Cai Liutengzi, Zhang Mishuai, Shao Tianci, He You, Li Jingyi, Ren Bingjie, Zhou Chenyan
School of Life Science and Technology, Xinxiang Medical University.
Synthetic Biology Engineering Lab of Henan Province.
J Gen Appl Microbiol. 2019 Dec 19;65(5):240-245. doi: 10.2323/jgam.2018.11.002. Epub 2019 Mar 22.
In this study, a mutant xylanase of high thermostability was obtained by site-directed mutagenesis. The homologous 3D structure of xylanase was successfully modeled and the mutation sites were predicted using bioinformatics software. Two amino acids of XynZF-2 were respectively substituted by cysteines (T205C and A52C) and a disulfide bridge was introduced into the C-terminal of XynZF-2. The mutant gene xynZFTA was cloned into pPIC9K and expressed in P. pastoris. The optimum temperature of the variant XynZFTA was improved from 45°C to 60°C, and XynZFTA retained greater than 90.0% activity (XynZF-2 retained only 50.0% activity) after treatment at 50°C for 5 min. The optimum pH of mutant xylanase was similar to XynZF-2 (pH = 5.0). The pH stability span (5.07.0) of the mutant xylanase was increased to 3.09.0. Overall, the results implied that the introduction of a disulfide bridge in the C-terminal structure improved the thermostability and pH stability of XynZF-2.
在本研究中，通过定点诱变获得了一种具有高热稳定性的突变木聚糖酶。成功构建了木聚糖酶的同源三维结构，并使用生物信息学软件预测了突变位点。XynZF-2的两个氨基酸分别被半胱氨酸取代（T205C和A52C），并在XynZF-2的C端引入了一个二硫键。将突变基因xynZFTA克隆到pPIC9K中并在巴斯德毕赤酵母中表达。变体XynZFTA的最适温度从45℃提高到60℃，并且在50℃处理5分钟后，XynZFTA保留了大于90.0%的活性（XynZF-2仅保留50.0%的活性）。突变木聚糖酶的最适pH与XynZF-2相似（pH = 5.0）。突变木聚糖酶的pH稳定范围（5.07.0）增加到3.09.0。总体而言，结果表明在C端结构中引入二硫键提高了XynZF-2的热稳定性和pH稳定性。
