一种来源于Phialophora attae的玉米赤霉烯酮内酯水解酶ZHD11F的酶学表征和结构特点

Enzymatic and structural characterization of a zearalenone lactone hydrolase ZHD11F from Phialophora attae

摘要:

玉米赤霉烯酮(zearalenone，ZEN)是一种雌激素类真菌毒素，可以与动物的雌激素受体竞争性结合，导致动物生殖系统内的激素紊乱。ZEN内酯水解酶(ZEN lactone hydrolase，ZHD)可以水解ZEN中的内酯键，进而使其转化为无雌激素毒性的产物。【目的】利用生物信息学分析以及酶学性质探索，鉴定出一个具有新特性的ZEN内酯水解酶。【方法】构建pET28a-zhd11f表达载体，在大肠杆菌BL21(DE3)中诱导表达ZHD11F，利用Ni-NTA纯化得到ZHD11F，对其酶学性质进行分析，并通过结构模拟和分子动力学分析阐明ZHD11F低温活性的机制。【结果】ZHD11F以ZEN为底物，比酶活为40.04 U/mg，最适反应温度与pH值分别为35 °C和8.0，在pH 6.0–9.5的范围内具有超过60%的酶活力，在35 °C以下具有较好的热稳定性，能够耐受多种金属离子。ZHD11F在10 °C和20 °C时，其活性分别保持20%和40%。更多的loop区增加了结构的柔韧性是该酶稳定性较差、在低温活性比较高的主要原因。【结论】Phialophora attae是瓶霉属的一种真菌，目前此真菌来源的酶极少被鉴定。关于本研究将Phialophora attae来源的ZEN内酯水解酶ZHD11F，在大肠杆菌中成功可溶性表达并得到纯酶，表征分析显示该酶是目前报道的第一个低温ZEN内酯水解酶，为研究此类酶的耐冷机制、广温度范围提供了候选，同时拓展了Phialophora attae来源酶的功能研究。

关键词:玉米赤霉烯酮;ZEN内酯水解酶;低温活性;异源表达;酶学性质

Abstract:

Zearalenone (ZEN) is an estrogen-like mycotoxin that can competitively bind to animal estrogen receptors, resulting in hormonal disorders in the reproductive system of animals. ZEN lactone hydrolase (ZHD) can hydrolyze the lactone bond of ZEN to non-toxic products. [Objective] By bioinformatics analysis and enzymatic property exploration, a ZEN lactone hydrolase with new properties was identified. [Methods] The pET28a-zhd11f expression vector was constructed and introduced into Escherichia coli BL21(DE3). ZHD11F was obtained by purification with Ni-NTA, and its enzymatic properties were analyzed. Furthermore, the mechanism of its low-temperature activity was elucidated by structural simulation and molecular dynamics. [Results] With ZEN as the substrate, the specific activity of ZHD11F was 40.04 U/mg, and its optimum reaction temperature and pH value were 35 °C and 8.0 respectively. The enzyme activity of ZHD11F exceeded 60% in the range of pH 6.0–9.5, and it showed good heat stability below 35 °C and could endure a variety of metal ions. The activity of ZHD11F remained 20% and 40% at 10 °C and 20 °C, respectively. A number of loop regions led to increased structural flexibility, which was the main reason for the poor stability of the enzyme and its high activity at low temperature. [Conclusion] Phialophora attae was a fungus belonging to the genus Phialophora, and the enzymes derived from this fungus were rarely identified. In this study, the ZEN lactone hydrolase ZHD11F from P. attae was successfully expressed in E. coli and pure enzyme was obtained. Characterization analysis showed that this enzyme was the first low-temperature ZEN lactone hydrolase reported so far, which provided reference for studying the cold-tolerance mechanism and wide-temperature range of such enzymes, and also expanded the functional study of enzymes derived from P. attae.

Key words:zearalenone;zearalenone lactone hydrolase;low-temperature activity;heterologous expression;enzymatic properties