| Vanillin with rich aroma is one of the most popular food flavours and fragrances around the world,widely used in all sides of life.At present there are mainly chemical synthesis vanillin,natural vanillin and biological vanillin in the market.Recently,food safety and environmental pollution have emerged in continously.The chemical synthesis method has polluted environment seriously by the excessive use of organic solvents and hazardous chemical reagents,and a large number of wastes were generated.Besides,chemical synthesis vanillin has a great potential safety hazard to the human body.The natural Vanillin exists in vanilla beans,but only accounts for 2%of the total dry weight.The direct extraction cost is high.And it is also affected by the planting environment and government policies.It is difficult to meet the market demand for natural vanillin.People are increasingly eager to develop a green synthetic method to industrialize the production of natural vanillin.The United Nations Codex Alimentarius Commission defined in its statute that perfumes prepared by physical or enzymatic or microbial processes using natural raw materials as substrates can be regarded as natural perfumes since 2018.Isoeugenol is an ideal natural raw material for vanillin production.Isoeugenol monooxygenase is the only key enzyme for the conversion of isoeugenol to vanillin.Biological vanillin produced by this enzyme from the engineering bacteria is almost identical to natural vanillin,but one of the bottlenecks of this transformation technique is the low enzymatic activity and the long transformation period.Directed evolution is a technical mean to improve the performance of enzymes and even create new enzymes.In this project,the auto-induced expression system of isoeugenol monooxygenase was explored.And a high throughput screening method for isoeugenol monooxygenase under auto-induced expression system was established.The engineering bacteria named E.coli BL21（DE3）-pET30a-iem-F281Q（phenylalanine was mutated to glutamine at 281 positions）was obtained by point mutation of the bacteria E.coli BL21（DE3）-pET21a-iem previously,which yield was increased by 76.9%.Item gene iem-F281Q expressing isoeugenol monooxygenase was chosen to be the start template.Error-prone PCR was used for constructing the random mutation library of item gene.Based on the reaction of thiobarbituric acid（TBA）with vanillin and the large capacity of 96-well plate,a high throughput screening method of 96-well plate-TBA for isoeugenol monooxygenase under auto-induced expression system was established,and then re-screened by high-performance liquid chromatography（HPLC）.The main results are as follows:1)SDS-PAGE analysis showed that isoeugenol monooxygenase could be successfully expressed by the auto-induced expression system.In order to establish the auto-induced expression system of isoeugenol monooxygenase,the main components and induced expression conditions of the auto-induction medium for isoeugenol monooxygenase were further optimized.The optimum formulation of the auto-induction medium was as follows:ZY stock solution was prepared with tryptone1.5%and yeast extract 0.25%;50×5052 stock solution was prepared with lactose 5%,glycerol 25%and glucose concentration 2.0%;50×M and 1000×trace elements were prepared with the original formulation.Then 20 mL 50×M and 50×5052 were added to 958 mL ZY,sterilized under high pressure.And sterilize 0.75 mol/L MgSO4·7H2O（final concentration is 1.5 mmol/L）and 0.2 mL 1000 x trace elements（final concentration is 0.2 x）were also added to the liquor,respectively.The optimum technological conditions were that 4%of the inoculation into a 50 mL conical bottle with 7.5 mL medium,and the expression was induced at 200 rpm,30℃for 10 hours.The yield of vanillin by conversion of isoeugenol can reach 2.04 g/L,which is 1.5times higher than that before.By simplifying the steps of enzyme activity determination under automatic induciton and optimizing the testing methods,the standard deviation was adjusted from 16%to 12%,basically meeting the requirements of high throughput screening.2)A high-throughput screening method based on reactive oxygen species（ROS）was explored.This method is simple to operate and has a short detection time.However,the residual substrate isoeugenol will seriously affect its absorbance value,so it is not suitable for high-throughput screening method in this subject.However,isoeugenol had little effect on the product vanillin-based visible light spectrophotometry,which was consistent with the trend of high performance liquid chromatography.It can be used as a screening method for isoeugenol monooxygenase.3)Using the isoeugenol monooxygenase gene named iem-F281Q as the template of error-prone PCR,with optimized concentration of gene mutation factor Mn2+in error-prone PCR reaction system.The optimal concentration was determined as 0.05mmol/L.A random mutation library with 80%positive rate was constructed by error-prone PCR.4)Based on the above 96-well plate-thiobarbituric acid high-throughput screening method,940 strains of bacteria were screened,and then the vanillin concentration was determined by HPLC.A mutant strain F10 was obtained.Its activity increased to 154%,which was 2.4 times higher than that of the original strain E.coli BL21（DE3）-pET21a-iem before transformation.Compared with the parent strain F281Q（phenylalanine at 281 positions was mutated to glutamine）,four mutations occurred in the mutant strain F10,one of which was a nonsense mutation.The methionine（ATG）at 354 positions was mutated to leucine（TTG）.Leucine and methionine both are non-polar amino acids,but there was no sulfur atom in the structure of leucine.It is presumed that sulfur atom is liable to form disulfide bonds which affect the binding of substrates or the release of products.The glutamic acid（GAA）at 414 positions was mutated into valine（GTA）.That is the negative polar amino acid is mutated into non-polar amino acids.The arginine（CGG）at 437positions was mutated into leucine（CTG）.That is the positive polar amino acid is mutated into non-polar amino acids,indicating that non-polar amino acid can promote the binding of enzymes and substrates or dissociation of products. |
