玉米赤霉烯酮水解酶ZHD101胰蛋白酶抗性的理性分子设计

Rational design improves the resistance of zearalenone hydrolase ZHD101 to trypsin

摘要:

【背景】 玉米赤霉烯酮(zearalenone, ZEN)是一种由镰刀菌属(Fusarium)真菌产生的毒素，可引起动物体生殖功能的紊乱，破坏其免疫系统，对人类的健康和动物的生产性能造成严重的危害。来源于粉红螺旋聚孢霉(Clonostachys rosea)的内酯水解酶ZHD101，又称玉米赤霉烯酮降解酶，能够通过水解反应高效地将玉米赤霉烯酮毒素降解为无毒产物，可作为饲用酶制剂减少霉菌毒素暴露对动物的危害。【目的】 动物胃肠道消化酶的水解作用会显著降低饲用酶制剂的使用效率。因此，研究如何提高玉米赤霉烯酮水解酶的消化酶抗性，以提高酶制剂的使用效率是非常必要的。【方法】 通过理性分子设计，突变位于与胰蛋白酶相互作用界面的关键氨基酸，减弱ZHD101与胰蛋白酶的结合，提高ZHD101对胰蛋白酶的耐受性，以达到提高ZHD101使用效率的目的。【结果】 突变体ZHD101^K254Q和ZHD101^K254Q/K262Q在人工肠液中半衰期较野生型分别延长了61.8%和57.8%。突变体酶最适pH值均为9.0−10.0，最适反应温度约为40 ℃，酶学性质与野生型无显著差异。【结论】 本研究对ZHD101的胰酶抗性理性设计提升了其在饲料工业中的应用价值，也为其他饲用酶的分子改良提供了策略。

Abstract:

[Background] Zearalenone (zearalenone, ZEN), a toxin produced by Fusarium, poses a serious risk to human health and animal production performance as it can disrupt the reproduction and damage the immune system of the body. The lactonase ZHD101 from Clonostachys rosea, also known as ZEN hydrolase, degrades ZEN into non-toxic products by hydrolysis and can be used as a feed enzyme additive to reduce the mycotoxin exposure in animals. [Objective] The digestive enzymes of animals significantly reduce the efficacy of feed enzyme additives, and it is therefore crucial to investigate the methods enhancing the trypsin resistance of feed enzymes to improve their utilization efficiency. [Methods] Rational design was employed to mutate the key amino acid residues of ZHD101 at the interaction interface with trypsin, thereby weakening the binding affinity of ZHD101 to trypsin and enhancing its trypsin resistance. [Results] Compared with that of the wild type, the half-life of ZHD101^K254Q and ZHD101^K254Q/K262Q in simulated intestinal fluid was prolonged by 61.8% and 57.8%, respectively. The mutated enzymes showcased the best performance at pH 9.0−10.0 and about 40 ℃, with similar enzymatic properties to the wild type. [Conclusion] The rational design of ZHD101 for improving the trypsin resistance enhances the application of this enzyme in the feed industry and provides clues for molecular modification of other feed enzymes.