The Study In Directed Evolution And Application Of Enoate Reductases

Biocatalysis refers to the process of catalyzing chemical reactions using enzymes,microbial cells,or animal and plant cells as catalysts.Compared with chemical catalysts,biocatalysts have many advantages,such as mild reaction conditions,high catalytic efficiency,and low by-products.Biocatalysis meets the requirements of green development in the current era,and will have a wider application range and bright development prospects.Adipic acid is often used as an intermediate in the synthesis of nylon 6.6 and is one of the most important dicarboxylic acids.It is estimated that by 2022,the global adipic acid market will reach 8 billion pounds,so the commercial value of adipic acid cannot be underestimated.At present,the production of adipic acid is mainly chemically synthesized through the petrochemical route,such as the oxidation of KA oil (cyclohexanol/cyclohexanone mixture) catalyzed by nitric acid.During the synthesis process,a large amount of N2O will be produced,which will cause a greenhouse climate impact that is greater than CO2 298 times higher.N2O emissions from adipic acid production account for about 80% of total industrial N2O emissions,and its industrial emission reduction potential accounts for about 96.2%.The use of green renewable raw materials to produce adipic acid can effectively reduce the environmental pollution caused by the production process.As there is no reliable natural synthetic pathway,it is necessary to integrate foreign genes by genetic engineering and metabolic engineering to construct a biological method for adipic acid synthesis,but the biological synthesis of adipic acid by biological methods is still not economically possible.More efforts are needed to construct a thermodynamically feasible and economically viable green bioadipate pathway.Most of the currently established adipic acid biosynthetic pathways involve carbon-carbon double bond reduction of adipic acid.Therefore,the catalytic activity of olefinic reductase is an important factor affecting the industrialization of adipic acid synthesis by biological methods.Enoate reductases (ERs,EC 1.3.1.31) can selectively reduce C=C in ?,?-unsaturated compounds with electron withdrawing groups,which is one of the important enzymes in biocatalytic synthesis.The research object of this project is the enoate reductase (ER-BC) derived from Bacillus coagulans,which is required in the adipic acid biosynthesis pathway.The aim is to improve its catalytic activity through directed evolution and to study its enzymatic properties such as its catalytic mechanism.1?Existing research on enoate reductase derived from Bacillus coagulans is relatively weak,and its crystal structure,active site and catalytic mechanism have not been reported,and it does not have the conditions for direct site-directed mutation.Therefore,the subject first performed random mutation experiments on the ER-BC.Random mutation is a more effective method for the directed evolution of enzymes with less biological information.Random mutations using error-prone PCR can cause random mutations in gene sequences,resulting in positive mutations with increased enzyme activity,and provide more information about the structure of the enzyme.Based on the mutation library constructed by error-prone PCR,two strains with significantly improved enzyme activity were obtained(ER-BC-1/ER-BC-2).ER-BC-1 has four mutation sites Leu181Pro/Ile187Asn/Phe207Tyr/Phe229I1e,and ER-BC-2 has two mutation sites Point Gly 111 Asp/Gly53 1 Ser.2?ER-BC does not have an analytical protein crystal structure,so it needs to be modeled to get its structure.There are three main protein modeling methods:homology modeling,threading,and ab initio algorithms.Because the sequence identity of the ER-BC homology structure found after BLAST alignment can only reach 29%,the homology modeling method is not applicable.Finally,the I-ATSSER prediction server is used to model the ER-BC structure.The ER-BC model constructed by the threading method was molecularly docked with the substrate 2-ene-1,6-adipic acid through autodock4.The docking results showed that the substrate formed hydrogen bonding with Va161,His71 and Tyr180.The ligand FMN is located near the substrate and the active site,but it has a certain distance from the substrate.Tyr180 can form a hydrogen bond with the carboxyl O- of the substrate.3?After reading the literature,it was found that ER-BC and YqjM(enoate reductase from Bacillus subtilis) are enzymes from the same family,Based on the sequence comparison of the superfamily enzymes,it was found that the catalytic sites H164,H167,and Y169 of YqjM correspond to H174,H177,and Y180 of ER-BC,respectively.Therefore,it is speculated that H174,H177,Y180 of ER-BC are the binding site and catalytic site of the enzyme,and their catalytic steps should be roughly the same.Based on the literature information,random mutation results and homology modeling results,a total of 21 mutant proteins were designed,and the mutant protein G111D+L181P with 12 times higher enzyme activity was obtained.Based on the above experimental results,the catalytic mechanism of ER-BC was conjectured and verified.4?Enzymatic properties of wild-type olefinic reductase ER-BC and mutant protein with increased enzyme activity,such as optimal reaction temperature,optimal reaction pH,oxygen tolerance,and kinetic parameters were determined.The results showed that the Km/Kcat of the mutant proteins S109N+G111D+L181P and G111D+L181P were increased 9 times and 14 times respectively compared with wild type ER-BC.