Improving Hydrogen Peroxide Tolerance Of Lipase From Bacillus Subtilis Using Semi-rational Design

In the process of lipase-catalyzed hydrolysis,in addition to activating water molecules,lipase could also activate hydrogen peroxide.The activated water molecules or?hydrogen peroxide?could attack the lipase-ester transition state complex?second transition state complex?and released free fatty acids?or peracetic acid?.Catalytic activity of lipase to activate hydrogen peroxide to synthesize peracetic acid was the perhydrolysis catalytic activity of lipase.It was one of the promiscuity activity of lipase;The development of perhydrolysis catalytic activity of lipase and the enzymatic synthesis of peracetic acid had important economic and social significance?safe production?.The presence of an oxidizing agent?such as hydrogen peroxide?in the reaction system or in the reaction medium would destroy the structure of the lipase,resulting in deactivation of the lipase.The semi-rational design improved the tolerance of lipase to hydrogen peroxide and reduced the negative influence of hydrogen peroxide on the structure and function of lipase,which was of great significance for further expanding the application field and application value of lipase.In this paper,Bacillus subtilis lipase?abbreviated as LipA?,which had relatively simple molecular structure and no disulfide bond and good biosafety,was selected as the research object.Potential amino acid residues that were more sensitive to hydrogen peroxide were selected by mass spectrometry and alanine scanning;Then mutants with good hydrogen peroxide tolerance were further screened by site-directed mutation and superposition mutation.Mutant protein was purified,and its enzymatic properties and hydrogen peroxide tolerance values were characterized and compared with wild-type lipase;Finally,combined with computational simulation and 3D structural analysis,the mechanism of hydrogen peroxide tolerance improvement was described.The specific experimental results were as follows:1?B.subtilis lipase was subjected to hydrogen peroxide treatment and analyzed by mass spectrometry.The molecular weight increase was found to be 64.Combined with the related literature,it could be inferred that the methionine residue and the tryptophan residue in the lipase molecule were oxidized after the treatment with hydrogen peroxide.Site-directed mutagenesis of four methionine residues and two tryptophan residues was followed by alanine scanning.Two positive mutants were obtained by screening:LipA-W⁴²A and LipA-M¹³⁴A.2?Trp⁴22 and Met¹3434 were replaced by hydrogen peroxide non-sensitive amino acid residues?Ile,Val,Glu and Asp?through site-directed mutation,and five positive mutants were obtained by screening.The positive mutants screened were subjected to superposition mutation to construct a Mini mutant library.After hydrogen peroxide treatment and enzymatic determination,the residual enzyme activity of the superimposed mutants was enhanced to varying degrees compared to the wild type.Among them,LipA-W⁴²I/M¹³⁴I,LipA-W⁴²I/M¹³⁴V,LipA-W⁴²A/M¹³⁴E and LipA-M¹³⁴A/W⁴²I showed the most significant increase in tolerance to hydrogen peroxide.The above four superimposed mutants and wild type lipase were purified by affinity chromatography purification and to obtain target protein which had single electrophoresis band.And the4-Nitrophenyl laurat was used as substrate to determine the relevant enzymatic properties.It was determined that the C₅₀0 values of wild type and four superimposed mutants(LipA-W⁴²A/M¹³⁴E?LipA-W⁴²I/M¹³⁴A?LipA-W⁴²I/M¹³⁴I and LipA-M¹³⁴I/W⁴²V)for hydrogen peroxide were:22 mmol/L?200 mmol/L?105 mmol/L?110 mmol/L and 80mmol/L.The half-lives of hydrogen peroxide inactivation were 24 min,3.8 h,27 min,36min and 30 min,respectively.3?Gromacs software analysis showed that the lipase in hydrogen peroxide/water mixing system,and the RMSD and RMSF values were increased compared with the pure water system.Analysis of the contact values of hydrogen peroxide with amino acid residues revealed that the Trp⁴²site had the highest contact value.The corresponding contact value was significantly reduced after Trp⁴22 replaced by Ala⁴²(or Ile⁴²).Amber software analysis showed that the RMSD values of the four superimposed mutants were reduced to varying degrees compared to the wild type.YASARA software analysis showed that the LipA-M¹³⁴E produced a salt bridge effect,while LipA-M¹³⁴A,LipA-M¹³⁴I,and LipA-M¹³⁴V promoted the improvement of oxidative tolerance by reducing the hydrophobic surface area.