基于易错PCR技术的红酵母D-氨基酸氧化酶的定向进化

Directed evolution of D-amino acid oxidase from Rhodotorula gracilis by error-prone PCR

采用易错PCR技术对来源于红酵母Rhodotorula gracilis的D-氨基酸氧化酶基因(RgDAAO)进行突变,构建并优化了突变株文库;结合48深孔板的高通量筛选方法,获得突变株M3217,其V_(max)相对于野生型提高了16.8%。对测序结果进行分析,发现突变酶基因序列中有5处点突变,其中3处发生了氨基酸置换,分别为:D242V/Q253R/D304V。利用Swiss-Model对突变株M3217进行三维结构模拟,结果显示所有突变位点都不在催化活性中心的附近,特别是V304的位置在连接F5和F6两个β折叠股的长loop环上。推测D304V这一突变位点很可能增强了RgDAAO二聚体形态的稳定性,或是增强了与辅酶FAD的结合能力,从而间接提高了全酶的催化活力。

By error-prone PCR, a random mutation library of D-amino acid oxidase gene from Rhodotorula gracilis （Rg- DAAO） was constructed and optimized. Through 48-well-plate based high-throughput screening method, a mutant named M3217 was obtained and its Vm^was 16.8% higher than that of the wild-type. The sequence of M3217 showed that five nucleotide substitutions occurred, and three of them （D242V/Q253R/D304V） caused amino acid changes. According to the three-dimension structure of M3217 simulated by Swiss-Model, none of the mutations was located in the vicinity of the active site. In particular, V304 was located on the loop connecting β-strands F5 and F6, and appeared to play an impor- tant role in monomer-monomer interaction. It was speculated that this substitution improved the stability or FAD binding ability of RgDAAO protein.