Application of isoeugenol monooxygenase mutant in production of isoeugenol vanillin

Abstract

The invention discloses an application of an isoeugenol monooxygenase mutant in production of isoeugenol vanillin, which comprises the steps of recombinant strain construction, fermentation culture of recombinant strains and catalytic reaction of fermentation broth, and the isoeugenol monooxygenase mutant is mutated through an isoeugenol monooxygenase amino acid sequence of wild fungi WT. And the thalli are expressed by escherichia coli, so that a series of thalli with a promoting effect on the catalytic efficiency of vanillin production are formed. The mutant super-efficient enzyme catalyst with the catalytic conversion rate reaching up to 98.5% or above is obtained through screening, the defects of low efficiency, pollution, high energy consumption and the like of traditional chemical catalyst production are overcome, and the mutant super-efficient enzyme catalyst is more suitable for green industrial production requirements and worthy of being popularized to industrial use.

Description

Technical Field

[0001] The present invention relates to the technical field of engineered bacteria and their industrial applications, and particularly to the application of isoeugenol monooxygenase mutants in the production of vanillin from isoeugenol. Background Art

[0002] Vanillin, as a key natural fragrance, is widely used in the fields of food, beverage, cosmetics, and medicine. Its traditional production method is mainly chemical synthesis. However, with the increasing emphasis on environmental protection and sustainable development, the biological synthesis method has received extensive attention due to its clean and efficient characteristics. The production of vanillin by the catalysis of isoeugenol monooxygenase is one of the important biological synthesis pathways.

[0003] Isoeugenol, with the chemical formula C10H12O2, is a natural compound with a special aroma. The phenolic hydroxyl group and methoxy group in its structure make it easily catalyzed and oxidized by enzymes. Vanillin, scientifically named 3-methoxy-4-hydroxybenzaldehyde, is one of the most widely produced and used fragrances globally, with a strong vanilla bean aroma.

[0004] Monooxygenases are a class of enzymes that can catalyze the introduction of an oxygen atom into a substrate molecule, and they participate in various metabolic processes in living organisms. Isoeugenol monooxygenase is an enzyme that can specifically catalyze the oxidation of isoeugenol to produce vanillin. This enzyme exists widely in nature, such as in bacteria, fungi, and plants, but enzymes from different sources vary in catalytic efficiency, substrate specificity, and stability.

[0005] The reaction of isoeugenol monooxygenase catalyzing the oxidation of isoeugenol to produce vanillin usually includes two steps: First, the enzyme catalyzes the oxidation of the phenolic hydroxyl group of isoeugenol to a quinone structure; second, the quinone structure is further hydrolyzed to produce vanillin. In this process, the enzyme binds to an oxygen molecule and activates it into a highly reactive oxygen free radical, which then attacks specific chemical bonds in the substrate molecule.

[0006] In recent years, domestic and foreign scholars have made remarkable progress in the production of vanillin by the catalysis of isoeugenol monooxygenase. For example, some studies have reported the construction of yeasts that can highly express isoeugenol monooxygenase using genetic engineering techniques, and through optimizing the fermentation conditions, high yields of vanillin have been achieved. Under the optimal reaction conditions, the conversion rate of isoeugenol can reach over 80%, and the molar yield of vanillin exceeds 70%.

[0007] The production of vanillin by the catalysis of isoeugenol monooxygenase, as a green and efficient biological synthesis method, has broad application prospects. By continuously optimizing enzyme engineering, fermentation technology, and immobilization technology, it is expected to further improve the yield and purity of vanillin, reduce production costs, and meet the market demand for high-quality vanillin. Summary of the Invention

[0008] The object of the present invention is to solve the drawbacks existing in the prior art. By mutating the amino acid sequence of isoeugenol monooxygenase from wild fungus WT and expressing the obtained cells through Escherichia coli, a series of cells that promote the catalytic efficiency of vanillin production are formed, that is, the application of isoeugenol monooxygenase mutants in the production of isoeugenol vanillin is proposed.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] The isoeugenol monooxygenase mutants proposed by the present invention are mutants obtained by mutating and optimizing codons on the basis of the amino acid sequence shown in SEQ ID NO.1. The amino acid sequence shown in SEQ ID NO.1 is derived from the amino acid sequence of isoeugenol monooxygenase of wild fungus WT. The obtained mutants include the following types:

