基于易错PCR的黄曲霉毒素解毒酶体外分子定向进化

Directed evolution of aflatoxin detoxifzyme in vitro by error-prone PCR

运用定向进化-易错PCR方法,提高黄曲霉毒素解毒酶的活力及稳定性,并结合辣根过氧化物酶(HRP)-隐性亮绿(RBG)快速高通量筛选系统,构建了库容约为104的突变体库。经过两轮易错PCR,最终分别获得了耐高温70℃突变酶A1773、pH 4.0稳定性的突变酶A1476,pH 4.0和pH 7.5均表现稳定性的突变酶A2863,其酶活力比野生酶分别提高了6.5倍、21倍和12.6倍。经序列分析表明,发现突变酶A1773发生了Glu127Lys和Gln613Arg突变;突变酶A2863发生了Gly73Ser、Ile307Leu、Val596Ala、Gln613Arg突变;突变酶A1476发生了Ser46Pro、Lys221Gln、Ile307Leu和Asn471Ile突变。结果为进一步了解黄曲霉毒素解毒酶的结构与功能之间的关系提供了参考。

The experiment was conducted by directed evolution strategy（error-prone PCR） to improve the activity of aflatoxin detoxifzyme with the high-throughput horse radish peroxidas and recessive brilliant green（HRP-RBG） screening system.We built up a mutant library to the order of 104.Two rounds of EP-PCR and HRP-RBG screening were used to obtain three optimum mutant strains A1773,A1476 and A2863.We found that mutant A1773 had upper temperature tolerance of 70 °C and that its enzyme activity was 6.5 times higher than that of the parent strain.Mutant strains A1476 worked well at pH 4.0 and its enzyme activity was 21 times higher than that of the parent strain.Mutant A2863 worked well at pH 4.0 and pH 7.5,and its enzyme activity was 12.6 times higher than that of the parent strain.With DNA sequencing we found that mutant A1773 revealed two amino acid substitutions,Glu127Lys and Gln613Arg.Mutant A1476 revealed four amino acid substitutions： Ser46Pro,Lys221Gln,Ile307Leu and Asn471Ile.Mutant A2863 revealed four amino acid substitutions： Gly73Ser,Ile307Leu,Val596Ala and Gln613Arg.The results provided a useful illustration for the deep understanding of the relationship between the function and structure of aflatoxin detoxifzyme.