Gene Resource Mining And Improvement In Catalytic Efficiency Of Mycotoxin Zearalenone Hydrolase

As a kind of mycotoxin like estrogen produced by Fusarium sp.,zearalenone(ZEN)not only damages the liver and kidney of animals,reduces the immune function,but also affects the reproductive performance of animals,and brings serious harm to livestock breeding and food safety.Lactone hydrolase can remove the mycotoxin ZEN in the feed with an efficient and environmentally friendly way,which can effectively promote the green and healthy breeding of the livestock industry.The purpose of this study is to obtain the enzyme that can hydrolyze ZEN efficiently,to study its biochemical characteristics and catalytic mechanism,to explore the efficient expression scheme,and to promote its application in the livestock breeding industry.In order to mining the enzyme gene resources with ZEN hydrolyze activity,a retrieval and comparison analysis were conducted in the NCBI database using the amino acid sequence of ZHD101,which was a widely studied lactone hydrolase with ZEN hydrolysis activity.18 potential ZEN hydrolase genes with low similarity were obtained,and the activity was screened after heterologous expression in Pichia pastoris.The results showed that all of genes were successfully heterologous expressed and 5 of them showed the significant activity.Among them,the hypothetical protein ZHD607 from Phialophora americana showed the 100% of degradation efficiency to ZEN,the high specific activity was 4940 U/mg.In order to further study the catalytic mechanism of ZHD607,the lactone hydrolase RmZHD from Rhinocladiella mackenziei with high similarity with ZHD607(74%)was selected as the comparative study material.RmZHD showed the 2.5-fold higher specific activity(12288 U/mg)than ZHD607.Through homologous modeling of ZHD607 and the structure and sequence comparison analysis which shared the RmZHD,the key amino acid sites and regions which binding to the substrate were identified,and 8 mutants related to ZHD607 catalysis were designed.The results showed that the catalytic efficiency of mutants ZHDM1(14419 U/mg)and I160Y(16652 U/mg)were significantly increased,which were 2.9 and 3.4 times higher than that of wildtype,respectively.Furthermore,the mechanism of the improvement in catalytic efficiency was analyzed by molecular dynamics simulation.The results showed that the change of side chain at position 160 and the torsion of Pro135 and Leu138 on loop 136-142 significantly enhance the hydrophobic interaction with the substrate,thus improving the binding ability of the enzyme to substrate.The low level expression of lactone hydrolase has always been the bottleneck problem restricting its application.Based on the fusion expression strategy,we explored the efficient expression of ZHD101 in P.pastoris.An acid xylanase XYNB from Aspergillus niger and an alkaline xylanase A11 from Bacillus sp.were fused with ZHD101 and expressed in P.pastoris X33,respectively.The results showed that the ZEN hydrolytic activity of the fusion protein XYNB + ZHD101 was 2.2-fold higher than that of ZHD101.After 48 h induction,the enzyme activity of XYNB + ZHD101 was reached to 983.77 U/mL.The results of qPCR and specific activity were consistent with the SDS-PAGE analysis,which indicating that the fusion expression can significantly improve the expression of ZHD101.In addition,xylanase activity of the fusion protein XYNB + ZHD101 has a significant linear relationship with ZEN hydrolysis activity,thus the activity of ZHD101 can be characterized by detecting the xylanase activity in the fermentation process,which lays a foundation for its large-scale industrial production.In this study,the lactone hydrolase ZHD101 with ZEN hydrolytic activity which has crystal structure reported was taken as the research background,several novel genes of ZEN hydrolase with high activity were obtained,which enriched the ZHD family members.The catalytic efficiency of ZHD607 was studied based on the modeling structure of ZHD607 for molecular improvement,and two mutants with significantly increased activity were obtained.And their catalytic mechanisms for the efficient hydrolysis of ZEN were explained.According to the fusion expression strategy,the expression of ZHD101 in P.pastoris was significantly increased.Combined with the indirect enzyme activity assay method,the study provided a strong support for large-scale production in industrial.