来源于麦氏喙枝孢霉的玉米赤霉烯酮水解酶在毕赤酵母中的高效表达及活性分析

High expression and characterization of a novel zearalenone hydrolase from Rhinocladiella mackenziei in Pichia pastoris

摘要:

【背景】玉米赤霉烯酮(zearalenone，ZEN)及其衍生物是一群具有雌激素活性的霉菌毒素，广泛存在于被霉菌污染的谷物中，造成食品业和畜牧业的巨大损失。利用专一性高的水解酶进行生物转化可有效去除玉米赤霉烯酮。【目的】构建高效表达玉米赤霉烯酮水解酶的酵母系统，以促进玉米赤霉烯酮水解酶的研究和工业应用。【方法】将来源于麦氏喙枝孢霉(Rhinocladiella mackenziei CBS 650.93)的Rmzhd基因转入毕赤酵母中，筛选获得高效表达菌株，通过高效液相色谱分析发酵液中重组酶的性质。【结果】发酵液中RmZHD对ZEN的酶活力为16.67 U/mL，对α-ZOL的酶活力为9.85 U/mL。SDS-PAGE检测表达产物的分子量，与理论值30.7 kD符合，且发酵上清液蛋白纯度高。RmZHD的最适pH值为9.6，最适温度为45 °C，并具有较好的耐热性。【结论】研究结果为玉米赤霉烯酮水解酶的异源表达及其潜在的工业应用提供了一定的指导。

Abstract:

[Background] Zearalenone (ZEN) and its derivatives are a series of mycotoxins with estrogenic activity which are widespread in mold contaminated cereals. Mycotoxin contamination has caused huge loss to food and feed industries. To develop an efficient solution is an urgent task. [Objective] In order to promote the research and application of zearalenone hydrolase, we constructed the recombinant plasmid that highly expressed zearalenone hydrolase in Pichia pastoris. [Methods] The Rmzhd gene from Rhinocladiella mackenziei CBS 650.93 was transformed into Pichia pastoris GS115 for heterologous expression. The high expression strain was screened and the characterization of recombinant RmZHD was analyzed by high performance liquid chromatography (HPLC). [Results] The ZEN and α-ZOL degradation activities of the recombinant RmZHD from the supernatant were 16.67 U/mL and 9.85 U/mL, respectively. The molecular mass of RmZHD was about 30.7 kD, and the purity of protein was very high, as shown by SDS-PAGE. The optimal pH and temperature of recombinant RmZHD were determined as pH 9.6 and 45 °C, and RmZHD has a good thermostability. [Conclusion] The results can provide guidance for the heterologous expression and potential industrial application of zearalenone hydrolase.