Study On Screening And Identification Of Esterase EstXT1 And Molecular Simulations

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 are commonly found in microorganisms,plants and animals and participate in various reactions such as ester hydrolysis,esterification and transesterification.Esterase has high catalytic activity,few side reactions,and has a wide range of substrate specificity and enantiomeric selectivity.It is used in food,medicine,detergent,papermaking and other fields.Traditional methods for screening microbial esterase genes rely on the cultivation and purification of microorganisms,but only 0.1%-1%of microorganisms in nature can grow under laboratory conditions,and more than 99%of uncultured microorganisms containing rich esterase genes resources have not been exploited.The metagenomic technology bypasses the process of isolation and culture of microorganisms,directly extracts total DNA from environmental samples,constructs a metagenomic library,and obtains esterase genes from the library through sequence or function-based screening methods,expanding the source of functional genes.It provides a new way to obtain new esterases.In this study,metagenomic technology was used to construct a soil metagenomic library.A functional screening method was used to obtain a novel esterase gene estXTl.Heterologous expression of estXTl was achieved in E.coli BL21(DE3).The enzymatic properties of EstXTl were characterized and the molecular dynamics of the enzyme-ligand complex was simulated.The main results are as follows:1.Construction of soil metagenomic library and screening of esterase genes.Using soil microorganisms as the research object,soil environmental DNA was extracted,and the method of phage packaging infection was used to construct a Fosmid metagenomic library with E.coli as the host.A novel esterase gene estXTl was screened from the library by functional screening.The length of nucleotide sequence is 1143 bp and it encodes 380 amino acid residues.The predicted molecular weight of the protein is 40.0 kDa and the isoelectric point is 4.60.Multi-sequence alignment results showed that EstXT1 has multiple motifs of Family Ⅷ esterases.Phylogenetic analysis showed that EstXT1 and FamilyⅧ esterases were grouped into one branch.Therefore,Ser56,Lys59,and Tyr165 were presumed to be catalytic triplets of EstXTl.2.Heterologous expression of esterase.Using the positive subclone as a template,the estXTl gene was amplified and cloned into the pET-28a(+)expression vector.The pET-estXTl recombinant plasmid was constructed and induced to express at low temperature in E.coli BL21(DE3).Recombinase was purified by nickel column affinity chromatography.The protein band was verified by SDS-PAGE and the molecular weight was consistent with the prediction.3.Characterization of enzymatic properties.EstXT1 has hydrolytic activity on ferulic acid esters.The optimal pH for catalytic reaction is 8.0,the optimal temperature is 55℃.After incubation for 3 hours in the pH range of 7-10.6,EstXT1 can maintain more than 80%of the activity,with strong alkali resistance,and maintain more than 70%of the activity within 3 hours of incubation at 30℃.Zn2+ and Co2+ have a certain activation effect on EstXT1.Cu2+and CTAB have a strong inhibitory effect on the activity of EstXT1.In addition to DMF and DMSO,the organic reagent has a significant inhibitory effect on enzyme activity.In addition,EstXT1 had a certain tolerance to the deionized surfactants Triton X-100 and Tween 80.4.EstXT1 is docked with the molecules of the ligand.The 3D model of EstXT1 was constructed with protein 4IVK as the template.The software AutoDock Vina was used to conduct flexible docking between EstXT1 and the substrate MFA to obtain the protein-substrate complex.There were multiple hydrogen bonds and hydrophobic interactions between proteins and substrates,which provided support for the stable binding of ligands.5.Research on molecular dynamics simulation of EstXT1.The protein ligand complex was solvated using Amber and NAMD software,and the energy of the entire system was minimized and balanced.Finally,dynamics simulation was performed.The last equilibrium configuration of NAMD molecular dynamics simulation was simulated by QM/MM.The catalytic triplet,substrate molecule and two-molecule water were divided into QM region,and the rest were divided into MM region.Quantum mechanics and molecular mechanics calculations were conducted respectively.The total energy of MM-MD calculated by Amber is lower,which is-91385.2304 kcal/mol,while MM-MD calculated by NAMD is-511381.1862 kcal/mol,and QM/MM is-453299.7295 kcal/mol.The free binding energies of EstXT1 and substrate MFA were-32.1722 kcal/mol and-4.1049 kcal/mol,respectively,calculated by MM-GBSA and MM-PBSA methods,indicating that the enzyme and substrate are tightly bound and the catalytic activity is high.Phe128 near the active pocket was mutated into alanine forming mutant F128A,and the difference was calculated by MM-GBSA and MM-PBSA with-1.7485 kcal/mol and-1.3438 kcal/mol,respectively,compared with that before the mutation.The free binding energy increased after the mutation,indicating that the substitution of alanine was disadvantageous,while Phe128 near the active pocket was beneficial for the close binding of MFA.EstXT1 is a Family Ⅷ esterase,which has strong alkali resistance,good thermal stability,certain tolerance to deionized surfactants Triton X-100 and Tween 80,and has hydrolytic activity on ferulic acid esters.These features make it have potential industrial application value.The research results show that using metagenomic technology can obtain novel esterases from genetic resources of uncultured microorganisms,providing a new research strategy for finding new esterases with special enzymatic properties,expanding the resources of functional genes,and providing biocatalysts with potential application value for industry.