草生欧文氏菌中酪氨酸酚裂解酶的体外定向进化及结构模拟  

Directed Evolution in vitro and Structural Simulation of Tyrosine Phenol Lyase from Erwinia herbicola

作　　者：郭小雷 GUO Xiaolei(Fuzhou Kehong Biotechnology Co.,Ltd.,Fuzhou 350108,China)

机构地区：[1]福州科宏生物技术开发有限责任公司,福建福州350108

出　　处：《化学与生物工程》2023年第7期34-39,44,共7页Chemistry & Bioengineering

基　　金：福建省中科院STS院省合作重大项目(2022T3004)。

摘　　要：以来源于草生欧文氏菌(Erwinia herbicola)的酪氨酸酚裂解酶(Eh-TPL)为目的基因,通过易错PCR定向进化技术构建突变体文库,利用高通量筛选方法获得了比酶活提高的突变体S20C和E202R,再通过定点突变得到突变体S20C/E202R/N161S。经摇瓶发酵培养发现,突变体S20C/E202R/N161S比野生型Eh-TPL的比酶活提高了30.61%;酶学性质研究结果表明,突变体S20C/E202R/N161S的最适温度仍为37℃,最适pH值从8.2升至8.5,热稳定性、pH稳定性均得到明显提高;结构模拟结果表明,突变体S20C/E202R/N161S的三维结构无明显变化,突变位点附近氢键和表面电势有所变化。体外定向进化技术能有效提高Eh-TPL的催化效率,对酶法生产L-酪氨酸具有重要应用价值。Taking tyrosine phenol lyase from Erwinia herbicola(Eh-TPL)as the target gene,we constructed mutant library by using error-prone PCR directed evolution technique,and obtained the mutants S20C and E202R with enhanced specific enzyme activities by a high-throughput screening method.Finally,we obtained the mutant S20C/E202R/N161S by site-directed mutagenesis.The specific enzyme activity of the mutant S20C/E202R/N161S is 30.61%higher than that of the wild-type Eh-TPL.The results of enzymatic properties show that the optimum temperature of the mutant S20C/E202R/N161S is still 37℃,the optimum pH value increases from 8.2 to 8.5,and the thermal stability and pH stability are significantly improved.The results of structural simulation show that the three-dimensional structure of the mutant S20C/E202R/N161S dose not change significantly after the mutation,while the hydrogen bond and surface potential around the mutation site change.This study indicates that directed evolution in vitro technique can effectively improve the catalytic efficiency of Eh-TPL,which has important application value for enzymatic production of L-tyrosine.

关 键 词：易错PCR 定向进化 草生欧文氏菌 酪氨酸酚裂解酶 L-酪氨酸 

分 类 号：Q78[生物学—分子生物学] Q814