半理性设计提高产碱杆菌KS-85来源的肌酸酶催化活性  

Improving the activity of creatinase from Alcaligenes sp.KS-85 through semi-rational design

作　　者：卞佳豪 郝俊尧 杨广宇[1] BIAN Jiahao;HAO Junyao;YANG Guang-Yu(State Key Laboratory of Microbial Metabolism,School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China)

机构地区：[1]上海交通大学生命科学技术学院、微生物代谢国家重点实验室,上海200240

出　　处：《生物工程学报》2022年第12期4601-4614,共14页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(32030063)。

摘　　要：肌酐水平是指示肾脏功能的重要临床指标。肌酸酶(creatinase,CRE)是肌酐的酶促检测体系中的关键酶之一,也是整个体系的限速酶,较差的活性限制了它在临床检测和工业上的应用。针对此问题,采用半理性设计策略对产碱杆菌属(Alcaligenessp.)KS-85来源肌酸酶Al-CRE的活性进行了改造,通过对挑选出的突变热点进行饱和突变筛选和组合,最终获得多个活性提升的突变酶,活性最高的五点突变酶I304L/F395V/K351V/Y63S/Q88A比活力相较于野生型提升了2.18倍。同时对相关突变位点进行了结构分析,为肌酸酶的实际应用及对其机理的进一步解析提供了基础。Creatinine levels in biological fluids are important indicators for the clinical evaluation of renal function.Creatinase(CRE,EC3.5.3.3)is one of the key enzymes in the enzymatic measurement of creatinine concentration,and it is also the rate-limiting enzyme in the whole enzymatic cascade system.The poor catalytic activity of CRE severely limits its clinical and industrial applications.To address this issue,a semi-rational design is applied to increase the activity of a creatinase from Alcaligenes sp.KS-85(Al-CRE).By high-throughput screen of saturation mutagenesis libraries on the selected hotspot mutations,multiple variant enzymes with increased activity are obtained.The five-point best variant enzyme(I304L/F395V/K351V/Y63S/Q88A)were further obtained by recombine the improved mutations sites that to showed a 2.18-fold increased specific activity.Additionally,structure analysis is conducted to understand the mechanism of the activity change.This study paves the way for a better practical application of creatinase and may help further understand its catalytic mechanism.

关 键 词：肌酐 肌酸酶 半理性设计 饱和突变 

分 类 号：Q814[生物学—生物工程]