Screening And Identification Of Esterase By Metagenomic Technology And Study Of Its Enzymatic Properties

Esterases(EC 3.1.1.x,Esterases)mainly include carboxylesterases(EC 3.1.1.1,Carboxylesterases)and lipases(EC 3.1.1.3,Lipases),which catalyze the cleavage and formation of ester bonds and amide bonds.It has a wide range of applications in the oil,food,chemical synthesis,pharmaceutical,detergent,paper and pulp,leather and other industries.Traditional enzyme and gene identification method involve culture-based screening of pure microorganisms,which are limited to cultured microorganism,a huge number of genes are still not identified as so far with as much as 99%of these microbial being recalcitrant to culturing under laboratory conditions.Alternatively,metagenomic approaches circumvent the cultural process of microorganisms,extracting the total microbial DNA to constuct library from an environmental sample followed by sequential or functional screening.Recently,the metagenomic technology has become an effective tool for discovering novel esterases.In this study,a novel esterase gene dlfae4 was screened from the soil metagenomic library by function-driven method.dlfae4 was analyzed by bioinformatics,and it was transformed into E.coli BL21(DE3)for heterologous expression.The enzymatic properties of DLFae4 were characterized and kinetic parameters for various substrates were calculated.Molecular dynamics was simulated with complexes of enzyme and ligand.The main research contents are listed as follows:1.Screening of esterase gene.The novel esterase gene dlfae4 was screened out with function-driven method.dlfae4 displayed the highest sequence similarity(54%)by Blast and it is composed of 1017 base pairs encoding 338 amino acid residues with a predicted molecular mass of 37.2 kDa and the isoelectric point is 7.77.The multiple sequence alignment showed that DLFae4 contained a SXXK motif which is conserved in class Cβ-lactamases and family Ⅷ esterases.Futhermore,the other conserved motifs of family Ⅷesterases all correspond to DLFae4.The phylogenetic analysis showed that DLFae4 clustered together with family Ⅷ esterases.2.Heterologous expression of esterase.dlfae4 gene was amplified,digested and ligated into an expression vector pET-28a and the recombinant plasmid pET-dlfae4 was transformed and overexpressed in E.coli BL21(DE3).The recombinant enzyme was purified by Ni-column affinity chromatography.It was confirmed by SDS-PAGE and showed a single band.The molecular mass was consistent with the prediction.The site-directed mutated gene was amplified by PCR using pET-dlfae4 vector as the template,the mutation sites were introduced by primers.The enzyme activity of three site-directed mutants S11A,K14N and T121A were almost lost.The results indicated the residues Ser11,Lys14,Tyr121 were essential for enzyme activity of DLFae4.Nature DLFae4 was determined to be a dimer based on the inverse relationship between the logarithm of protein molecular weight and the retention volumes.3.Biochemical characterization of DLFae4 and substrate specificity.The optimum pH and temperature of DLFae4 are 8.6 and 50℃,respectively.It was stable at pH 6-9.5.It showed remarkable thermostability and remained over 50%residual activity at 60℃ for 3 h.DLFae4 was strongly inhibited by Cu2+.Ethanol,methanol and SDS also have inhibitory effect on the enzyme activity.Other tested additives displayed little effect on enzyme activity.DLFae4 revealed hydrolytic activity against the p-nitrophenyl ester(p-NP)and the four methyl hydroxycinnamate in the C2-C8 side chain length,showing the highest activity for MFA(123.78 U/mg).Additionally,it has hydrolysis activity towards five phthalates(PAEs).4.The hydrolysis activity of DLFae4 against lactam substrates.Due to high sequence similarity with β-lactamases,DLFae4 also displayed hydrolytic activity against benzylpenicillin(21.24U/mg),ampicillin(35.78 U/mg)and cefazolin(11.33U/mg).The hydrolysis activity of ampicillin is higher than that of the partially specific Class-Cβ-lactamase.5.Molecular dynamics simulation of DLFae4.The complexes of protein and ligand were obtained using molecular docking as input files.Molecular dynamics simulations by Gromacs showed that the O2 of MFA could structure hydrogen bond with the amino group H of Gly102.The whole system was stable in the early stage of the 10 ns simulation process,but it was fluctuant in the later stage.Finally,the total energy of the non-bonding interaction between DLFae4 and MFA was calculated to be-129.89 kJ/mol,it indicated the combination of the two molecule is tight and DLFae4 has high catalytic activity towards MFA..DLFae4 is a family VIII carboxylesterase containing a conserved SXXK motif with high sequence similarity to Class C β-lactamase and have high ampicillin hydrolysis activity.It displayed remarkable thermostability remained over 50%residual activity at 60℃ for 3 h.It can hydrolyze short-chain p-nitrophenyl esters and four methyl hydroxycinnamates.It is the first member of family VIII carboxylesterase reported to hydrolyze PAEs.These features suggest that DLFae4 is a potential biocatalyst in industry and environmental PAEs-biodegradation.These new findings are of great importance for further in-depth research and engineering development of esterases.