Rational Modification To Improve The Esterification Ability Of Lipase MAS1 And Its High-Density Fermentation

Lipase（EC 3.1.1.3）is an important industrial enzyme for industrial use.It is widely used in the food,medicine and chemical industries.The lipase MAS1 derived from marine actinomycetes（Streptomyces sp.）W007,which has good heat resistance and organic solvent resistance,has an important application prospect.To improve the catalytic performance of this lipase,protein engineering technology is a research hotspot.This study focused on the catalytic pocket of lipase MAS1,selected key sites to mutate individually,and performed expression,purification and enzymatic properties characterization of the lipase mutants.We focused on the effect of point mutations on the esterification ability of the lipase.This study may provide a good research basis for the rational modification of important enzymes for industrial use.The main research contents are as follows:1.Design and preparation of mutants based on the structure of lipase MAS1Through the structural analysis of lipase MAS1,residues G40,T237 and V233 in the alcohol binding pocket are associated with water molecule concentration during the esterification reaction.G40,T237,and V233 were mutated to hydrophobic amino acids G40F,G40L,T237A,T237F,T237L,and V233F,respectively,to reduce access of water molecules to the alcohol binding pocket and then facilitate the esterification.The mutant genes were obtained by PCR site-directed mutagenesis and verified by sequencing.Then the mutant genes were transferred into the engineered expression strain BL21（DE3）,and induced expression performed in a 7L fermenter to obtain the bacteroids containing the protein of interest.After collecting the bacteria and purification by affinity chromatography,high purity MAS1-WT,G40F,G40L,T237A,T237F,T237L and V233F proteins were obtained.2.Catalytic characterization and basal biochemical characterization of lipase MAS1 mutantsMAS1 wild type and its mutants were evaluated for their ability to catalyze the synthesis of glycerol from glycerol and oleic acid.When the reaction reached the equilibrium by 72 h,the esterification rate catalyzed by MAS1-G40L reached 76.49%,which was 9.52%higher than that of the wild-type.Among the products,the triglycerides（TAGs）obtained catalyzed by MAS1-G40L were higher than that of the wild type.G40F,V233F and T237F of the remaining mutants exhibited a slightly higher esterification rate than the wild type.Mutants T237A and T237L showed significantly lower esterification rate than wild type.With the reaction system using lauric acid and n-propanol to produce propyl laurate under the optimal reaction conditions,the esterification viability of MAS1-G40L was 1.38 folds higher than that of the wild type.The enzymatic properties of MAS1 wild-type and mutants were further characterized.The results showed that MAS1 wild type and its mutants catalyzed the hydrolysis reaction with an optimal temperature of 65℃and an optimal p H of 7.0.The mutant G40L exhibited better thermal stability than the wild type at the temperature range between 40-70℃.In the range of p H 3.0～9.0,both the wild type and its mutants were p H tolerant.Zn²⁺,Cu²⁺and Ni²⁺inhibited the MAS1 and its mutants’activity.Except for T237F,the rest of the MAS1 proteins exhibited good tolerance to organic solvents.In terms of surfactant tolerance,SDS inhibited the enzymatic activity all MAS1 proteins.While Tween-80 promoted the hydrolase activity of all MAS1 proteins.Take together,MAS1-G40L with the highest esterification viability also has the best enzymatic properties.3.Optimization of process parameters for high-density fermentation conditions for BL21（DE3）/p ET22b-MAS1-G40LBy single factor-experiments,the effects of induction time,Isopropyl-β-D-thiogalactoside（IPTG）concentration,induction p H and induction temperature on expression level of MAS1-G40L were investigated in a 7L fermenter.Then the IPTG concentration,induction p H and induction temperature were considered as variables,respectively,and further optimized by the Support Vector Machine（SVM）and genetic algorithm optimization,and validated by the experimental method.Finally,the effects of different feeding parameters on the production level of MAS1-G40L in the E.coli system were investigated.The optimal fermentation conditions were determined as follows:IPTG at a final concentration of 0.65 m M was added for induction at 37°C,p H 7.0,7 h grow.Then the E.coli would be induced at 23°C,p H 6.7.Under p H-STAT feeding mode,the highest enzyme activity of expressed MAS1-G40L could reach 4123 U/m L,which was increased2.38-fold compared with that before optimization.