Alcaligenes sp.腈水解酶组成型毕赤酵母表达条件的优化  

Optimization of expression conditions of nitrilase from Alcaligenes sp.in constitutive Pichia pastoris

作　　者：张新红 朱富成 常飞 徐涛[1] Zhang Xin-hong;Zhu Fu-cheng;Chang Fei;Xu Tao(Hefei University,Hefei 230630 China;West Anhui University,Lu′an 237012 China)

机构地区：[1]合肥学院生物食品与环境学院,安徽合肥230601 [2]皖西学院生物与制药工程学院,安徽六安237012

出　　处：《新余学院学报》2022年第6期24-30,共7页Journal of Xinyu University

基　　金：国家青年科学基金项目“大肠杆菌光控诱导表达系统中光敏蛋白YF1的定向进化”(31800042);安徽省自然科学基金“基于功能模块共进化的蛋白酶结合域分子改造研究”(2008085QB96);安徽省高等学校自然科学研究项目“腈水解酶逆性分子改造及光控诱导表达系统的构建”(KJ2021A1007)。

摘　　要：以实验室前期构建的组成型胞外表达Alcaligenes sp.腈水解酶重组菌株Pichia pastoris X33/pGAPza-rDNA-NIT为研究对象,对该菌株的发酵培养基进行筛选,以最适发酵培养基为基础,通过对发酵培养基组分和表达条件进行优化,提高腈水解酶的表达量并检测其催化性能。结果表明,甘油培养基d效果最佳,腈水解酶体积酶活可达1.03 U/L,OD600为18.36。经发酵表达条件优化后,在蛋白胨25 g/L、酵母粉10 g/L、甘油20 g/L、MgSO41 g/L、磷酸盐20 mmol/L、培养基初始pH 8.0、装液量20%、诱导温度26℃、发酵时间72 h条件下,腈水解酶体积酶活达1.75 U/L,OD600为45.7,分别是优化前的1.7倍和2.5倍。在此基础上,利用腈水解酶进行扁桃腈催化形成R-扁桃酸的进程研究,反应2h产率可达95.1%,e.e.值达100%,此结果可为工业化应用提供参考,可为腈水解酶的研究进一步奠定基础。A recombinant Pichia pastoris X33/pGAPza-rDNA-NIT harboring Alcaligenes sp.nitrilase was used as the experimental strain.The culture media were screened,and the components and expression conditions were optimized based on the selected media to improve the expression of nitrilase.Besides,the catalytic performance was detected.The results showed that the glycerin medium d was the best,and the specific activity was 1.03 U/L,and OD600 was 18.36.After the optimization of fermentation expression,the specific activity was 1.75 U/L,and OD600 was 45.7 under the optimal conditions including tryptone 25 g/L,yeast powder 10 g/L,glycerol 20 g/L,MgSO41 g/L,phosphate 20 mmol/L,initial pH 8.0,loaded liquid 20%,induction temperature 26℃and fermentation time 72 h,which were 1.7-fold and 2.5-fold higher than before,respectively.On this basis,the time course of R,S-mandelonitrile was studied,and the yield was up to 95.1%,and e.e.was 100%after 2 h.The results provided references for the industrial application,and futher lay the foundation for the nitrilase study.

关 键 词：腈水解酶 毕赤酵母 发酵优化 胞外表达 R-扁桃酸 

分 类 号：Q814.9[生物学—生物工程] Q815Q819