| Zearalenone(ZEN)is a regenerative cyclic lactone that was first isolated by Stob in 1962 from moldy corn.ZEN is a mycotoxin biosynthesised by the polyketide pathway of various Fusarium sp.The high content of ZEN is always present in crops and grain by-products,including corn,barley and wheat,especially under environmental conditions conducive to fungal growth.ZEN can be converted into ?-zearalenol,?-zearalenol,?-zearalanol and ?-zearalanol.ZEN and its derivatives can enter into humans and animals,then they compete with estrogen receptors and lead to many reproductive health problems.It is reported that many methods have been invented to solve this problem,including physical methods,chemical methods,and biological methods.The principles of the biological methods are degrade ZEN to produce non-toxic products by the microbial cell adsorption,the microorganisms themselves or their enzymes.Biological methods are safer,more effective,more specific,and more economical.The present study focus on the investigation of new ZEN-degrading enzymes..The reported ZEN-degrading enzymes are Zhd101,ZEN-JJM and Zhly-6,whose encoding genes presented 99%and 98%sequence identity.The crystal structure of Zhd101 has been resolved.These enzymes act on the opening and deacidification of ester bonds to produce an alkyldiphenyl ring.Another kind enzyme with detoxification function for ZEN is peroxiredoxin(EC 1.11.1.15),which has detoxification effect of ZEN in the presence of hydrogen peroxide.However,the presence of hydrogen peroxide may affect the application of the enzyme in food and feed.1,Cloning,enzymatic characterization and mutation of neutral ZEN-degrading enzyme Zhd518.After the NCBI sequence alignment,a gene derived from Rhonocladiella mackenziei CBS 650.93 was artificially synthesized and named as zhd518,and protein Zhd518 presented 65%identity to protein Zhd101.The strain BL21-pET28a-zhd518 was constructed for heterologous expression.The purified protein was detected to be 29 kDa by SDS-PAGE.Feature analysis showed that the optimum temperature and optimum pH of Zhd518 were 40? and pH 8.0,respectively.However,the optimum temperature and optimum pH of Zhd101 in the previous report were 40? and pH 9.5,respectively.Therefore,the ZENdegrading enzyme Zhd518 in this study was the first reported neutral ZEN-degrading enzyme.The effects of various metal ions on the activity of Zhd518 towards ZEN were determined.The addition of EDTA did not affect its activity.These results suggested that Zhd518 is a metal-independent enzyme that same with Zhd101.One unit of ZEN-degrading enzyme activity was defined as the amount of enzyme that degrade 1?g of substrate per minute under the assay conditions.Zhd518 showed different hydrolytic activities towards five measured substrate.Xu et al.solved the complex structure of Zhd101,and found that the mutant V153H of Zhd101 exhibited 3.7-fold activity against ?-ZOL compared to the wildtype enzyme.Sequence alignment displayed that the 153rd site of Zhd101 corresponding to the 156th site of Zhd518,then the 156th site of N of Zhd518 was mutated to H,followed by the activity determination.The mutant N156H exhibited 3.3-fold activity against ?-ZOL compared to the wild-type enzyme,which is corresponding with the reports.On this basis,we screened five amino acids within the 3 A of catalytic triad and on the flexible loops region through the software,then each amino acid were mutated to alanine(A)respectively,resulting in five mutants:Zhd518(D34A),Zhd518(G35A),Zhd518(G214A),Zhd518(T217A),Zhd518(N242A).The results showned that the activities of Zhd518(D34A)and Zhd518(G35A)completely disappeared,the activity of Zhd518(G214A)lost 75%and the activities of Zhd518(T217A)and Zhd518(N242A)did not change.Therefore,mutation on 34site,35site,and 214 site may have severe effect on enzyme activities,and on 217site and 242site may have no effect on enzyme activity.This experiment provided a basis for further directed evolution.2,Expression of neutral ZEN-degrading enzyme Zhd518(N156H)in P.pastoris.In our study,based on the zhd518(N156H)gene,a modified gene was synthesized based on Pichia pastoris codon bias,named zhd518s.We then constructed the recombinant plasmid pHBM905M-zhd518s in vitro as following steps,the pHBM905M-zhd518s plasmid was firstly linearized and were excised of E.coli replication initiation zone and ampicillin resistance gene though Sal I restriction enzyme digestion,and then it was converted into P.pastoris GS115.The shake-flask fermentation of positive clone showed that the expression of target protein increased with the fermentation days,which implied that zhd518(N156H)gene can be effectively expressed in P.pastoris.3,Cloning,enzymatic characterization and mutation of ZEN-degrading enzyme ZhdAY3.After the NCBI sequence alignment,a gene derived from Exophiala aquamarina CBS 119918 was artificially synthesized and named as zhdAY3,and protein ZhdAY3 presented 63%identity to protein Zhd101.The strain BL21-pET28a-zhdAY3 was constructed for heterologous expression.The purified protein was detected to be 29 kDa by SDS-PAGE.Feature analysis showed that the optimum temperature and optimum pH of ZhdAY3 were 40? and pH 9.5,respectively.The effects of various metal ions on the activity of ZhdAY3 towards ZEN were determined.The addition of EDTA did not affect its activity.These results suggested that ZhdAY3 is a metal-independent enzyme that same with Zhd101.ZhdAY3 showed different hydrolytic activities towards five measured substrate.Sequence alignment displayed that the V153rd site of Zhd101 corresponding to the N153th site of ZhdAY3,then the 153th site of N of ZhdAY3 was mutated to H,followed by the activity determination.The mutant N153H exhibited 2.1-fold activity against ?-ZAL compared and 1.4-fold activity against ?-ZAL to the wild-type enzyme that different from Zhd101 and Zhd518.This is a completely new feature and worth studying in-depth.In all,two new ZEN-degrading enzyme were cloned and heterologous expressed.The molecular modification were also investigated,which provide a foundation for ZEN detoxification. |
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