Composition and method for producing piceatannol

Abstract

The present invention provides a composition for producing fisetanol using a tyrosinase derived from Bacillus megaterium and a method for producing fisetanol. Using the composition and method of the present invention, it is possible to produce fisetanol in a high yield.

Description

Technology Field

[0001] The present invention relates to a method for converting resveratrol into ficetanol using tyrosinase. Background Technology

[0003] Picetannol is a metabolite of resveratrol and is a stilbenoid compound found in grapes, red wine, passion fruit, and white tea. Picetannol has been shown to have anticancer effects against various cancers, including leukemia, lymphoma, breast cancer, prostate cancer, colorectal cancer, and skin cancer. In addition to its anticancer effects, it is known to possess anti-aging, anti-inflammatory, antioxidant, diabetes resistance, inhibition of neurodegenerative diseases, inhibition of cardiovascular diseases, and anti-obesity effects. Like resveratrol, picetannol is a natural product and possesses similar anticancer effects, but exhibits superior anticancer efficacy over a broader range.

[0004] Currently, although fisetanol can be extracted from nature or chemically synthesized, there are limitations due to low yields and complex processes, so there is currently no method for manufacturing fisetanol that can be utilized industrially.

[0005] The inventors have completed the present invention by discovering a method to produce ficetanol from resveratrol using tyrosinase derived from Bacillus megaterium as a biocatalyst. Prior art literature

[0007] Korean Registered Patent No. 10-0878394 The problem to be solved

[0008] The present invention aims to provide a composition for producing picetanol.

[0009] Another objective of the present invention is to provide a method for mass-producing picetanol.

[0010] Another objective of the present invention is to provide a method for producing fisetanol using tyrosinase derived from Bacillus megaterium.

[0011] Another objective of the present invention is to provide a method for producing picetanol using a biocatalyst under conditions of ethanol and oxygen supply.

[0012] Another objective of the present invention is to provide a method for producing biological physetanol that can be utilized in the manufacturing process of natural materials. means of solving the problem

[0014] 1. A composition for producing fisetanol comprising a tyrosinase having an amino acid sequence of any one of SEQ ID NOs 1 to 6, a microorganism expressing the same, a culture or pulverized product of the microorganism.

[0015] 2. A composition for producing fisetanol, wherein the tyrosinase is derived from Bacillus megaterium, in accordance with 1 above.

[0016] 3. A method for producing picetanol comprising the step of reacting resveratrol with the composition of 1 above.

[0017] 4. A method for producing picetanol according to 3, further comprising the step of dissolving resveratrol in ethanol or an aqueous solution thereof.

[0018] 5. A method for producing picetanol according to 3 above, further comprising the step of supplying oxygen.

[0019] 6. A method for producing picetanol according to 3 above, wherein the reaction is carried out at 30 to 50 ℃.

[0020] 7. A method for producing picetanol according to 3 above, wherein the reaction is carried out at a pH of 3 to 9.

[0021] 8. A method for producing picetanol, wherein, in the above 3, the step of reducing (E)-4-(3,5-dihydroxystyryl)cyclohexa-3,5-diene-1,2-dione generated by the peroxidation reaction of resveratrol to picetanol.

[0022] 9. A method for producing picetanol, wherein the reaction in the above 3 is carried out in the presence of any one reducing agent selected from the group consisting of NADH, NADPH, ascorbic acid, cysteine, p-coumaric acid, hydroxylamine, catechol, pyrogallol, and caffeic acid.

[0023] 10. A method for producing picetanol according to 3 above, further comprising the step of separating or purifying picetanol from the reaction solution. Effects of the invention

[0025] The composition and method for producing picetanol according to the present invention enable the industrial mass production of picetanol.

[0026] The tyrosinase of the present invention can function as a biocatalyst under ethanol conditions, thereby increasing the solubility of the substrate resveratrol.

[0027] Using the tyrosinase of the present invention improves the productivity of fisetanol.

[0028] The tyrosinase of the present invention has ethanol resistance and can be utilized in the ethanol extraction process of natural products.

