Study On The Molecular Modification And Enzymatic Properties Of Lipase SMG1

Mono-and diacylglycerol lipases,a unique group of lipases,display chemical selectivity against monoglycerides and diglycerides instead of conventional triglycerides.The unique substrate specificity makes it have promising application prospects in oil modification and functional oil production.Great thermostability and enzyme activity are the prerequisites for the applications of enzymes in industrial production.Therefore,it has been a hot topic in science and industry to explore strategies to improve the thermostability and activity of enzymes.In this study,mono-and diacylglycerol lipase SMG1 was taken as the research object,its rational molecular modification was carried out through molecular simulation,protein crystallography and molecular biology,aiming to explore an effective molecular modification strategy for improving its thermostability and activity,and paved the way for its industrial application.In addition,the study provided an available approach for the engineering of other lipases of the same family.The main findings of this paper are as follows:（1）Molecular design through enhancing the rigidity of the inactive centers and the determination of enzymatic properties.Based on structural analysis and computational screening,disulfide bonds were designed to be introduced into lipase SMG1 to improve the thermostability of the enzyme,and designed disulfide bonds were located at the inactive central region 10?away from the catalytic S171.Six effective disulfide bond mutants were obtained through screening.Compared with the wild type,the disulfide bond mutant G28C-P206C displayed a 9.0°C increase in the melting temperature（T_m）,5°C in the optimal temperature,and 64.8-fold in the half-life(t_1/2)at 50°C.In addition,the specific activity of the mutant G28C-P206C was also improved,which was 3.1 times higher than that of the wild type.Through the analysis of the predicted structure of mutant G28C-P206C,we assumed that the disulfide bond G28C-P206C enhanced the rigidity of the enzyme’s overall structure,which improved the thermostability of the enzyme.In addition,the enhanced rigidity of the overall structure indirectly stabilized the enzyme activity center,which made the substrate bind more strongly to the enzyme,resulting in the improvement of enzyme activity.Then,the disulfide bond G28C-P206C was combined with the point mutations that improved the thermostability of the enzyme screened in our previous study,and the mutant M5D（Q34P/A37P/M176V/G177A/M294R/G28C-P206C）was obtained.Compared with the wild-type,the T_m value and t_1/2 at 60℃of mutant M5D was enhanced by 15.0°C and 576.9-fold,and its specific activity was similar to that of mutant G28C-P206C.（2）Molecular design through expanded the pocket of enzyme active center and the determination of enzymatic properties.To improve enzyme activity,the active center of M5D was further designed.Our previous study expanded the space above the catalytic triad of lipase SMG1 to make the substrate more accessible to the catalytic residue,and obtained the single-point mutant F278N with improved enzyme activity after the experimental screening.However,in this study,the mutation site F278N was introduced into the mutant M5D to obtain mutant M5DN,and the enzyme activity of mutant M5DN decreased.After analyzing the experimental results combined with the predicted structure,a series of mutations were designed for the mutant M5D.Among them,the specific activity of the mutant M5DL（M5D+F278L）was further improved,which was 4.4 times higher than that of the wild-type SMG1.In addition,the thermostability of the mutant M5DL was also improved,and its T_m value and t_1/2 at 60°C were 15.0°C and 922.5 times higher than those of the wild-type lipase SMG1,respectively.In mutant M5DN,the combination of mutations F278N and M294R might destabilize the active center of the enzyme,resulting in a decrease in enzyme activity.In the mutant M5DL,the higher hydrophobicity of mutation F278L might stabilize the active center of the enzyme,thereby improving the enzyme activity;in addition,the stable active center also improved the temperature tolerance of the enzyme.（3）The mechanism verification of molecular modification affecting enzymatic properties.Mutant M5DN with a purity of more than 95%were obtained by using Ni-NTA affinity chromatography and gel filtration chromatography.It crystallized under the conditions of 150 m M potassium bromide and 30%PEG MME 2000.The crystal structure of mutant M5DN with a resolution of 1.8?was solved using the molecular replacement method.The crystal structure of the mutant M5DN with multiple mutations was similar to the predicted structure,which implied that the predicted structure of the single disulfide bond mutant G28C-P206C was reliable.It suggested that the molecular mechanism of the disulfide bond G28C-P206C and the mutagenesis of residue 278 affecting the enzymatic properties based on the predicted structure has the theoretical significance.Then disulfide bond G28C-P206C and point mutation F278L were selected as the research objects,and their effect on enzymatic properties was verified by using the molecular dynamics simulation.Results of the molecular dynamics simulation showed that the disulfide bond G28C-P206C might improve the thermostability and activity by improving the rigidity of the enzyme and the reduction of the binding free energy between the enzyme and the substrate.Mutation F278L decreased the distance between the carbonyl of the substrate and the catalytic residue serine,indicating that the substrate may be more accessible to the catalytic residue;it enhanced the rigidity of loop V272-G283 and stabilized the hydrogen bond between the catalytic residues,suggesting that the Enzyme active center.Therefore,mutation F278L has improved the activity and thermostability of the enzyme.Based on the results of molecular dynamics simulations,more screening criteria were added into the design strategy of the non-enzyme active center and the enzyme active center,and the design strategy was further applied to the molecular modification of lipases Mg MDL2 and Pri Lip with similar structures to SMG1.Compared with wild-type Mg MDL2,the t_1/2and specific activity of mutant Mg MDL2-F278L were enhanced by 85.7%and 1.4 times,respectively,while those of mutant Mg MDL2-A26C/P206C were enhanced by 47.0 times and 91.8%,respectively.Compared with wild-type Pr Lip,the t_1/2and specific activity of mutant Pr Lip-F258L were enhanced by 1.9 times and41.9%,respectively,while those of mutant Pr Lip-E1C/K234C were enhanced by 95.9 times and 87.3%,respectively.