Modification And Mechanism Study Of Bacillus Subtilis Lip A Stability Via Computational Design

The engineering of natural enzymes into higher thermostability industrial catalysts through laboratory evolution was the focus of extensive research in protein engineering.Many mature algorithms based on sequence and structure have been successfully used to design thermostable enzymes mutants efficiently.However,the obvious flexibility of enzyme structure and the high complexity of enzyme catalysis easily lead to activity lost in the process of thermostability modification,which was difficult to the efficient and low-cost application of thermostable enzymes.Therefore,obtaining thermostability mutants without activity loss was a major challenge in engineering.In this paper,Bacillus subtilis Lipase A(BSLA)was used as a model protein,the thermostable mutants were constructed by experiments,and the analysis of the conformational dynamics was introduced to explore the relationship between enzyme activity,thermostability,and conformational changes.Combined with experimental verification,the mutations were screened for optimization and combinatorial sequence design.The main findings are as follows:(1)In this study,the seven mutants were obtained by the Protein Repair One Stop Shop(PROSS)strategy,the mutant D7 had an improved thermal melting temperature of 13.6℃ and relative activity of 106.4%.Based on molecular dynamics simulations,local interaction analysis suggests that the introduction of π-π interactions and the improvement of surface hydrophilicity were the molecular mechanisms for enhanced thermal stability.Conformational analysis of the active sites in D2 indicated that the drift of the catalytic triad hydrogen bond network and the increase in the inactive state of S77 were responsible for the decrease in relative activity.(2)Based on the dynamic cross-correlation network,the shortest correlation distance analysis explored the allosteric mechanism of the active sites cause by the distal mutation sites.By removing the residues with smaller shortest correlation distances to active sites,the thermal melting temperature of the optimized mutant RD3 was improved to 8.10℃ and the relative activity was increased to 149.6% compared to the wild type.(3)Further,based on the thirteen single-point mutation designs obtained by the PROSS strategy,four mutation sites A15 S,F17S,G155 S,and I157 M were selected for full-path combination.The four-point combinatorial mutant with an increased thermal melting temperature of 10.51℃ and relative enzyme activity of 118%.Interaction analysis indicated that the epistasis interaction of adjacent residues and the improvement of surface hydrophobic packing were responsible for negative and positive epistatic effects,respectively.(4)Based on the discussion in(1)(2)(3),by selecting the fifteen positive single-point mutations in directed evolution,a mutation library was constructed.Seven-dimensional parameters such as thermostability contribution,structural information,and conformational dynamics parameters were selected.K-means analysis was used to divide the data points into clusters containing 3-9 data points with similar properties.The thermal melting temperature of the mutant C31 with 9 mutations was about 22℃ higher than that of the wild type.This strategy could be effectively used for combinatorial sequence design and improved screening efficiency.