嗜热酯酶APE1547催化活性的定向进化研究

对来源于嗜热古菌Aeropyrum pernix的酯酶(APE1547)催化活性进行定向进化研究。利用APE1547特殊的稳定性,建立了准确的高通量高温酯酶筛选方法。对第一代随机突变库筛选获得了催化活性较野生型提高1.5倍的突变体M010,序列分析表明其氨基酸突变为R526S。从第二代突变库中筛选出的总活力提高5.8倍突变体M020,突变位点为R526S/E88G/A200T/I519L,其比活力与M010一致,但表达量比野生型提高约4倍。对M020酶学性质表征发现,其最适pH为8.5,比野生型向碱性偏移0.5;活性中心残基酸性基团的解离常数(pK1)由野生型的7.0提高至7.5。晶体结构分析表明,突变位点R526距离活性中心较近,将其突变为Ser降低了活性中心的极性,抑制了催化残基His的解离,使酸性基团的解离常数升高。

Directed evolution of thermophilic esterase from the archaeon Aeropyrum pernix K1

Thermophilic esterase(APE1547) from Aeropyrum pernix K1 was subjected to error-prone PCR(epPCR) to enhance activity.For the screening of mutants,an efficient and reliable assay suitable for high throughput screening was developed based on the enzyme thermostability.Two successive rounds of random mutagenesis by epPCR resulted in a four amino acid substitution variant M020 with significantly increased activity(six-fold under the screening condition.Further assay for the purified enzymes showed that the mutant possess 1.5-fold higher specific activity and nearly 4-fold higher expressed level than the wild-type.The mutant has an optimal activity at pH 8.5,corresponding to an alkaline shift of 0.5 pH unit compared to the wild type.The structure analysis suggests that R526S may contribute to the enhanced activity and the shift of pK-1.