Rational design improves the resistance of zearalenone hydrolase ZHD101 to trypsin

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.

Key words:zearalenone hydrolase;trypsin resistance;rational design;molecular simulation