对菌株Rhodobacter sphaeroides的RSP-2728酯酶基因进行立体选择性定向进化研究,建立了有效的定向进化策略,以及通过扁桃酸甲酯水解反应所产生的酸根离子浓度来监测酶活的高通量筛选方法.利用易错PCR技术,进行了三轮突变,获得了突变株A6A5,B7F11和C8G1,对映体选择性(E值)从3.13分别提高到5.32,8.77和14.01;提高了1.7倍,2.8倍和4.5倍.序列分析表明,突变体A6A5有一个62位上的氨基酸发生了改变,由原先的天冬酰胺变成了酪氨酸;突变体B7F11的62位的天冬酰胺变成了酪氨酸,145位上的亮氨酸变成了组氨酸;突变体C8G1的62位的天冬酰胺变成了半胱氨酸,145位的亮氨酸变为组氨酸.利用定向进化策略能够有效地提高酶的立体选择性.%The enantioselecttivity evolution of esterase gene RSP-2728 from Rhodobacter sphaeroides was investigated. An efficient directed evolution strategy and a high-throughput screening method based on the pH sensitivity of the indicator, bromothymol blue, to the acid formation occurring by the hydrolysis of (R,S)-methyl mandelate were established. After three cycles of evolution, three mutants, A6A5, B7FI 1 and C8G1,were obtained. Compared with the wild-type esterase, the enantioselectivities of the three mutants are improved respectively by 1.7, 2.8 and 4.5 times (from E=3.13 to E=5.32, 8.77 and 14.01). According to the sequence analysis, the mutant A6A5 shows that its amino acid Asn 62 is changed into Tyr 62; the mutant B7Fll shows that its amino acid Asn 62 is changed into Tyr 62, and its amino Leu 145 is changed into His 145; the mutant C8Gl shows that its amino acid Ash 62 is changed into Cys 62, and its amino acid Leu 1454 is changed into His 1454. The conclusion can be drawn that the enzyme directed evolution strategy is able to be used to improve enantioselectivity of enzyme.
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