巨大芽孢杆菌细胞色素P450酶的表达纯化及定向进化研究

Expression Purification and Directed Evolution of Cytochrome P450 from Bacillus megaterium

为了获得高效的重组来自巨大芽孢杆菌ALA2的细胞色素P450酶(CYPs),将CYPs基因(cyp)克隆到大肠杆菌表达载体p ET20b(含T7启动子)和p Trc99A(含trc启动子)中,转入大肠杆菌表达,发现trc启动子更适合CYPs的表达。优化了Ni-NTA纯化CYPs的条件,在p H值8.1时,用60 mmol/L的咪唑缓冲液洗脱得到电泳纯的重组CYPs,酶活达到2 095 U/mg,纯化回收率为31%,回收倍数为2.32。通过理性设计确定突变位点为269位苏氨酸(T)和394位苯丙氨酸(F),通过筛选获得了突变酶T269S和T269R,2种突变酶以软脂酸为底物,酶活提高32.0%和29.0%,以油酸为底物酶活提高11.6%和9.1%。

In order to express the cytochrome P450 enzyme （ CYPs） from Bacillus megaterium ALA2 effectively, the CYPs genes （cyp） were cloned into pET20b （T7 promoter） and pTrc99A （Trc promoter） to yield pET20b-cyp and pTrc99A-cyp. Then they were transferred into the Escherichia coli BL21 （DE3） and Top10. The pTrc99A-cyp produced more recombinant CYPs than pET20b-cyp. The Ni-NTA purification of CYPs was optimized. The recombinant CYPs were eluted by 60 mmol/L imidazole at pH 8. 1. The enzyme activity of purified recombinant CYPs was 2 095 U/mg and recovery rate reached 31%, and the recovery ratio was 2. 32. The mutation positions of the threonine （T） and phenylalanine （F） were measured as 269 loci and 394 loci by reasonable experimental design. The activities of mutant enzyme T269S and T269R were increased by 32. 0% and 29. 0%respectively with palmitic acid as substrate, and 11. 6% and 9. 1% with oleic acid as substrate.