Heterologous Expression Of Zearalenone Degrading Lactonases From Gliocladium Spp.and Its Degradation Characteristics

Zearalenone（ZEA）is an estrogenic mycotoxin,which is widely distributed in cereals and their processed products and poses a serious threat to livestock and poultry breeding and human health.Therefore,it is urgent to study the detoxification technology of zearalenone and elucidate its detoxification mechanism.According to the literature,lactonase,laccase,and peroxidase are the three kinds of enzymes with ZEA degradation functions.The lactone hydrolase ZHD101 has good application potential since it can degrade ZEA into non-toxic products without causing any damage to crops.In this study,the lactone hydrolase gene zhd101 sequence in Clonostachys rosea was used as a template to screen homology genes from the genome-wide database of five strains of Gliocladium spp.,and heterologous expression was performed in E.coli to obtain novel zearalenone degrading lactone hydrolases ZHD2 and ZHD4.The ZEA degradation ability of the novel enzymes was analyzed by cell degradation and enzyme degradation tests.The mechanism of ZEA degradation by lactonase ZHD2 and ZHD4 was preliminarily explored by enzymatic properties analysis,protein structure analysis,and degradation products analysis.This study laid a theoretical foundation for zearalenone enzymatic detoxification and provided theoretical guidance for the application and promotion of zearalenone enzymatic detoxification technology.The main contents and results of this study are as follows:1.Based on the zhd101 sequence and the whole genome sequences of G.Rosea,G.Catenulatum,G.Penicilloides,G.Deliguescens,and G.Viride constructed in our laboratory,6 homology genes of zhd101 were screened,including 3 genes with high similarity（percentage of identity>95%）and 3 genes with low similarity（sequence percentage of identity>50%）.According to the percentage of identity,four genes（zhd1,zhd2,zhd3,and zhd4）were selected to construct expression vectors for heterologous expression;2.ZHD1,ZHD2,ZHD3,and ZHD4 were found to have the ability to degrade ZEA,it was found that ZHD2 and ZHD4 had degradability,while ZHD1 and ZHD3did not.According to the key amino acid position and sequence alignment analysis of ZHD101,the mutant enzymes ZHD1m（Y33L,M135L,S153V,K158V）and ZHD3m（Y33L,M135L,S153V,K158V）were designed and expressed.The results showed that ZHD1m and ZHD3m still had no degradation effect on ZEA;3.ZHD2,ZHD4 and ZHD101 were purified by affinity chromatography and purified by SDS-PAGE.Results show that ZHD2 and ZHD4 optimal p H value of 9,optimal temperature 45℃,and the degree of inhibition of enzyme activity by the metal ions from strong to weak,in order:Zn²⁺>Cu²⁺>Ni²⁺>Fe³⁺>Mg²⁺.In addition,the divalent metal ion chelator EDTA inhibited the enzymatic activities of ZHD2 and ZHD4,indicating that some kind of divalent metal ion could promote the degradation of ZEA by these enzymes.The Km value of ZHD2 was 39.72±14.72μmol L^-1,and the Kcat value was 1.6150 s^-1;ZHD4 has a Km value of 32.00±7.38μmol L^-1 and a Kcat value of 1.2744 s^-1;The positive control ZHD101 had a Km value of 41.71±9.42μmol L^-1 and a Kcat value of 1.536 s^-1;4.The products of ZEA degraded by ZHD2,ZHD4 and ZHD101 were analyzed by TLC.The results showed that ZEA could be degraded by ZHD2,ZHD4,and ZHD101 and form new products.The ZEA content in the reaction solution was determined by HPLC and LC-MS,and the results showed that ZEA was almost completely removed.Further protein structure analysis showed that ZHD2,ZHD4,and ZHD101 had the same catalytic triad Ser102-His242-Glu126,and the ester bond of the ZEA lactone ring was attacked by Ser102 at position 102.After the ester bond of ZEA was cleaved,ZEA loses estrogenic toxicity and degrades into non-toxic products.Finally,cytotoxicity experiments also proved that ZHD2,ZHD4,and the positive control ZHD101 can degrade ZEA into products of non-estrogenic activity.In conclusion,two novel zearalenone degrading lactone hydrolases ZHD2 and ZHD4 were screened,and their degradation characteristics were studied.Compared to ZHD101,ZHD2 and ZHD4 catalyze ZEA degradation at similar rates,but have a higher affinity for ZEA.This study enriched the zearalenone-degrading enzyme resource pool and was of great significance to the zearalenone detoxification research and its application.