[0011] WT as shown in SEQ ID NO.1, (expression record as strain 1);

[0012] E47A as shown in SEQ ID NO.2: Mutate the 47th glutamate E of WT to alanine A, (expression record as strain 2);

[0013] T51A as shown in SEQ ID NO.3: Mutate the 51st threonine T of WT to alanine A, (expression record as strain 3);

[0014] S122A as shown in SEQ ID NO.4: Mutate the 122nd serine S of WT to alanine A, (expression record as strain 4);

[0015] F216A as shown in SEQ ID NO.5: Mutate the 216th phenylalanine F of WT to alanine A, (expression record as strain 5);

[0016] W279A as shown in SEQ ID NO.6: Mutate the 279th tryptophan W of WT to alanine A, (expression record as strain 6);

[0017] F281A as shown in SEQ ID NO.7: Mutate the 281st phenylalanine F of WT to alanine A, (expression record as strain 7);

[0018] F355A as shown in SEQ ID NO.8: Mutate the 355th phenylalanine F of WT to alanine A, (expression record as strain 8);

[0019] C361A as shown in SEQ ID NO.9: The cysteine C at position 361 of WT was mutated to alanine A, (the expression record is strain 9);

[0020] C473A as shown in SEQ ID NO.10: The cysteine C at position 473 of WT was mutated to alanine A, (the expression record is strain 10);

[0021] T51A / F216A / W279A as shown in SEQ ID NO.11: The amino acids at positions 47, 51, 122, 216, 279, 281, and 355 of WT were all mutated to alanine A, (the expression record is strain 11);

[0022] C361A / C473A as shown in SEQ ID NO.12: The amino acids at positions 361 and 473 of WT were both mutated to alanine A, (the expression record is strain 12);

[0023] T51A / F216A / W279A / C361A / C473A as shown in SEQ ID NO.13: The amino acids at positions 47, 51, 122, 216, 279, 281, 355, 361, and 473 of WT were all mutated to alanine A, (the expression record is strain 13);

[0024] The present invention also provides the application of the above isoeugenol monooxygenase mutants in the production of isoeugenol vanillin, including the following steps:

[0025] S1. Construction of recombinant strains:

[0026] The isoeugenol monooxygenase mutants are expressed through Escherichia coli BL21(DE3) (preserved by this laboratory), and recombinant engineering strains are screened; the recombinant engineering strains are cultured to obtain glycerol bacteria;

[0027] S2. Fermentation culture of recombinant strains:

[0028] S201. Preparation of seed liquid:

[0029] Take 6 glycerol bacteria, and inoculate 100 μl of each glycerol bacteria into 100 ml of M1 medium, and culture at 35°C and 220 rpm for 9 hours to obtain a resuscitated seed liquid;

[0030] Subsequently, take 1 ml of the resuscitated seed liquid and inoculate it into 100 ml of M2 medium, and continue to culture at 35°C and 220 rpm for 4 hours to prepare an expanded seed liquid;

[0031] S202. Fermentation culture:

[0032] Prepare 2 L of M3 medium, weigh 20 g of glucose, make up the volume to 25 ml, sterilize it independently, and then add it to the medium; the initial fermentation conditions are set as a stirring speed of 200 rpm, an aeration volume of 2, a tank pressure of 0.25 MPa, and a pH value of 7.0; maintain the stirring-related dissolved oxygen at 30%, control the pH value at 7.0 by adding ammonia water, and use an 80% glucose solution for feeding;

[0033] S203. Cooling induction:

[0034] When OD600 reaches 30, lower the temperature of the fermenter to 25 °C, and add IPTG with a final concentration of 0.6 mM / L to induce the expression of the target gene; the fermentation process lasts for about 24 hours until OD600 no longer increases, at which point the fermentation is stopped to obtain the fermentation broth; subsequently, take the fermentation broth for protein gel electrophoresis analysis, and the specific final OD value and protein concentration are shown in Table 3.

[0035] S3. Catalytic reaction of the fermentation broth:

[0036] Adjust the fermentation broth to pH = 10.5 with NaOH, add isoeugenol to a substrate concentration of 25 g / L for catalysis, and the catalytic conditions are: 35 °C, 180 rpm, maintain pH = 10.5, and continuously catalyze for 16 h overnight. After the reaction, samples are taken for HPLC to detect the vanillin yield and conversion rate, as shown in Table 4.

