点饱和突变提高腈水解酶不对称合成L-2-氨基丁酸的酶活  

Increasing activity of nitrilase by site-saturation mutagenesis for improved asymmetric synthesis of L-2-aminobutyric acid

作　　者：周海岩[1] 张旺[1] 徐建妙[1] 柳志强[1] 郑裕国[1] 

机构地区：[1]浙江工业大学生物工程研究所,浙江杭州310014

出　　处：《工业微生物》2015年第6期1-8,共8页Industrial Microbiology

基　　金：国家基金面上项目(编号:21476210)

摘　　要：为了提高腈水解酶催化外消旋2-氨基丁腈不对称合成L-2-氨基丁酸的能力,首先从实验室的腈水解重组大肠杆菌中筛选获得了一株立体选择性较高的菌株E.coli BL21(DE3)/p ET-28bbll6402。通过对其腈水解酶Nitbll6402进行三维结构同源模建和"热点"氨基酸分析,选择了6个位于"疏水口袋"附近的氨基酸残基(Y198、P239、P272、K251、E240和M234)进行点饱和突变,得到酶活力明显提高的突变型腈水解酶E240G和K251C。在60 mmol/L底物下,生成的L-2-氨基丁酸的e.e值均为100%,浓度分别为5.43 g/L和5.29 g/L,比原始酶分别提高了63%和59%。此结果表明,E240G或K251C的单点突变均能在不影响立体选择性的前提下不同程度的提高腈水解酶Nitbll6402合成L-2-氨基丁酸的能力,这将为利用腈水解酶催化合成L-2-氨基丁酸的进一步研究奠定重要的理论基础。In order to increase nitrilase activity of catalysing racemic 2-aminobutyronitrile for an improved asymmetric synthesis of L-2-aminobutyric acid, one strain named E. coliBL21 （DE3）/pET-28b-b116402 with high stereoselective nitrilase Nitbl16402 was screened and obtained from nitrilase producing recombinant Escherichia coli strains constructed by our laboratory. Through the homology modelling of the structure of nitrilase Nitbl16402 and then the analysis of ＂Hotspot＂ amino acids, 6 residues near ＂hydrophobic pocket＂ including Y198 ,P239,P272,K251 ,E240 and M234 were selected for site-saturation mutagenesis. 2 strains of mutant nitrilases E240G and K251C with higher activity were obtained. Single mutation of E240G and K251C in nitrilase Nitbl16402 resulted in titers of 5.43 g/L and 5.29 g/L L-2-aminobutyric acid at the substrate concentration of 60 mmol/L, 63% and 59% higher than those of parental nitrilase respectively. Furthermore, the e. e value of L-2-aminobutyric acid was maintained at 100% in both cases. The results indicated that the single mutation of E240G and K251C in nitrilase Nitbl16402 could increase its catalytic activity in L-2-aminobutyric acld biosynthesis without affecting its stereoselectivity, which established an important theoretical foundation for the further investigation of L-2-aminobutyric acid biosynthesis with nitrilase.

关 键 词：腈水解酶 2-氨基丁腈 L-2-氨基丁酸 不对称合成 点饱和突变 

分 类 号：O629.8[理学—有机化学]