基于定点突变改善中性植酸酶催化特性及结构-效应分析  

Improvement of Catalytic Properties and Structure-Activity Relationship of Neutral Phytase Based on Site-Directed Mutagenesis

作　　者：王祖鹏[1,2] 许伟[2] 邵荣[2] 韦萍[1] 

机构地区：[1]南京工业大学生物与制药工程学院,江苏南京211816 [2]盐城工学院海洋与生物工程学院,江苏盐城224051

出　　处：《食品科学》2015年第19期118-123,共6页Food Science

基　　金：国家自然科学基金青年科学基金项目(31101912);江苏省自然科学基金项目(BK2011420);江苏省"青蓝工程"项目

摘　　要：对前期设计的来源于淀粉液化芽孢杆菌DSM 1061的3种中性植酸酶突变体(D148E/H149R、Q67E/N68R、D191E)的稳定性及催化效率等进行研究,并对影响其性质的结构因素进行分析。结果表明:与野生酶相比,在85℃条件下,突变体D191E半衰期延长了4.3 min,但催化效率下降为野生酶的48.9%;突变体D148E/H149R催化效率为野生酶的229%,半衰期与野生酶基本一致;突变体Q67E/N68R催化效率为野生酶的93%,半衰期延长了0.7 min。圆二色光谱分析结果显示,D148E/H149R催化效率提高最为显著,α-螺旋含量由野生酶中11.79%降至2.90%,无规卷曲含量增加;Q67E/N68R性质与野生酶接近,其二级结构变化最小;D191E催化效率降低,α-螺旋含量增加至24.59%。对野生酶和突变酶的同源模型分析发现,残基D191具有较大的B因子值,不利于酶的热稳定性,各突变残基周围的氢键有所变化。同源模型的分析也将为植酸酶的进一步改良提供理论依据。Three mutations（D148E/H149 R,Q67E/N68 R and D191E） of neutral phytase from Bacillus amyloliquefaciens DSM 1061 have designed in our previous study.Enzymatic properties such as thermostability and catalytic efficiency were studied in this paper.The structural changes were analyzed to explore the relationship with properties of mutant phytases.The results showed that the half-life of D191 E was 4.3 min longer than that of wild-type phytase at 85 ℃,but its catalytic efficiency reduced to 48.9%of wild-type phytase.The catalytic efficiency of mutant D148E/H149 R was increased to 229%of wild-type phytase although the half-life was similar.The catalytic efficiency of Q67E/N68 R was 93%of wild-type phytase and the half-life was extended by 0.7 min.Circular dichroism showed that the catalytic efficiency of D148E/H149 R was improved most obviously while the content of α-helix decreased from 11.79%to 2.90%,and the content of random coil was increased.The structure of Q67E/N68 R changed little and its properties were closed to those of wild-type phytase.Theα-helical content of D191 E increased to 24.59%while the catalytic efficiency decreased a lot.Using homologous models to analyze the factors which may affect the thermal stability of phytases,we found that the B-factor value of D191 was high so that it was unfavorable to the stability of phytase.The hydrogen bonds around mutation residues also changed.Homologous model analysis will provide the theoretical basis for further study of phytase.

关 键 词：中性植酸酶 定点突变 催化效率 圆二色光谱 

分 类 号：Q55[生物学—生物化学]