[0037] Preferably, the preparation process of the recombinant engineering strain is as follows: the WT amino acid sequence is retrieved from the NCBI database (http: / / www.ncbi.nlm.nih.gov / ), with the number C3VA26.1; after mutation and codon optimization, this sequence is synthesized by GenScript Biotech Corporation and the corresponding plasmid is constructed; as Figure 1 shown in Table 2, these recombinant plasmids are transformed into Escherichia coli BL21(DE3) [purchased from Huayueyang Biotechnology, product number NRR01210, named Escherichia coli BL21(DE3) strain, stored in our laboratory] by heat shock method; through ampicillin resistance screening, we obtained the recombinant engineering strains, and these recombinant strains are listed in Table 2; their correctness is verified by colony PCR method.

[0038] Preferably, the culture process of the glycerol bacteria is as follows: transfer the recombinant engineering strain from the plate to LB medium with an inoculation loop and culture it at 35 °C and 220 rpm. When OD600 reaches 1.0, add glycerol and mix well, then aliquot it into cryotubes and store it at -80 °C for later use.

[0039] Further, the LB medium comprises components at the following concentrations: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0040] Preferably, the M1 medium comprises components at the following concentrations: peptone 10 g / L, yeast extract powder 5 g / L, sodium chloride 10 g / L, and the remainder is purified water;

[0041] Preferably, the M2 medium comprises components at the following concentrations: peptone 16 g / L, yeast extract powder 10 g / L, sodium chloride 10 g / L, and the remainder is purified water;

[0042] Preferably, the M3 medium comprises components at the following concentrations: disodium hydrogen phosphate dodecahydrate 17 g / L, potassium hydrogen phosphate 3 g / L, sodium chloride 0.5 g / L, magnesium sulfate heptahydrate 0.5 g / L, glucose 10 g / L, ammonium sulfate 15 g / L, ammonium chloride 1 g / L, and the remainder is purified water, and the pH is adjusted to 7.0 with ammonia water.

[0043] Preferably, according to the catalytic yield and conversion rate, the isoeugenol monooxygenase mutants are specifically E47A, T51A, S122A, W279A, F355A, C361A, C473A, C361A / C473A, and T51A / F216A / W279A / C361A / C473A. The catalytic conversion rates of the above mutants are all > 96%, and among them, the catalytic conversion rates of C361A / C473A and T51A / F216A / W279A / C361A / C473A reach the highest (> 98.5%). It is a project of highly efficient catalytic engineering bacteria and is worthy of promotion to industrial use.

[0044] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0045] By mutating the amino acid sequence of isoeugenol monooxygenase and expressing it in Escherichia coli, the present invention improves the catalytic efficiency of the reaction of oxidizing isoeugenol to vanillin, making it more suitable for the requirements of green industrial production; by screening mutant ultra-high-efficiency enzyme catalysts with a catalytic conversion rate of up to more than 98.5%, the present invention avoids the disadvantages of low efficiency, pollution, and high energy consumption in the production of traditional chemical catalysts and is worthy of promotion to industrial use. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is the plasmid transformation map obtained in Example Group 1 of the present invention;

[0047] Figure 2 It is the protein gel electrophoresis map of the fermentation broth in Example Group 2 of the present invention;

[0048] Figure 3This is the HPLC detection chromatogram of vanillin production and conversion rate in Example Group 3 of the present invention. Detailed implementation manners

[0049] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the existing well-known technologies. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0050] The raw materials used in the present invention (all are mixed under sterile conditions):

[0051] The formula of LB medium is: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0052] The formula of M1 medium is: peptone 10 g / L, yeast extract powder 5 g / L, sodium chloride 10 g / L, and the rest is purified water;

[0053] The formula of M2 medium is: peptone 16 g / L, yeast extract powder 10 g / L, sodium chloride 10 g / L, and the rest is purified water;

[0054] The formula of M3 medium is: disodium hydrogen phosphate dodecahydrate 17 g / L, potassium hydrogen phosphate 3 g / L, sodium chloride 0.5 g / L, magnesium sulfate heptahydrate 0.5 g / L, glucose 10 g / L, ammonium sulfate 15 g / L, ammonium chloride 1 g / L, and the rest is purified water, and the pH is adjusted to 7.0 with ammonia water.

