结构指导的玉米赤霉烯酮水解酶的热稳定性改造

Structure-guided engineering for improving the thermal stability of zearalenone hydrolase

玉米赤霉烯酮是世界上污染最为广泛的一种镰刀菌(Fusarium)毒素,严重危害牲畜以及人类的健康。来源于粉红螺旋聚孢霉(Clonostachys rosea)的玉米赤霉烯酮水解酶(zearalenone hydrolase,ZHD)能有效降解饲料中的玉米赤霉烯酮,然而饲料加工中的高温环境限制了该酶的应用。基于结构特征的理性设计可为酶的热稳定性改造提供指导。本研究首先基于蛋白质结构比对(multiple structure alignment, MSTA)筛选ZHD的结构灵活区,随后基于序列保守性打分以及构象自由能计算设计突变文库,得到基于136号和220号残基的9个单点突变设计。结果表明,9个突变体的热熔融温度(Tm)提高了0.4–5.6℃,其中S220R和S220W热稳定性表现最好,Tm分别提高了5.6℃和4.0℃,45℃下的热半失活时间分别延长了15.4倍和3.1倍,相对酶活分别为野生型的70.6%和57.3%。分子动力学模拟分析表明突变位点及附近区域的作用力得到了增强,突变体S220R和S220W的220-K130氢键成键概率分别增加了37.1%和19.3%、K130-D223盐桥成键概率分别增加了30.1%和12.5%,为ZHD热稳定性的提高作出了贡献。这项工作表明结合天然酶的结构比对、序列分析及自由能计算的热稳定性改造策略的可行性,并获得了热稳定性增强的ZHD变体,为ZHD在工业上的应用打下基础。

Zearalenone is one of the most widely polluted Fusarium toxins in the world,seriously endangering livestock and human health.Zearalenone hydrolase(ZHD)derived from Clonostachys rosea can effectively degrade zearalenone.However,the high temperature environment in feed processing hampers the application of this enzyme.Structure-based rational design may provide guidance for engineering the thermal stability of enzymes.In this paper,we used the multiple structure alignment(MSTA)to screen the structural flexibility regions of ZHD.Subsequently,a candidate mutation library was constructed by sequence conservation scoring and conformational free energy calculation,from which 9 single point mutations based on residues 136 and 220 were obtained.The experiments showed that the thermal melting temperature(Tm)of the 9 mutants increased by 0.4–5.6℃.The S220R and S220W mutants showed the best thermal stability,the Tm of which increased by 5.6℃and 4.0℃compared to that of the wild type.Moreover,the thermal half-inactivation time at 45℃were 15.4 times and 3.1 times longer,and the relative activities were 70.6%and 57.3%of the wild type.Molecular dynamics simulation analysis showed that the interaction force at and around the mutation site was enhanced,contributing to the improved thermal stability of ZHD.The probability of 220-K130 hydrogen bond of the mutants S220R and S220W increased by 37.1%and 19.3%,and the probability of K130-D223 salt bridge increased by 30.1%and 12.5%,respectively.This work demonstrated the feasibility of thermal stability engineering strategy where the structural and sequence alignment as well as free energy calculation of natural enzymes were integrated,and obtained ZHD variants with enhanced thermal stability,which may facilitate the industrial application of ZHD.