Study On The Directed Evolution And Immobilization Of Esterase From Marine Microorganism

Esterases?EC3.1.1.1?are widely distributed in microorganisms,plants and animals,which can catalyze the reaction while formation and cleavage of diverse ester bonds.The advantages of catalyzed reactions were mild reaction conditions,single product,low energy utilization,without coenzyme,separation and purification of the product was easily.The esterases has wide application in the fields of food,chemical industries,environmental protection,transesterification,and medicine.The diverse types of genes occurred in the microbes in different extreme environmental conditions and it seems to be a large number of infrequent genetic resources.The present study deals to screen more than 400 strains of marine microorganisms of different species that have been stored in the laboratory,clone a novel cold-adapted esterase gene with high activity,combine two rounds molecular evolution,protein engineering techniques to advancement the target gene and significantly improving its catalytic activity.The enzyme properties,kinetics,structure and function were determined.The results provide information of structure and function.Immobilization method of this research using organic-inorganic hybrid nanoflower technology.The optimal conditions for synthesizing Y193G-Fe₃?PO₄?₂ hybrid nanoflower were investigated.The shaps of nanoflowers formed under different conditions was observed.The properties and stablity of nanoflower were greatly increased.At the same time the Y193G-Fe₃?PO₄?₂ hybrid showd similar catalytic activity to the free enzyme,which broke the limitation that the traditional immobilization technology damaged the enzyme activity easily and lay the foundation for industrial and transesterification applications.In this study,29 bacterial strains were screened for their hydrolytic activity and they were preserved in the China Marine Microorganisms Culture Collection Management Center.In secondary screening,one of the marine strains has the highest hydrolytic activity and was identified by 16S rRNA.The high active marine strain was Enterobacter cloacae?ZS825?.A novel low-temperature esterase gene was obtained by the Lip cloning and expression,which can still sustain 40%activity at 0°C against to p-nitrophenyl acetate.Phylogenetic analysis shown that it belongs to Esterase family IV and kinetics studies displays that Km and K_cat was 0.643 mM^-1 and9.8,and catalytic efficiency(K_cat/Km)was 15.25 S^-1·mM^-1.In order to improve the catalytic efficiency of Lip,conducted Error-prone PCR for establishing a random mutation library and 2000 mutants were obtained and the activity was checked via the high-throughput screening method based on T7 phage lyses.From the mutant library we obtained a mutant V29A/Y193C.The enzyme kinetics analysis of the mutant shown as Km 0.363 mM^-1 and K_cat/Km was 71.08 S^-1·mM^-1.Kinetic studies signifies that mutant Val29Ala/Tyr193Cys shown43.5%decrease in Km,2.6-fold increase in Kcat,and 4.7-fold increase in K_cat/Km comparative to the wild type.To investigate the relationship between two mutation sites and enzyme protein function in beneficial mutants obtained by error-prone PCR,site-directed mutagenesis was used to create single mutants V29A and Y193C and analyze their activity individually.The results showed that Km and K_cat/Km values of the mutant V29A were approximately same as those of the wild type.In case of Y193C showed 52.7%decrease in Km,2.7-fold increase in K_cat,and 5.6-fold increase in K_cat/Km contrast to wild type.The kinetics results indicates no change in the enzyme activity of the29^th site,however the key mutation was 193^th site.In determining the 193 amino acid is directly related to the function of the enzyme protein,saturation mutations were designed for 193^th amino acid,and also near 18 other amino acids,respectively,resulting that a highly active mutant Y193G was obtained.The kinetics values,Km0.311 mM^-1,and K_cat/Km 104.2S^-1·Mm^-1.Y193G showed 51.6%decrease in Km,3.3-fold increase in K_cat,and 6.8-fold increase in K_cat/Km compared with the wild type.After two rounds mutation,a highly active mutant strain Y193G was obtained.Which was used to increase the application value of Y193G,the enzyme-inorganic hybrid nanoflowers were prepared using FeSO₄ and esterase Y193G through organic-inorganic hybridization.The optimum enzyme and Fe²⁺concentration for formation of nanoflower was 0.05 mg/mL,1.2 mM.The optimum temperature and pH for nanoflowers formation was 50°C and 8.5.Kinetic analysis of the immobilized enzyme showed that the catalytic activity of the immobilized enzyme was not an immense different from free enzyme.The stability of the immobilized enzyme was ensuring at 50-70°C for 3 hours,more than 80%relative activity could be retained.However,stability at 90°C for 3 hours,it retains only 55%.The pH stability of the immobilized enzyme was checked overnight at pH 4 and retained its activity more than 60%.Subsequently in an alkaline condition at pH 10 incubated overnight,the immobilized enzyme was able to retain its activity 90%.The relative activity 80%was gained until 9 cycles,although the relative activity of 30%can be retained after 9 cycles.The stability of the immobilized enzyme was significantly improved,provided that a theoretical base for industrial application.The immobilized enzyme was used to synthesize geranyl acetate with geraniol and p-nitrophenylacetate as the reaction substrates.GC-MS analysis showed that after 12 hours of reaction,the conversion of geraniol reached 82%,which provided a theoretical basis for industrial application.