Improving The Thermal Stability Of Bacillus Subtilis Lipase A Via Computational Protein Designing

Enzyme plays a very important role in biocatalysis and synthetic biology.The structure of natural enzymes is mostly unstable.It is difficult to meet the requirements of high temperature resistance in industrial application,so the transformation of enzyme thermal stability has always been a long-term goal in the field of enzyme engineering.Although the traditional directed evolution can screen enzymes according to the specific requirements of industrial applications,it usually requires several rounds of mutation and screening,and requires high-throughput screening methods,which is time-consuming and labor-consuming.On the other hand,rational design is more purposeful,more efficient and faster.Based on the structure of enzyme,computer-aided rational design is more and more widely and deeply used in the field of enzyme engineering.Because of its simple structure and low molecular weight,Bacillus subtilis lipase A(BSLA)has great application potential in the production of biodiesel and detergents,and it is often used as a model protein for computational protein design.In this research,the multiple calculation design strategy based on enzyme cavity analysis,protein solvent accessible surface area and consensus sequence analysis successfully improved the thermal stability of BSLA,and preliminarily explored the calculation and design method and expression of non-natural covalent bonds,which laid a foundation for the related research of non-natural amino acids.The main results are as follows:(1)Cavity of BSLA was analyzed by Rosetta-VIP,the mutation which was beneficial to the filling of the structure cavity(??E<0)was selected,followed by the solvent accessible surface area and conservation analysis.The thermal stabilities of 6 out of 16 designed single-point mutants were improved,with a maximum?T_m value of 3.18°C.The thermal stability of mutation with lower energy value,reduced accessible surface area,while conformed conservation,was more likely to be improved.(2)These 6 mutations were further used for combination mutation,the maximum?T_m of the two-point and three-point combination mutants were 4.04°C and 5.13°C,respectively.The T_m of the four-point combination mutant was increased by 7.30°C.The T_m of the six-point combination mutant was increased by 7.43°C.(3)Green fluorescent protein(GFP)as a model protein,the expression system of unnatural amino acids in double plasmids was constructed successfully.11 BSLA mutants containing non-natural covalent bonds were obtained by Rosetta calculation and design.Through energy value evaluation and near-attack conformation analysis,3 well-designed mutants:Z6C173,Z157C14 and Z31C174 were obtained,and their expression conditions were optimized..