随机突变提高单胺氧化酶活性  

Increasing activity of a monoamine oxidase by random mutation

作　　者：陈雪君[1] 马元慧 邵建华 赖敦岳[2] 王志国[4] 陈振明[2] 

机构地区：[1]杭州师范大学生命与环境科学学院,浙江杭州310036 [2]杭州师范大学生物催化研究室,浙江杭州311121 [3]浙江台州清泉医药化工有限公司,浙江台州317300 [4]杭州师范大学衰老研究所,浙江杭州311121

出　　处：《生物工程学报》2014年第1期109-118,共10页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.31100584)资助~~

摘　　要：前期获得了一个对底物美西律具有一定活性的单胺氧化酶突变体A-1(F210V/L213C)。为进一步提高其酶活性,利用MegaWHOP PCR构建了库容约为104的随机突变库。筛选后获得了一个最优突变酶ep-1,比活力为A-1的189%。选择性测定结果表明,酶的对映体选择性有较大提高,E值由101提高到282;动力学常数测定揭示,酶催化效率有较大提高,kcat/Km由0.001 51 mmol/(L?s)提高到0.002 89 mmol/(L?s)。和A-1酶相比,在所测定的11种胺类底物中,ep-1对其他7种底物的比活力有较明显提高,对其他4种底物的比活力变化不大。序列分析表明,ep-1的突变为T162A。分子动力学模拟结果提示,该突变主要通过修正通道氨基酸的二级结构和扩大活性口袋来发挥作用。The monoamine oxidase mutant A-1 （F210V/L213C） from Aspergillus niger showed some catalytic activity on mexiletine. To futher improve its activity, the mutant was subjected to directed evolution with MegaWHOP PCR （Megaprimer PCR of Whole Plasmid） and selection employing a high-throughput agar plate-based colorimetric screen. This approach led to the identification of a mutant ep-1, which specific activity was 189% of that for A-1. The ep-1 also showed significantly improved enantioselectivity, with the E value increased from 101 to 282; its kinetic kcat/Km value increased from 0.001 51 mmol/（L＇s） to 0.002 89 mmol/（L＇s）, suggesting that catalytic efficiency of ep-1 had been improved. The mutant showed obviously higher specific activities on 7 of all tested 11 amines substrates, and the others were comparable. Sequence analysis revealed that there was a new mutation T162A on ep-1. The molecular dynamics simulation indicated that TI62A may affect the secondary structure of the substrate channel and expand the binding pocket.

关 键 词：单胺氧化酶 易错PCR 酶活力 分子动力学模拟 

分 类 号：TQ464.8[化学工程—制药化工]