Molecular Modification And Mechanism Study Of Improving Zearalenone Hydrolase's Thermal Stability Via Multiple Computational Design

The zearalenone hydrolase(ZHD101)derived from Clonostachys rosea can effectively degrade the mycotoxin zearalenone(ZEN)in agricultural by-products of grain and feed.However,the inherent low thermal stability of native enzyme limits its application.Traditional directed evolution can improve the specific properties of proteins,but it requires a lot of work to establish and screen mutation libraries.With the rapid development of structural biology,computational biology,and computer technology,advanced protein-related algorithms continue to emerge,and computational protein design has become an important direction in protein engineering.In this paper,ZHD101 is used as a model protein.Combined with a variety of computational methods,its molecular structure is designed to improve thermal stability through two different strategies,and the properties are verified by experiments.The main findings are as follows:(1)Based on the dynamic characteristics of enzyme,the flexible and temperature sensitive areas in ZHD101 were screened and modified to improve its structural stability.By comparing the molecular dynamics simulation trajectories at different temperatures,32 flexible sites were selected.Then by the position-specific score and enzyme conformation free energy calculation,12 mutants were screened from 608 virtual saturation mutants on the 32 flexible sites.Experiments have verified that three of the mutants,N156F,S194T and T259F,show a significant increase in their thermal melting temperature(?T_m>4°C)and their enzyme activity is similar to or even higher than that of the wild type(relative enzyme activity is 95.8%,131.6%,169.0%).Molecular dynamics simulation analysis showed that the improvement of the thermal stability of the mutant structures will be affected by the introduction of NH-?interaction,the rearrangement of the salt bridge network,and the filling of holes on the molecular surface.The3 mutants were combined iteratively,and N156F/S194T showed the highest thermal stability(?T_m=6.7°C).(2)Based on the combined mutant design provided by the PROSS strategy,the properties of the sites are optimized and recombined to reduce the loss of enzyme activity during the thermal stability modification process.The thermostability and enzyme activity of 7 combined mutants(D1-D7)provided by the PROSS computational strategy were tested and found that their thermal melting temperature increased sequentially,but the relative enzyme activity decreased sequentially with the addition of more mutation sites.In order to achieve an efficient combination of mutation sites,33 mutation sites were constructed and tested for single-point mutations.Among them,we re-screened 4 mutants S8T,K66D,T138L and V251E.They have an increased thermal melting temperature(?T_m>2°C),and the relative enzyme activity(>90%)and residual enzyme activity(>40%)are close to or even higher than the wild type.The thermal melting temperature of the combined four-point mutant abcd(S8T/K66D/T138L/V251E)increased by 17.6°C,and the relative enzyme activity and residual enzyme activity reached118.4%and 103.6%,respectively.The molecular dynamics simulation showed that the fluctuation of abcd is lower,the structure is more compact,and there are fewer transitions of subconformation states.These conformational changes may be due to the four mutations,which are caused by the salt bridge interaction and CH-?interaction,respectively,and the thermal stability is improved.This study successfully achieved the improvement of the thermal stability of ZHD101through computational design,which provided relevant gist and theoretical basis for improving its application potential.At the same time,this study shows the feasibility of the modification of enzyme stability based on the virtual saturation mutation in the flexible region and the screening and optimization of automated computational design strategies.The information of the conformational free energy calculation and conformational dynamics in the molecular modification is helpful to further explore virtual modification based on computational design and realize efficient enzyme modification.