[0055] The genes involved in the present invention are shown in Table 1:

[0056] Table 1. Gene sequences of isoeugenol monooxygenase used in the present invention

[0057] Name Amino acid sequence Nucleotide sequence WT SEQ ID NO.1 SEQ ID NO.14 E47A SEQ ID NO.2 SEQ ID NO.15 T51A SEQ ID NO.3 SEQ ID NO.16 S122A SEQ ID NO.4 SEQ ID NO.17 F216A SEQ ID NO.5 SEQ ID NO.18 W279A SEQ ID NO.6 SEQ ID NO.19 F281A SEQ ID NO.7 SEQ ID NO.20 F355A SEQ ID NO.8 SEQ ID NO.21 C361A SEQ ID NO.9 SEQ ID NO.22 C473A SEQ ID NO.10 SEQ ID NO.23 E47A / T51A / S122A / F216A / w279A / F281A / F355A SEQ ID NO.11 SEQ ID NO.24 C361A / C473A SEQ ID NO.12 SEQ ID NO.25 E47A / T51A / S122A / F216A / w279A / F281A / F355A / C361A / C473A SEQ ID NO.13 SEQ ID NO.26

[0058] Example Group 1:

[0059] Construction of recombinant strains containing the isoeugenol monooxygenase gene sequence:

[0060] The amino acid sequence of the wild strain WT required for the present invention was retrieved from the NCBI database (website: http: / / www.ncbi.nlm.nih.gov / ), numbered C3VA26.1; after mutation and codon optimization, this sequence was synthesized by Suzhou GeneScript Biotechnology Co., Ltd., and the corresponding plasmid was constructed;

[0061] As Figure 1 and shown in Table 2, these recombinant plasmids were transformed into Escherichia coli BL21(DE3) by heat shock method [product catalog number: Beijing Huayueyang Biology NRR01210, product name: BL21(DE3) Escherichia coli strain, preserved by this laboratory];

[0062] Through ampicillin resistance screening, we obtained recombinant engineering strains, and these recombinant strains are listed in Table 2, and their correctness was verified by colony PCR method;

[0063] Thirteen strains were separately transferred from the plate to LB medium with an inoculation loop and cultured at 35 °C and 220 rpm. When OD600 reached 1.0, glycerol was added and mixed evenly, then aliquoted into cryotubes and stored at -80 °C for later use.

[0064] Table 2. Recombinant Bacterial Plasmids and Gene Names

[0065]

[0066] Example Group 2:

[0067] Fermentation Culture of Recombinant Strains:

[0068] 1. Preparation of Seed Solution

[0069] Six glycerol bacteria were taken, and 100 μl of each was inoculated into 100 ml of M1 medium and cultured at 35 °C and 220 rpm for 9 hours to obtain a resuscitated seed solution; subsequently, 1 ml of the resuscitated seed solution was inoculated into 100 ml of M2 medium and continued to be cultured at 35 °C and 220 rpm for 4 hours to prepare an expanded culture seed solution.

[0070] 2. Fermentation Culture

[0071] Prepare 2 L of M3 medium, weigh 20 g of glucose, make up the volume to 25 ml and sterilize it independently, then add it to the medium. The initial fermentation conditions were set as a stirring speed of 200 rpm, an aeration volume of 2, a tank pressure of 0.25 MPa, and a pH value of 7.0. Maintain the stirring-related dissolved oxygen at 30%. Control the pH value at 7.0 by adding ammonia water and use an 80% glucose solution for feeding.

[0072] 3. Cooling Induction

[0073] When OD600 reached 30, the temperature of the fermenter was reduced to 25 °C, and IPTG with a final concentration of 0.6 mM / L was added to induce the expression of the target gene. The fermentation process continued for about 24 hours until OD600 no longer increased, and then the fermentation was stopped. Subsequently, the fermentation broth was taken for protein gel electrophoresis analysis (refer to Figure 2 Taking the fermentation broth of strain 13 as an example), the specific final OD value and protein concentration are shown in Table 3.

