Fundamental Research On The Application Of Thermal Stability Modification Of Lipase In SMG1 And PCL Based On Biological Calculation

Mono-and di-acylglycerol lipase(DGL)specially catalyze the mono-or di-glycerides to produce glyceride and free fatty acids.It has great application potential in oil modification and chemical industry.However,the optimal temperature of all reported microorganism DGLs range from 15 to 40 °C,which hinders their usage in industry;therefore,improving the thermostability of enzymes is urgent to DGLs.Lipase SMG1 and PCL were studied and their thermal stability was modified by computational biology.This study mainly included the following contents: 1.Computer-aided design for thermal stability of lipase SMG1.Using Rosetta,FoldX and ABACUS to calculate thermal stability mutants of SMG1,18 mutants with enhanced Tm value were obtained.By combining the above 18 mutants,the best mutant S5(Q34P+A37P+M176V+G177A+M294R)was obtained.The Tm value increased by 6 °C,and the optimum temperature increased by 5 °C.Baseding on the mutant S5,a second round of computer-aided design was carried out and a mutant Q282 F with a 6 °C increase was obtained.However,when the enzyme activity was measured,the mutant was found to have negative effect on the enzyme activity.Structural analysis may be steric hindrance caused by amino acid phenylalanine or steric hindrance of π-π interaction with F278.2.Improving thermostability of PCL by amino acids substitution and structural analyses.By comparing the amino acid composition of the lipase PCL with that of the thermostable protein,the amino acids A,D,S,and T in PCL were selected to be mutated to E,K,R,I,and L,resulting in 93 mutants.Comparison of homologous sequences excluded conservative amino acid positions and distribution of amino acid left 28 mutants.Comparison of these candidate mutants with PCL-WT,the results showed that only R25 could form new salt bridge interactions and hydrogen bonding interactions.The mutant PCL-D25 R was constructed and had a 3 °C increase in Tm compared to that of PCL-WT,a 5°C increase in the optimum temperature,and a 4-fold increase in half-life at 45°C.Dynamics analysis revealed that mutant PCL-D25 R was more stable than that of the PCL-WT because of a salt bridge forming between R25 and D32.3.Comparative analysis and summary of two protein thermal stability modification methods.Comparing and analyzing the thermal stability modification methods of the two enzymes,it can be seen that the computer-based protein stability design method is relatively comprehensive,but it needs to build a larger mutant library and the experimental period is longer.Based on the computational biology method,the amino acid composition characteristics of thermostable proteins were summarized.Using amino acid substitution method to construct a virtual mutant library,combined with sequence homology and structural analysis,can effectively reduce the number of mutants that need to be constructed.It may be a new exploration of the rational dedign of enzymes based on the biochemical properties of lipases.