Characterization Of Enzymatic Properties And Structural Analysis Of Lipase CoMGL From Cytobacillus Oceanisediminis

Monoglyceride lipase(MGL) is a class of lipase that can specifically hydrolyze glyceride substrate.MGL is a key enzyme in the regulation of biological functions,involved in nerve signal transduction,cancerization and anti-inflammatory response,etc.Studying the structure-function relationship of MGL can provide the foundation for further understanding of its physiological functions.Microbial-derived MGL has industrial application prospects and has broad application value in the fields of oil modification,pharmaceutical synthesis and biochemistry.In order to meet the needs of industrial application,digging out novel MGL,research on structure and function and rational reconstruction should be carried out continuously.The development of monoglyceride lipase with higher thermal stability can further expand its industrial application value.In this study,a monoglyceride lipase CoMGL was mined from the genome of Cytobacillus oceanisediminis.It was studied including its heterogeneous expression,enzymatic properties and crystal structure,combined with the construction of genetic mosaic and structure alignment,elaborates the molecular structural basis of regulating its catalytic activity.The main research contents are as follows:(1)Preparation and enzymatic properties of CoMGL.the comgl gene was obtained by gene synthesis technology and was cloned into p ET-30a(+).After being expressed in E.coli BL21(DE3),the target protein was obtained and purified by nickel column affinity chromatography,172.6 mg of pure target protein could be obtained per liter of fermentation broth.The enzymatic properties of the purified enzyme solution showed that the optimal reaction temperature was 50?,the optimal reaction p H was 9.0,and the enzyme activity remained more than 60% after incubation at p H 10.0 for 12 hours.It can only specifically hydrolyze monoglyceride,but not diglycerides and triglycerides,so it was classified into the glycerol monoester lipase family(2)Crystal preparation and structure analysis of CoMGL wild type.CoMGL was further purified by gel filtration chromatography to meet the crystallization conditions.Through screening and optimization,the optimal crystallization conditions were as followed:18? for 5 days,0.2 M Magnesium acetate tetrahydrate,0.1 M Sodium cacodylate trihydrate,p H 6.8,19% w/v PEG 8000.The crystal structure with resolution of 1.25 ?(PDB ID :7E04)was obtained through molecular replacement.CoMGL is a typical ?/? hydrolase structure.The "cap" structure covers Asn120-Val171,the catalytic triad is Ser97-Asp194-His224,and the oxygen anion hole is composed of-NH-on the main chain of Phe29 and Met98.(3)Chimeras construction of CoMGL and thermal stability Analysis.Five N-terminal replacement chimeras of CoMGL and GMGL were constructed.By comparing the thermal stability and enzyme activity,it was found that the optimal reaction temperature of chimeric GMGL-160-CoMGL was 20? higher than that of CoMGL,and the enzyme activity was 3.9times higher than that of CoMGL under the optimal conditions.The crystal of chimeric GMGL-160-CoMGL was further prepared and analyzed(PDB ID:7E0N).Through sequence and structure analysis,?5 region was found to be the key region for the improvement of chimeric performance,and amino acids such as Asn149,Asp151 and Ile152 may play a key role in regulating the enzyme thermostability.