种简单经济的Ni亲和层析纯化方法,以获得高纯度的木聚糖酶及其突变体。十个突变体(Xyn-A,Xyn-B,Xyn-C,E45T,Q93R,E45T/Q93R,A161P,Xyn-D,Xyn-E,Xyn-F)被鉴定。在十个突变体中,四个(Xyn-A,Xyn-C,A161P,Xyn-F)提出了改进的热稳定性和活性,Xyn-F(A161P/E45T/Q93R)是最热稳定和活性。与Xyn-WT相比,在55°C和60°C下热处理10分钟后,Xyn-F的剩余酶活性是Xyn-WT的12和6倍,分别,当不进行热处理时,Xyn-F大约是Xyn-WT的1.5倍。Xyn-F的pH适应性也显著增强。总之,已经报道了设计变体Xyn-F的改进的催化活性和热稳定性。
Xylanase plays the most important role in catalyzing xylan to xylose moieties. GH11 xylanases have been widely used in many fields, but most GH11 xylanases are mesophilic enzymes. To improve the catalytic activity and thermostability of Aspergillus niger
xylanase (Xyn-WT), we predicted potential key mutation sites of Xyn-WT through multiple computer-aided enzyme engineering strategies. We introduce a simple and economical Ni affinity chromatography purification method to obtain high-purity
xylanase and its mutants. Ten mutants (Xyn-A, Xyn-B, Xyn-C, E45T, Q93R, E45T/Q93R, A161P, Xyn-D, Xyn-E, Xyn-F) were identified. Among the ten mutants, four (Xyn-A, Xyn-C, A161P, Xyn-F) presented improved thermal stability and activity, with Xyn-F(A161P/E45T/Q93R) being the most thermally stable and active. Compared with Xyn-WT, after heat treatment at 55 °C and 60 °C for 10 min, the remaining enzyme activity of Xyn-F was 12 and 6 times greater than that of Xyn-WT, respectively, and Xyn-F was approximately 1.5 times greater than Xyn-WT when not heat treated. The pH adaptation of Xyn-F was also significantly enhanced. In summary, an improved catalytic activity and thermostability of the design variant Xyn-F has been reported.