The Principle Of P450 Enzyme Hydroxylation And Detection Of Key Products In Fermentation Broth By SERS

The cytochrome P450(P450)enzyme is an oxidation catalyst widely found in nature,and its catalytic cycling mechanism has been extensively studied.Hydroxylation is a reaction that introduces a hydroxyl group into a molecule,and many valuable industrial monomers can be produced by this reaction.Stearic acid can be hydroxylated by P450 enzyme to form a cyclopentadecanolide which can be used to synthetic fragrances.The formation of cyclopentadecanolactone requires the hydroxylation positions of stearic acid at the ?-2 position.Therefore,the principle of P450 enzymes hydroxylation can guide experiments to obtain better performance products.Microbial fermentation is a technology that utilizes microorganisms to transform a given raw material into fermented products needed in daily life through a specific way under certain conditions.Microbial fermentation technology is low cost,wide-ranging,environmentally friendly,which is consistent with the current sustainable development idea,and it is an important direction of scientific research.1,3-propanediol(1,3-PDO)is one of the oldest known fermentation products.It can be used as a monomer to synthesize polyterephthalate with flexibility,bulkiness,anti-fouling and elasticity.Microbial fermentation technology is developed from small workshop-style family production.It has the characteristics of empiricism.There are some neglects in understanding the principle of microbial fermentation technology.Due to the non-linear and time-varying characteristics of it,many detection methods can not be used for real-time quantitative detection.Surface-enhanced Raman scattering(SERS)is an enhancement of the Raman effect that extends the application range of Raman technology.The SERS detection method has the advantages of high sensitivity,selectivity,real-time,rapidity and non-destructive.It has good detection results from small molecules of organic matter to macromolecular enzymes and proteins.Real-time and quantitative detection of key products in fermentation broth by SERS technology and mastering the microbial fermentation process are of great significance for optimizing raw material replenishment,improving conversion rate and yield.The main research content of the this thesis is divided into the following two aspects:1.The principle of stearic acid hydroxylation catalyzed by P450 enzymeIn order to synthesize cyclopentadecanolide,three mutants of P450,F87V,L188Q,188 and 87 double mutations(188/87),were selected to study.Through the experiment of stearic acid hydroxylation with P450 enzyme,the conversion rate of hydroxylation products of ?-2 position in L188Q mutant was the most obvious than that of wild type,followed by 188/87,and the result of F87V was not as good as wild type.Molecular dynamics(MD)simulation analysis was carried out according to the experimental results.In the process of stearic acid hydroxylation of MD simulation by P450 enzyme,the distances between stearic acid ?-2 H and P450 en2yme hemoglobin central part of the oxygen of L188Q,wild type,F87V,188/87 four kinds less than 5A were 17%,4%,0%and 10%respectively,which were consistent with the experimental results.Through the statistical analysis of the hydrogen bonds formed during MD simulation process,the L188Q mutant forms a unique hydrogen bond between GLN188 and GLN73,the 188/87 mutant forms a unique hydrogen bond between GLN188 and ARG73.The unique hydrogen bond formed by L188Q is close to the inside of the pocket,and the unique hydrogen bond formed by 188/87 is close to the outside of the pocket.Two different hydrogen bonds have different effects on the hydroxylation process.At the same time,the 87 site of P450 enzyme is a large residue phenylalanine,which will eliminate the hindrance after mutation.Stearic acid is a long-chain amino acid,so the disappearance of steric hindrance would cause the key site of stearic acid is far away from the central part of heme,which was the key part of P450 enzyme.The distances between stearic acid ?-1,?-2,?-3 H and P450 enzyme hemoglobin central part of the oxygen of wild type was 6%,4%and 1%,respectively.The ratio of L188Q was 16%,17%and 2%,respectively.It can be seen that the distance between stearic acid?-2 H of L188Q mutant and P450 enzyme hemoglobin central part of the oxygen is significantly closer than that of wild type,and obvious enhancement can be seen in three sites.This conclusion is proved by the calculation of flexibility force constant.2.Detection of key products in fermentation broth by SERS1,3-PDO can be produced by microbial fermentation from glycerol as raw material.The main by-product produced is lactic acid.The molecular structure of 1,3-PDO,glycerol and lactic acid is very similar,It is difficult to detect the actual fermentation broth.In this study,a Ag/Si SERS substrate with simple preparation and excellent enhancement effect was used as a biosensor.The concentration of 1,3-PDO,glycerol and lactic acid in the fermentation process was determined by SERS spectroscopy,and the progress of fermentation reaction was mastered.After testing the solution of pure,two-two mixing,and three mixtures,the calculated concentration compared with the actual addition which error is very small can be used for the detection of the actual fermentation broth.Through the continuous sampling of the fermentation broth for 9 to 20 hours in the actual fermentation process,the SERS spectroscopy analysis reflects the change of three substances content in the fermentation process.In order to verify the accuracy of the test results,the SERS test results of the actual fermentation broth were compared with those of high performance liquid chromatography(HPLC).The range of relative error can meet the needs of actual detection.