Zhong Xia, Yang Shaomin, Su Xinying, Shen Xiaoxia, Zhao Wen, Chan Zhi
College of Life Science and Technology, Jinan University, Guangzhou, Guangdong, P.R. China.
Department of Pain Medicine, Shenzhen Municipal Sixth People's Hospital, Shenzhen , Guangdong, P.R. China.
J Microbiol Biotechnol. 2019 May 28;29(5):749-757. doi: 10.4014/jmb.1901.01030.
Nitrilase is a valuable type of hydrolase that catalyzes nitriles into carboxylic acid and ammonia. Its applications, however, are severely restricted by the harsh conditions of industrial reaction processes. To solve this problem, a nitrilase from 72W was inserted into an shuttle vector for spore surface display. Western blot, enzyme activity measurements and flow cytometric analysis results all indicated a successful spore surface display of the CotB-nit fusion protein. In addition, the optimal catalytic pH value and temperature of the displayed nitrilase were determined to be 7.0 and 50°C, respectively. Moreover, results of reusability tests revealed that 64% of the initial activity of the displayed nitrilase was still retained at the 10 cycle. Furthermore, hydrolysis efficiency of upscale production of cyanocarboxylic acid was significantly higher in the displayed nitrilase-treated group than in the free group expressed by (pET-28a-nit). Generally, the display of 72W nitrilase on the spore surface of may be a useful method for immobilization of enzyme and consequent biocatalytic stabilization.
腈水解酶是一种重要的水解酶，可将腈催化转化为羧酸和氨。然而，其应用受到工业反应过程苛刻条件的严重限制。为了解决这个问题，将来自72W的腈水解酶插入到用于芽孢表面展示的穿梭载体中。蛋白质免疫印迹、酶活性测定和流式细胞术分析结果均表明CotB-nit融合蛋白在芽孢表面成功展示。此外，所展示腈水解酶的最佳催化pH值和温度分别确定为7.0和50°C。此外，重复使用性测试结果表明，在第10个循环时，所展示腈水解酶仍保留64%的初始活性。此外，在大规模生产氰基羧酸时，所展示腈水解酶处理组的水解效率明显高于（pET-28a-nit）表达的游离组。一般来说，在芽孢表面展示72W腈水解酶可能是一种固定化酶并实现生物催化稳定化的有用方法。
