| Zearalenone is one of the most widely polluted mycotoxins in the world,seriously endangering livestock and human health.Zearalenone hydrolase(ZHD101)derived from Clonostachys rosea can effectively degrade zearalenone in feed,but the high temperature environment in feed processing limits the application of this enzyme.With the development of computational biology,structural biology,and biophysics,there are a variety of computational design strategies in the field of protein engineering that can provide efficient guidance for the engineering of proteins.ZHD101 as the research object,this paper selects potential mutation libraries with improved thermal stability from structural,evolutionary and energy factors based on computational design strategies,and verifies them through biological experiments.Furthermore,protein crystallization experiments were carried out on enzyme mutation with significantly enhanced thermal stability,and the mechanism of improved thermal stability was analyzed by molecular dynamics simulation.The main findings are as follows:(1)First,the secondary structure of ZHD101 with high plasticity in evolution was screened out through protein structure comparison,and it was used as the target mutation region,and then the sequence conservation scoring and amino acid mutation free energy calculation were performed on the target mutation region,and 15 single-point mutations were obtained.Experiments showed that the thermal melting temperature(T_m)of the 15 mutants increased by0.4～9.4°C,among which T138L,S220R,and S220W performed the best thermal stability,and their T_m increased by 9.4°C,5.6°C,and 4.0°C;the thermal half-inactivation time was extended by 7.5 times,15.4 times and 3.1 times respectively;the relative enzyme activities of the mutants were 146.9%,70.6%and 57.3%.The improvement of thermal stability of S220R and S220W was accompanied by the loss of enzyme activity.According to the analysis,residue 220 is located in the active pocket,9.3(?)away from the catalytic center,and the increase of the amino acid side chain caused by the mutation may make it difficult for the substrate to enter the active region;(2)The mechanism of the improved thermal stability of the ZHD-T138L mutant was analyzed based on the crystal structure.The change of the B-factor of the crystal structure shows that the the mutation reduces the conformational fluctuation of the mutation region;further analysis showed that the interaction force at and around the mutation site was enhanced,and a new hydrogen bond R185-L141 and a salt bridge R185-D143 were introduced to the more flexible cap region of the protein,and the hydrophobic cluster area of the cap region increases by 415.8(?)~2.The enhanced force may be one of the reasons for the improved thermostability of the mutation.(3)The 200 ns molecular dynamics simulation experiment was carried out.Molecular dynamics simulation analysis showed that,compared with the higher conformational volatility of the wild-type cap region,the RMSF values of the cap region of the mutations T138L,S220R and S220W were reduced.Free energy landscape analysis found that the conformational states of the mutants were more compact than those of the wild type,indicating a more stable structure.At the same time,the force at the mutation site and nearby regions was enhanced,the 138-L141and R185-L141 hydrogen bonding probabilities of the mutation T138L increased by 13.4%and19.7%and the R185-D143 salt bridge bonding probabilities increased by 47.2%,respectively.The 220-K130 hydrogen bonding probability of mutations S220R and S220W increased by37.1%and 19.3%,and the K130-D223 salt bridge bonding probability increased by 30.1%and12.5%,respectively. |
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