Research On Detection Method Development And Molecular Modification For Catalytic Specificity Of Thermomyces Lanuginosus Lipase

Lipase（E.C.3.1.1.3）is one of the most widely used enzymes,which can catalyze esterification,hydrolysis and other reactions.Lipase with sn-1,3 selectivity can react specifically on the sn-1 and sn-3 sites of glycerol skeleton and can be used for efficient production of diacylglycerol（DAG）.However,there are few studies on the detection and separation methods of 1,2-diglycerol（1,2-DAG）and 1,3-diglycerol（1,3-DAG）,and the stability and specificity of sn-1,3 selective lipase used in industry are still insufficient.In this paper,Thermomyces lanuginosus lipase（TLL）with high thermal stability was selected as the research object,and a regional selectivity detection method was developed,and wild-type TLL was modified molecularly based on semi-rational design to improve its sn-1,3 regional selectivity.The results are as follows:（1）Heterologous expression and optimization of TLL.Pichia pastoris,which has higher enzyme activity and better secretion efficiency than Escherichia coli,was selected as the heterologous expression host of TLL.The secretion effects of SCW1,UTH-1,PAS＿chr＿0030and MFα-factor on TLL were compared.SCW1,UTH-1 and MFα-factor can significantly secrete TLL,and MFα-factor has better secretion ability.In order to improve the enzyme activity of TLL in fermentation broth for subsequent analysis,the culture conditions of Pichia pastoris were optimized at the shaking flask level,and the highest secretase activity reached213 U·m L^-1 at 1%methanol induction concentration,12.5%inoculation dosage and 4 d culture.（2）Construction and optimization of detection method to DAG.A semi-quantitative TLC method was established for the determination to oil by using n-hexane:ether:acetic acid（50:30:2）as the developing agent and primulin as the staining agent.Image J was used to analyze the fluorescence spots of the chromatographic plates.The standard curve between the gray area ratio and the actual concentration ratio of the two DAG was drawn,and the R² was greater than 0.97.A method for the analysis of DAG by ultra performance convergence chromatography（UPC²）combined with evaporative light scattering detector（ELSD）was established,and the parameters are optimized.Finally,1,2-DAG(0.025–0.2 g·L^-1)and 1,3-DAG(0.025–0.15 g·L^-1)could be isolated and quantitatively detected within 13 min.（3）Molecular modification of sn-1,3 regional selectivity of lipase.G82,S83,I86,I90,N92,L93,F95,I202,V203,and L206 in the substrate pocket were selected as mutation sites to construct a saturated mutation library by molecular docking of triacylglycerol（TAG）with TLL.The mutant library was screened by semi-quantitative TLC and UPC²-ELSD methods,and the re value of the optimal mutant I202V was 76%,which was 11.7%higher than that of WT-TLL.RMSD molecular dynamics simulations found increased structural rigidity in the mutant,which explained the slight increase in thermal stability of I202V,while RMSF analysis found that the increased flexibility of the Gly82-Ile100 region may promote the sn-1,3 region selectivity.In I202V,the distance required by the active site Ser-146 to attack the carbonyl carbon at sn-3 is reduced by 62.5%,which may be the reason why I202V shows higher sn-1,3 specificity.In addition,this study speculates that the conformational change of Loop 202-204 in I202V affects the binding mode of TAG,and its conformational changes may be potentially related to TLL region selectivity.