来源于Rhinocladiella mackenziei的玉米赤霉烯酮水解酶的表达纯化与酶学性质

Expression, purification and characterization of a novel zearalenone hydrolase from Rhinocladiella mackenziei

摘要:

【背景】玉米赤霉烯酮(Zearalenone，ZEN)是污染最广泛的霉菌毒素之一，对饲料行业和畜牧业造成了巨大的经济损失。目前研究最为广泛的玉米赤霉烯酮降解酶ZHD101因其热稳定性较差，无法满足工业应用上的要求。【目的】为实现玉米赤霉烯酮降解酶在工业上的应用，寻找酶学性质更突出的ZEN降解酶。【方法】基于对GenBank数据库的挖掘，发现一个来源于麦氏喙枝孢霉(Rhinocladiella mackenziei CBS 650.93)的Rmzhd基因，构建pET-46-Rmzhd质粒。利用大肠杆菌表达体系和亲和层析、离子交换纯化体系对蛋白进行表达和纯化，通过高效液相凝胶色谱分析酶学性质。【结果】发现一个新的ZEN水解酶RmZHD，RmZHD在pH 8.6和45 °C条件下的活性最高，而且具有较高的耐热性。结构分析表明，较高的盐桥数目和溶剂暴露脯氨酸含量可能是造成其高耐热性的原因。【结论】本研究为促进玉米赤霉烯酮降解酶在工业上的应用打下基础。

Abstract:

[Background] Zearalenone (ZEN) is one of the most widely contaminated mycotoxins, which causes huge economic losses to feed industry and animal husbandry. The lactonase ZHD101 is the most widely studied ZEN-detoxifying enzyme. However, its application in industry was limited by the low thermostability. [Objective] In order to realize the application of zearalenone-degrading enzyme in industry, this study explored a ZEN-detoxifying enzyme with more prominent enzymatic properties. [Methods] We found an Rmzhd gene from Rhinocladiella mackenziei CBS 650.93 in the GenBank database, and constructed the pET-46-Rmzhd plasmid. E. coli system was used to express the target protein. The protein was purified by using affinity chromatography and ion exchange purification system. Enzymatic properties were analyzed by high performance liquid chromatography (HPLC). [Results] In this study, we characterized a novel ZEN hydrolase, denoted as RmZHD. RmZHD exhibits the highest activity at pH 8.6 and 45 °C, and has a higher thermostability. Analyzing the structure of RmZHD, we found that the higher contents of salt bridges and solvent-exposed prolines might contribute to higher protein thermostability. [Conclusion] This study provides foundation for improving the industrial applications of zearalenone hydrolases.