Expression, functional analysis and mutation of a novel neutral zearalenone-degrading enzyme

Abstract

The crops and grains were often contaminated by high level of mycotoxin zearalenone (ZEN). In order to remove ZEN and keep food safe, ZEN-degrading or detoxifying enzymes are urgently needed. Here, a newly identified lactonohydrolase responsible for the detoxification of ZEN, annotated as Zhd518, was expressed and characterized. Zhd518 showed 65% amino acid identity with Zhd101, which was widely studied for its ZEN-degrading ability. A detailed activity measurement method of ZEN-degrading enzyme was provided. Biochemical analysis indicated that the purified recombinant Zhd518 from E. coli exhibited a high activity against ZEN (207.0 U/mg), with the optimal temperature and pH of 40 °C and 8.0, respectively. The Zhd518 can degrade ZEN derivatives, and the specific activities against α-Zearalenol, β-Zearalenol, α-Zearalanol and β-Zearalanol were 23.0 U/mg, 64.7 U/mg, 119.8 U/mg and 66.5 U/mg, respectively. The active sites of Zhd518 were predicted by structure modeling and determined by mutation analysis. A point mutant N156H exhibited 3.3-fold activity against α-Zearalenol comparing to Zhd518. Zhd518 is the first reported neutral and the second characterized ZEN-degrading enzyme, which provides a new and more excellent candidate for ZEN detoxifying in food and feed industry.

Keywords: Enzymatic characterization; Mutation; ZEN-degrading enzyme; Zearalenone.