Study On Crystal Structure,enzymatic Properties And The Synthesis Of Long-medium-long Type Structured Triacylglycerols

Lipase MAJ1 is a novel sn-1,3 specific lipase and has great potential in the synthesis of structural esters.However,its application is limited by the low optimum temperature and poor thermal stability.Therefore,In this paper,lipase MAJ1 derived from Janibacter SP was studied by combining biochemical,molecular and protein engineering techniques.In this work,we firstly completes the expression of lipase MAJ1 in E.coli and the characterization of its enzymatic properties.Then,its crystal structure was solved,and the key amino acid sites regulating the thermal stability and positional selectivity of lipase MAJ1 were determined by structural analysis combined with MD simulations.We evaluated its use in the synthesis of LML-type structured triacylglycerols as well.This study provided important theoretical support for the improvement of the enzymatic activity and thermal stability by structural modification without altering positional selectivity.The main research contents and resμLts are as follows:（1）MAJ1 was expressed in E.coli BL21（DE3）and purified with Ni²⁺colum.Details of the study showed that the recombing MAJ1 has a maximum catalytic activity at 30℃and p H7.0 and displayed great stability at 25-35℃or p H4-9.The PSI of MAJ1 was 96.5 and MAJ1was identified as novel sn-1,3 lipase.（2）To obtain the crystal,lipase MAJ1 was expressed in P.pastoris X-33 and purified by the one-step Nickel column.MAJ1 was further purified by gel filtration chromatography.The higher purity of MAJ1 was conducive to the growth of the crystal,and the final crystallization condition was 20%PEG3350,200 m M potassium citrate tribasic and a protein concentration of 10 mg/m L（molar ratio protein:oleic acid=5:1）.Crystal data were collected from synchrotron SSRF and the crystal structure was resolved with the resolution of 2.5（?）and space group P4 2₁2.The residues of the catalytic triad in MAJ1 consisted of Ser130,Asp227and His262.The“oxyanion hole”was constituted by backbone-NH from Thr54 and Gln131,which would stabilize the tetrahedral intermediates during hydrolysis.There are two disulfide bonds Cys38-Cys75 and Cys255-Cys289 in MAJ1.Residues Gly168 to Pro192 form the“lid”of MAJ1.The catalytic active center is exposed as a“lid opening”form.One asymmetric unit bound three molecules of oleic acid,two of which were located within the catalytic pocket and the other on its surface.（3）Structural alignment analysis and molecular docking revealed that the second site of conserved pentapeptide（G-X-S-X-G）W129 and residue F15 of the N-terminal of MAJ1lipase are involved in the alcohol binding pocket.They are critical in regulating the regiospecificity of lipase MAJ1.We designed mutants F15A,W129H,W129A and W129H-F15A to examine their effect on the regiospecificity.The results showed that mutants of F15A,W129H and W129A reduced sn-1,3 region-preference.And it even disappears in the mutant W129H-F15A.This result suggested that mutation of F15 to W129 will enlarge the alcohol binding pocket space,better accommodate substrates in different conformations and increase the preference for the sn-2 position.The geometry of the alcohol binding pocket would strongly influence the regiospecificity of MAJ1.Based on MAJ1 crystal structure analysis and molecular dynamics simulations,amino acids in the lid region were found to significantly affect lipase MAJ1’s thermal stability.Mutants in the lid hinge and loop region were designed.We obtained mutants L171A,A182P,P192A and T100C-P192C with thermal stability or specific activity improvements.The combined mutagenesis was performed to obtain the mutant M4（L171A/A182P/T100C-P192C）.The mutant M4 retained the strong sn-1,3 position selectivity of lipase MAJ1,increased 4.3-fold over wt enzyme activity,and increased the optimal reaction temperature by10°C.（4）Studies on the catalytic synthesis of LML type structural esters by immobilized lipase M4.We obtained the immobilized lipase M4.The effect of substrate molar ratio,reaction temperature,and enzyme addition on its catalytic transesterification of trioctanoate with methyl palmitate were investigated by single-factor experiments.The conditions optimized to obtain the most suitable ester for LML type structure were:substrate molar ratio（methyl palmitate:trioctanoate）of 4:1,reaction temperature of 45°C,and enzyme addition of 7.5wt%.After 24 h of reaction under this condition,the content of double long-chain glycerides reached 57.1%,which is the highest value reported so far.The immobilized lipase MAJ1catalyzed the reaction with only 44.3%final dolichol content,and the enzyme activity retained by immobilized lipase M4 after 13 cycles of use was 1.33 times that of immobilized lipase MAJ1.