Screening,Expression,Purification And Crystallography Study Of A Novel Zearalenone Hydrolase

Zearalenone（ZEN）is a non-steroidal mycotoxin with estrogenic activity produced mainly by Fusarium graminearum.It has a variety of derivatives:α/β-zearalenol（α-ZOL,β-ZOL）,zearalanone（ZAN）,α/β-zearalanol（α-ZAL,β-ZAL）.They are widely present in crop feeds contaminated by molds,causing great losses to the feed and livestock industries.Zearalenone hydrolase can hydrolyze ZEN to smaller non-toxic metabolites.α-ZOL exhibits higher toxicity than ZEN,but currently reported zearalenone hydrolases all exhibit less activity againstα-ZOL than against ZEN.Thus it is necessary to discover new zearalenone hydrolase that exhibits higher activity toα-ZOL.With this aim,the study explored the screening,expression purification,enzymatic property analysis,crystallographic studies of novel zearalenone hydrolases.We got four candidate lactone hydrolases after blast search in NCBI data base,MpZHD from Mycolicibacterium peregrinum,NmZHD from Nocarda mexicana,PsZHD from Paraburkholderia sp.7MH5 and EaZHD from Exophiala aquamarina CBS 119918.After expression,purification and enzymatic analysis,EaZHD showed the higher activity towardsα-ZOL than ZEN.Therefore,we performed further studies on EaZHD.Eazhd was expressed in E.coli.Target proteins were purified by using Ni-NTA affinity chromatography,2^ndNi-NTA affinity chromatography and DEAE ion exchange.In order to optimize the growth of protein crystals,different protein expression vectors,protein buffers and crystal culture conditions were screened.The Sso and Sac tags were added and produced constructs:EaZHD-C-Sso,N-Sso-EaZHD,EaZHD-C-Sac,N-Sac-EaZHD to help crystallization,besides EaZHDS102A was also constructed.The optimal crystallization precipitant conditions for co-crystallization of EaZHDS102A with the substrateα-ZOL were obtained:26%-28%PEG 5000 MME,0.1 M MES p H 6.5,and 0.1-0.2 M Ammonium Sulfate.The crystals were subjected to X-ray data collecting,and it can diffract to 2.9（?）resolution.It is identified several amino acid sites that play a key role in catalysis,after mutation and activity analysis of amino acids near the active center of EaZHD.