Wang Qiu-Yan, Yang Guang-Yu, Liu Yan-Li, Wang Yan-Ping, Feng Yan
Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130023, China.
Wei Sheng Wu Xue Bao. 2006 Apr;46(2):259-62.
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 pKi.
对来自嗜热栖热菌K1的嗜热酯酶（APE1547）进行易错PCR（epPCR）以提高其活性。为了筛选突变体，基于该酶的热稳定性开发了一种适用于高通量筛选的高效可靠检测方法。通过epPCR进行的两轮连续随机诱变产生了一个四氨基酸取代变体M020，其活性显著提高（在筛选条件下提高了6倍）。对纯化酶的进一步检测表明，该突变体的比活性比野生型高1.5倍，表达水平比野生型高近4倍。该突变体在pH 8.5时具有最佳活性，与野生型相比，pH值碱性偏移了0.5个单位。结构分析表明，R526S可能有助于活性增强和pKi的偏移。
