Characterization Of Enzymatic Properties And Catalyzation Mechanism Of Lipase From Geobacillus Sp.12AMOR1

Monoglyceride lipase is a class of hydrolases that specifically act on monoglyceride substrates,and its unique catalytic properties make it extremely useful in industrial applications.In addition,monoglyceride lipases also play a key role in the physiological regulation of mammals.The research on the three-dimensional structure of key enzymes for industrial demand or involved in physiological metabolism is helpful to understand the structure and function relationship of this kind of enzymes and enrich the theory of enzymology,which has scientific and practical application research value.This study studied the preparation,enzymatic properties and three-dimensional structure of recombinant enzymes by using the monoglyceride lipase Geobacillus sp.12AMOR1?GMGL?of Bacillus licheniformis,combined with site-directed mutagenesis and molecular dynamics simulation.Study and explain the process by which GMGL recognizes and hydrolyzes substrates.The main research contents are as follows:?1?Preparation of GMGL and study of enzymatic properties.The prokaryotic expression vector of GMGL was constructed and expressed in E.coli BL21?DE3?host.The GMGL recombinase protein was isolated and purified by metal chelate affinity chromatography.SDS-PAGE electrophoresis analysis It shows that the target protein band is uniform and the purity is greater than 90%.The results of enzymatic characterization showed that the optimum reaction temperature of GMGL was 60 ^oC,the T1/2 of the enzyme was 70 min at 70 ^oC,and the optimum pH was 8.0,which was stable in alkaline buffer environment.Experiments for hydrolyzing monoglyceride substrates and esterifying glycerol with fatty acids confirmed that GMGL is a monoglyceride lipase.?2?Analysis of the crystal structure of GMGL.Gel filtration chromatography was used to obtain higher purity GMGL protein,and the crystallization conditions for diffracting the enzyme protein were optimized:protein concentration 12 mg/mL,0.2 M Ammonium sulfate,0.1 M MES monohydrate?pH 6.6?,28%w/v Polyethylene glycol monomethyl ether 5,000,20 ^oC.The GMGL protein structure?PDB ID:5XKS?with a resolution of 2.19?was resolved by molecular replacement.The GMGL structure is a typical alpha/beta hydrolase class consisting of 9 alpha-helix and 9 beta-sheets.The catalytic triad of GMGL consists of Ser97,Asp196 and His226,and the oxygen anion hole consists of the main chain-NH-group of Phe29 and Met98.?3?GMGL substrate selectivity studies.Three amino acids affecting chain length selectivity were selected by crystallographic analysis and three mutants Leu142Ala,Ile145Ala and Ile170Phe were constructed.The k_cat/K_m values of wild-type and mutants were detected by using p-nitrophenol esters with different fatty acid chain lengths as substrates.The results showed that the selectivity of Leu142Ala,Ile145Ala and Ile170Phe for pNP-C6substrates was 2.3 folds compared with wild type,1.2 and 2.2 times improvement,respectively.Finally,the effects of these three mutants on the selectivity of GMGL substrates were analyzed by means of molecular docking.?4?Substance process for GMGL recognition and hydrolysis.Molecular dynamics analysis revealed that there is a unique"pore structure"in the structure of monoglyceride,and the S32,S35,S147 and E156 amino acid sites near the pore were selected,and 12 mutants were constructed?S32W/S32V/S32A,S35W/S35V/S35A,S147W/S147V/S147A,E156W/E156V/E156A?,and the hydrolysis activity of the enzyme mutant was detected by using monoglyceride stearate as a hydrolysis substrate.The results showed that all mutants showed different degrees of inhibition relative to the wild type,suggesting that the“hole structure”may be the release channel of glycerol involved in the hydrolysis product.Comprehensive biochemical experimental data and bio-computation analysis further proposed the process of GMGL to identify and hydrolyze substrates through the“induction fit”of enzymes.