[0074] Table 3. Final OD Value and Protein Concentration of Fermentation Broth ""

[0075] Strain <![CDATA[OD 600 > Enzyme concentration (g / L) Strain 1 140.53 6.13 Strain 2 138.42 6.25 Strain 3 143.45 6.41 Strain 4 140.03 5.82 Strain 5 138.31 5.73 Strain 6 139.03 5.82 Strain 7 143.56 5.93 Strain 8 142.11 6.04 Strain 9 139.26 5.83 Strain 10 139.41 5.93 Strain 11 140.32 6.24 Strain 12 141.21 6.38 Strain 13 143.53 6.50

[0076] Example Group 3:

[0077] Catalytic application of the recombinant strain fermentation broth:

[0078] Adjust each fermentation broth in Example Group 2 to pH = 10.5 with NaOH, add isoeugenol to a substrate concentration of 25 g / L for catalysis. Catalysis conditions: 35 °C, 180 rpm, maintain pH = 10.5, and continuously catalyze overnight for 16 h. Samples were taken after the reaction for HPLC detection. The vanillin yield and conversion rate are shown in Table 4.

[0079] Table 4. Yield and conversion rate of vanillin produced by catalytic fermentation broth

[0080] Strain Yield (g / L) Conversion rate (%) Strain 1 20.13 86.85％ Strain 2 22.32 96.30％ Strain 3 22.54 97.25％ Strain 4 22.46 96.91％ Strain 5 19.85 85.65％ Strain 6 22.42 96.74％ Strain 7 21.53 92.90％ Strain 8 22.43 96.78％ Strain 9 22.34 96.39％ Strain 10 22.85 98.59％ Strain 11 20.08 86.64％ Strain 12 22.87 98.68％ Strain 13 23.01 99.28％

[0081] As can be seen from Table 4, compared with the wild gene WT, the mutants C361A / C473A with amino acid conversions at positions 361 and 473 have a greater improvement in the catalytic reaction, that is, a higher catalytic efficiency improvement is achieved with less gene editing or modification, while the T51A / F216A / W279A / C361A / C473A with all characteristic amino acids changed even reaches a catalytic conversion rate of > 99%.

[0082] As described above, it is only the preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution of the present invention and its inventive concept, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.

Claims

1. Application of isoeugenol monooxygenase mutant in production of isoeugenol vanillin, characterized in that, The isoeugenol monooxygenase mutant is a mutant obtained by mutating and codon-optimizing the amino acid sequence shown in SEQ ID NO.1, and the amino acid sequence shown in SEQ ID NO.1 is derived from the amino acid sequence of isoeugenol monooxygenase of wild strain WT. The obtained mutants include the following types: E47A as shown in SEQ ID NO.2: Glutamic acid E at position 47 of WT is mutated to alanine A; T51A as shown in SEQ ID NO.3: Threonine T at position 51 of WT is mutated to alanine A; S122A as shown in SEQ ID NO.4: Serine S at position 122 of WT is mutated to alanine A; F216A as shown in SEQ ID NO.5: Phenylalanine F at position 216 of WT is mutated to alanine A; W279A as shown in SEQ ID NO.6: Tryptophan W at position 279 of WT is mutated to alanine A; F281A as shown in SEQ ID NO.7: Phenylalanine F at position 281 of WT is mutated to alanine A; F355A as shown in SEQ ID NO.8: Phenylalanine F at position 355 of WT is mutated to alanine A; C361A as shown in SEQ ID NO.9: Cysteine C at position 361 of WT is mutated to alanine A; C473A as shown in SEQ ID NO.10: Cysteine C at position 473 of WT is mutated to alanine A; T51A / F216A / W279A as shown in SEQ ID NO.11: Amino acids at positions 47, 51, 122, 216, 279, 281 and 355 of WT are all mutated to alanine A; C361A / C473A as shown in SEQ ID NO.12: Amino acids at positions 361 and 473 of WT are all mutated to alanine A; T51A / F216A / W279A / C361A / C473A as shown in SEQ ID NO.13: Amino acids at positions 47, 51, 122, 216, 279, 281, 355, 361 and 473 of WT are all mutated to alanine A; The application of the isoeugenol monooxygenase mutant in the production of isoeugenol vanillin includes the following steps: S1. Construction of recombinant strains: The isoeugenol monooxygenase mutant is expressed through Escherichia coli BL21, and recombinant engineering strains are screened; glycerol bacteria are prepared by culturing the recombinant engineering strains; S2. Fermentation culture of recombinant strains: S201. Preparation of seed liquid: Take 6 glycerol bacteria, and inoculate 100 μl of each glycerol bacteria into 100 ml of M1 medium, and culture at 35 °C and 220 rpm for 9 hours to obtain a resuscitated seed liquid; Subsequently, take 1 ml of the resuscitated seed liquid and inoculate it into 100 ml of M2 medium, and continue to culture at 35 °C and 220 rpm for 4 hours to prepare an expanded seed liquid; S202. Fermentation culture: Prepare 2 L of M3 medium. Weigh 20 g of glucose, make up the volume to 25 ml, and then sterilize it independently before adding it to the medium. Set the initial fermentation conditions as follows: stirring speed of 200 rpm, aeration volume of 2, tank pressure of 0.25 MPa, and pH value of 7.

