基于定点突变改善中性植酸酶催化特性及结构-效应分析

doi:10.7506/spkx1002-6630-201519021

食品科学

基于定点突变改善中性植酸酶催化特性及结构-效应分析

王祖鹏1,2，许伟2，邵荣2，韦萍1,*

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

出版日期:2015-10-15 发布日期:2015-10-20

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

WANG Zupeng1,2, XU Wei2, SHAO Rong2, WEI Ping1,*

1. College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, China;
2. School of Marine and Biological Engineering, Yancheng Institute of Technology, Yancheng 224051, China

Online:2015-10-15 Published:2015-10-20

摘要/Abstract

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。

关键词: 中性植酸酶, 定点突变, 催化效率, 圆二色光谱

Abstract:

Three mutations (D148E/H149R, Q67E/N68R 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 D191E 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/H149R was increased to 229%
of wild-type phytase although the half-life was similar. The catalytic efficiency of Q67E/N68R 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/H149R
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/N68R changed little and its properties were closed to those of wild-type phytase. The
α-helical content of D191E 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.

Key words: neutral phytase, site-directed mutagenesis, catalytic efficiency, circular dichroism

中图分类号:

王祖鹏，许伟，邵荣，韦萍. 基于定点突变改善中性植酸酶催化特性及结构-效应分析[J]. 食品科学, doi: 10.7506/spkx1002-6630-201519021.

WANG Zupeng, XU Wei, SHAO Rong, WEI Ping. Improvement of Catalytic Properties and Structure-Activity Relationship of Neutral Phytase Based on Site-Directed Mutagenesis[J]. FOOD SCIENCE, doi: 10.7506/spkx1002-6630-201519021.

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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

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