Improving The Thermal Stability Of Proteus Mirabilis Lipase Via Computational Design

The modification of enzyme thermal stability is one of the hot topics in protein engineering.Traditional protein engineering,such as directed evolution,often produces industrial variants at the cost of low screening efficiency.In recent years,the development of various computational design strategies provides more ideas for the improvement of enzyme thermal stability.In this study,lipase from Proteus mirabilis（PML）was used as a model protein.Based on its structural information and sequence information,two different multiple computational strategies were developed to improve its thermal stability,so as to further expand its industrial application and provide a more time-saving choice for industrial transformation of other biological enzymes.The main results are as follows:（1）Heterologous expression of PML.A recombinant Escherichia coli strain containing p ET28a（+）-PML was constructed.The enzyme activity and thermal stability of PML were characterized.The specific activity of PML was 33.48 U·mg^-1 whenρ-nitrophenyl laurate was used as substrate at 35℃.And its melting temperature（T_m）was 49.96℃.（2）Strategy 1:Improve the thermal stability of PML based on energy and evolution analysis.The complementary algorithm-based ABACUS,PROSS,and Fold X were employed for positive selection of PML mutations,and their pairwise intersections were further subjected to negative selection by PSSM and G_REMLIN to narrow the mutation library.Thereby,18potential single-point mutants were screened out.According to experimental verification,7mutants had improved T_m.Five of these with enhanced activity were selected for combination by greedy accumulation.Finally,the T_m of the five-point combination mutant MA10 increased by 10.63℃,and the relative activity was 140%that of the WT.（3）Strategy 2:Reconstruction of PML flexible region.By comparing molecular dynamics simulations at different temperatures and B-factor,the flexible sites of PML were determined,and 22 single point mutation designs were obtained by Rosetta free energy calculation and evolution analysis.The T_m of 7 mutants was improved,theΔT_m of G206E was the highest,which was 5.19℃.Four single point mutations were combined to obtain MB6,whose T_m was increased by 12.47℃and relative enzyme activity was 180%of that of the WT.The synergistic effect of G206E,E207A and K208G was the main reason for the improvement of its thermal stability.Two single point mutations unique to strategy 1 library were combined with MB6 to obtain a six-point combination mutant MC,whose T_m increased by 13℃and the relative enzyme activity reached 180%of that of the WT.The resistance of WT,MB6 and MC to organic solvents was characterized.PML and its mutants showed good resistance to six commonly used organic solvents such as methanol.（4）Molecular dynamics simulations were performed on the combined mutants MA10 and MB6.Molecular dynamics simulations showed that new forces were generated at and around the mutation site of MA10 and MB6,and the rearrangement of forces around 206-208 residues may stabilize the Ca²⁺binding site,which is critical to the stability of PML.