0. Maintain the dissolved oxygen associated with stirring at 30%, control the pH value at 7.0 by adding ammonia water, and use an 80% glucose solution for feeding. S203. Cooling induction: When OD600 reaches 30, lower the temperature of the fermenter to 25 °C and add IPTG with a final concentration of 0.6 mM / L to induce the expression of the target gene. The fermentation process lasts for about 24 hours until OD600 no longer increases. At this time, stop the fermentation to obtain the fermentation broth. Subsequently, take the fermentation broth for protein gel electrophoresis analysis to obtain the final OD value and protein concentration. S3. Catalytic reaction of the fermentation broth: Adjust the fermentation broth to pH = 10.5 with NaOH, add isoeugenol to a substrate concentration of 25 g / L for catalysis. Catalysis conditions: 35 °C, 180 rpm, maintain pH = 10.5, and continuously catalyze overnight for 16 h. Take samples after the reaction for HPLC detection of vanillin production and conversion rate.

2. Use of the isoeugenol monooxygenase mutant according to claim 1 in the production of vanillin from isoeugenol, characterized in that, The preparation process of the recombinant engineering strain is as follows: The WT amino acid sequence was retrieved from the NCBI database with the accession number C3VA26.

1. After mutation and codon optimization, this sequence was synthesized and the corresponding plasmid was constructed. The plasmid was transformed into Escherichia coli BL21 by heat shock method. Through ampicillin resistance screening, the recombinant engineering strain was obtained. The correctness was verified by colony PCR method.

3. Use of the isoeugenol monooxygenase mutant according to claim 1 in the production of isoeugenol vanillin, characterized in that, The culture process of the glycerol bacteria is as follows: Transfer the recombinant engineering strain from the plate to LB medium with an inoculation loop and culture it at 35 °C and 220 rpm. When OD600 reaches 1.0, add glycerol and mix well, then aliquot it into cryotubes and store it at -80 °C for later use.

4. Use of the isoeugenol monooxygenase mutant according to claim 3 in the production of vanillin from isoeugenol, characterized in that, The LB medium includes the following components at the following concentrations: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

5. Use of the isoeugenol monooxygenase mutant according to claim 1 in the production of vanillin from isoeugenol, characterized in that, The M1 medium includes the following components at the following concentrations: peptone 10 g / L, yeast extract powder 5 g / L, sodium chloride 10 g / L, and the rest is purified water.

6. Use of the isoeugenol monooxygenase mutant according to claim 1 in the production of isoeugenol vanillin, characterized in that, The M2 medium includes the following components at the following concentrations: peptone 16 g / L, yeast extract powder 10 g / L, sodium chloride 10 g / L, and the rest is purified water.

7. Use of the isoeugenol monooxygenase mutant according to claim 1 in the production of isoeugenol vanillin, characterized in that, The M3 medium includes the following components at the following concentrations: disodium hydrogen phosphate dodecahydrate 17 g / L, potassium hydrogen phosphate 3 g / L, sodium chloride 0.5 g / L, magnesium sulfate heptahydrate 0.5 g / L, glucose 10 g / L, ammonium sulfate 15 g / L, ammonium chloride 1 g / L, and the rest is purified water. Adjust the pH to 7.0 with ammonia water.

8. Use of the isoeugenol monooxygenase mutant according to claim 1 in the production of vanillin from isoeugenol, characterized in that, The isoeugenol monooxygenase mutants are specifically E47A, T51A, S122A, W279A, F355A, C361A, C473A, C361A / C473A, and T51A / F216A / W279A / C361A / C473A.

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