Abstract:Zearalenone (ZEN) is an estrogen-like mycotoxin that can competitively bind to animal estrogen receptors, resulting in hormonal disorders in the reproductive system of animals. ZEN lactone hydrolase (ZHD) can hydrolyze the lactone bond of ZEN to non-toxic products. [Objective] By bioinformatics analysis and enzymatic property exploration, a ZEN lactone hydrolase with new properties was identified. [Methods] The pET28a-zhd11f expression vector was constructed and introduced into Escherichia coli BL21(DE3). ZHD11F was obtained by purification with Ni-NTA, and its enzymatic properties were analyzed. Furthermore, the mechanism of its low-temperature activity was elucidated by structural simulation and molecular dynamics. [Results] With ZEN as the substrate, the specific activity of ZHD11F was 40.04 U/mg, and its optimum reaction temperature and pH value were 35 °C and 8.0 respectively. The enzyme activity of ZHD11F exceeded 60% in the range of pH 6.0–9.5, and it showed good heat stability below 35 °C and could endure a variety of metal ions. The activity of ZHD11F remained 20% and 40% at 10 °C and 20 °C, respectively. A number of loop regions led to increased structural flexibility, which was the main reason for the poor stability of the enzyme and its high activity at low temperature. [Conclusion] Phialophora attae was a fungus belonging to the genus Phialophora, and the enzymes derived from this fungus were rarely identified. In this study, the ZEN lactone hydrolase ZHD11F from P. attae was successfully expressed in E. coli and pure enzyme was obtained. Characterization analysis showed that this enzyme was the first low-temperature ZEN lactone hydrolase reported so far, which provided reference for studying the cold-tolerance mechanism and wide-temperature range of such enzymes, and also expanded the functional study of enzymes derived from P. attae.
