Study On The Biochemical Characterization And Molecular Modification Of Esterase SIEst From Solibacillus Isronensis B3W22

Lactonase is an ester hydrolase that can efficiently catalyze the hydrolysis of the ester bond of lactone compounds.Its unique catalytic properties are used in many fields such as the food industry,medicine and chemical industry.In nature,most lactonase belong to the metal?-lactamase superfamily,a few are the?/?-hydrolase fold superfamily,and a few are the phosphotriesterase superfamily.The members of the?/?hydrofoldase superfamily are generally esterases or lipases,but some of them have promiscuous lactonase activities,so they are not general esterases but lactonases.The lactonase from the superfamily of?/?hydrolyzing foldases has received extensive attention due to its good stability and non-metal dependence.However,the low catalytic efficiency of the existing lactonase in this family cannot meet all the needs of the industry.Therefore,it is necessary to understand the substrate recognition mechanism of the?/?hydrolase superfamily lactonase to improve its catalysis further.This topic takes Solibacillus isronensis esterase(SIEst)as the research object,and explores the bioinformatics,recombinant enzyme preparation and enzymatic properties of SIEst.SIEst is a member of the general esterase family,but it has a specific lactonase activity.We combined rational molecular design,site-directed mutagenesis,and enzymatic reaction kinetics to research and elaborate strategies to improve the activity of SIEst lactonase,and at the same time provide a scientific theory for improving the catalytic activity of the?/?hydrolase superfamily lactonase guide.The main research contents are as follows:(1)Bioinformatics analysis of SIEst.Using the reported lactonase MLH as a template,a potential lactonase gene from the genome of Solibacillus isronensis B3W22 was screened out from the NCBI database,and the sequence was named SIEst.The molecular evolution genetic analysis software MEGA-X,the multiple sequence alignment program Clustal Omega sequence alignment program and the Prot Param tool on the Ex PASy official website were used to analyze the SIEst gene sequence.The results show that SIEst is closely related to the acetyl esterase family,and it is likely to belong to a new branch of the acetyl esterase family.It is speculated that it has mixed catalytic activity,including the hydrolysis of short-chain ester compounds and the hydrolysis of partial lactone compounds.The SIEst-encoded protein has a typical conserved pentapeptide motif G-X-S-X-G,Ser150/Asp242/His272 constitutes a catalytic triad.The conserved HGGG motif(amino acids 76-79)is found upstream of the active site conserved motif,which participates in hydrogen bond interactions to stabilize the oxygen anion hole and plays a key role in catalysis.SIEst may be a heat-labile protein,which is prone to degradation in vitro.Its theoretical molecular weight is 33.87 k Da,the predicted isoelectric point is 6.08,the instability coefficient is 46.88,and the aliphatic coefficient is 86.74.The half-lives in mammalian reticulocytes,yeast and E.coli are 30 h,respectively.>20 h and>10 h.(2)Study on the heterologous expression of SIEst and its enzymatic properties.The SIEst gene was obtained using the whole gene synthesis technology,and the SIEst gene was successfully cloned into p ET28a using the seamless cloning technology.The successfully constructed p ET28a-SIEst recombinant plasmid was transformed into E.coli BL21(DE3)to achieve soluble expression.The protein was purified using a nickel affinity chromatography column.After the protein concentration was detected,each 1 L of fermentation broth could express 2.33 mg of purified protein.SDS-PAGE electrophoresis detection showed that most of SIEst was expressed in the form of inclusion bodies.Experiments have verified that the induction time significantly affects the protein expression of SIEst.The induction time is optimized,and the best induction time is 12 h.Enzymatic characterization results show that the optimal catalytic temperature of SIEst is 20?,and the T_1/2 of this enzyme is 90 min at 30?.When the temperature is higher than 30?,the enzyme activity will be rapidly attenuated.The optimal catalytic p H of SIEst is 8,which is relatively stable in a moderately alkaline environment.The determination of the substrate specificity of SIEst with 8 different lactones and 5 different ester compounds using the method of enzymatic kinetic determination showed that SIEst effectively catalyzed acyclic substrates,such as octyl acetate,but was Cyclic lactone-type substrates are less sensitive.Among the lactones,?-laurolactone has the highest catalytic efficiency.(3)Improve the lactonase activity of SIEst based on the rational structural design.Using the homology modeling program Modeller with PROCHECK and Verify 3D to construct a reliable SIEst protein three-dimensional model.The SIEst protein model and its homologues were analyzed and compared in detail using Pymol molecular visualization software.The results show that:SIEst is a typical?/?hydrolytic folding enzyme,and the whole is composed of two parts:a"lid"structure and a catalytic domain.SIEst is very similar to the two common lactonases.The overall framework of the structure of MLH and Aid H is very similar,and the catalytic triad is also highly conserved in space,but the shape of the active pocket is entirely different,which may result in the relatively high activity of SIEst lactonase.Weak reason.The molecular docking program Auto Dock was used to analyze the structure of the enzyme-substrate complex.After docking?-caprolactone to the SIEst active pocket,the five sites were F82,Y178,W198,W207,and F244,which were observed to be restricted.The expected?-caprolactone binding,mutating it to alanine is expected to expand the substrate-binding area while maintaining hydrophobic interactions.Besides,K204 may cause abnormal binding of the substrate in the binding pocket,and mutate it to the amino acid corresponding to the MLH lactonase to avoid abnormal conditions.Therefore,six mutants were constructed,namely F82A,Y178A,W198A,W207A,F244A and K204L.Combined with the analysis of the experimental results of the enzymatic reaction kinetics determination,4 out of the 6 mutants of SIEst improved the catalytic efficiency of the hydrolysis of?-caprolactone.For?-caprolactone,the best variant is F244A,with a 48 times k_cat/K_m that is higher than wt SIEst.It can be concluded that increasing the space of the lactone ring binding region in the lactonase activity pocket is the best way to improve the catalytic efficiency of the lactonase in the?/?-hydrolase folding enzyme;secondly,the lactonase activity pocket prefers a hydrophobic environment.The introduction of hydrophobic amino acids into the binding region of the lactone ring can enhance the hydrophobic interaction to increase the affinity of the enzyme and the substrate;finally,the extra space in the alcohol binding pocket is not conducive to the catalysis of the lactone substrate.