[0029] Pisetanol produced according to the present invention is suitable for use as a material for pharmaceuticals and health functional foods. Brief explanation of the drawing

[0031] Figure 1 shows the concentration of fisetanol produced according to the concentration of ethanol. Figures 2 and 3 show the concentration of fisetanol produced according to temperature. Figure 4 shows the concentration of fisetanol produced according to pH. Figures 5 and 6 compare the concentration of fisetanol produced according to aeration, reaction time, and substrate concentration. Figure 7 shows the results of a reaction using various tyrosinase variants as shown by HPLC analysis. Figure 8 confirms the fisetanol productivity of various tyrosinase variants derived from Bacillus megaterium. Figure 9 shows the TIC (Total Ion Chromatogram) figure obtained through GC-MS analysis of the fisetanol production reaction using tyrosinase. Figures 10 and 11 are the results of Mass Spectrum analysis of resveratrol and ficetanol in a reaction using the composition of the present invention. Specific details for implementing the invention

[0032] The present invention relates to a composition for the production of piceatannol.

[0033] Pisetanol may be represented by the following chemical formula 1.

[0034]

[0035] The composition of the present invention comprises a tyrosinase consisting of any one of the amino acid sequences of SEQ ID NOs 1 to 6, a microorganism expressing the same, a culture or pulverized product of said microorganism.

[0036] Tyrosinase composed of the amino acid sequences of SEQ ID NOs. 1 to 6 is Bacillus megaterium ( Bacillus megaterium It may be derived from ). Sequence numbers 1 to 6 are as shown in Table 1.

[0037] 티로시나아제 아미노산 서열 GJ803(서열번호 1) MSNKYRVRKNVLHLTDTEKRDFIRAVLILKEKGIYDRYVAWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFERDLQSINPEVTLPYWEWETDAQLQDPSQSPIWNADFMGGNGNPKKDFIVDTGPFAAGRWTTIDEQGNPSGGLKRNFGATKEAPTLPTRDDVLKALKITQYDTPPWDMTSQNSFRNQLEGFINGPQLHNRVHRWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQIVHRNQNYLPMKNGPFGQNFRDPMYPWNTTPEDVMNHRKLGYVYDIELRKSKRSS GJ804(서열번호 2) MSNKYRVRKNVLHLTDAEKRDFIRAVLILKEKGIYDRYIAWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFERDLQSINPEVTLPYWEWETDAQLQDPSQSPIWSADFMGGNGNPKKDFIVDTGPFAAGRWTTIDEQGNPSGGLKRNFGATKEAPTLPTRDDVLKALKITQYDTPPWDMTSQNSFRNQLEGFINGPQLHNRVHRWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQIVHRDQNYRPMKNGPFGQNFRDPMYPWNTTPEDVMNHRKLGYVYDIELRKSKRSS DY801(서열번호 3) MSNKYRVRKNVLHLTDTEKRDFVRTVLILKEKGIYDRYIAWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFERDLQSINPEVTLPYWEWETDAQMQDPSQSQIWSADFMGGNGNPKKDFIVDTGPFAAGRWTTIDEQGNPSGGLKRNFGATKEAPTLPTRDDVLNALKITQYDTPPWDMTSQNSFRNQLEGFINGPQLHNRVHRWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQIVHRNQNYQPMKNGPFGQNFRDPMYPWNTTPEDVMNHRKLGYVYDIELRKSKRSS DY802(서열번호 4) MSNKYRVRKNVLNLTDMEKRDFVRTVLILKEKGIYDRYIAWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFERDLQSINPEVTLPYWEWETDAQMQDPSQSQIWSADFMGGNGNPKKDFIVDTGPFAAGRWTTIDEQGNPSGGLKRNFGATKEAPTLPTRDDVLNALKITQYDTPPWDMTSQNSFRNQLEGFINGPQLHNRVHRWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQIVHRNQNYQPMKNGPFGQNFRDPMYPWNTTPEDVMNHRKLGYVYDIELRKSKRSS DY804(서열번호 5) MSNKYRVRKNVLHLTDTEKRDFIRAVLILKEKGIYDRYVAWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFERDLQSINPEVTLPYWEWETDAQLQDPSQSPIWNADFMGGNGNRKKDFIVDTGPFAAGRWTTIDEQGNPSGGLKRNFGATKEAPTLPTRDDVLKALKITQYDTPPWDMTSQNSFRNQLEGFINGPQLHNRVHRWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQIVHRNQNYLPMKNGPFGQNFRDPMYPWNTTPEDVMNHRKLGYVYDIELRKSKRSS DY805(서열번호 6) MSNKYRVRKNVLHLTVTEKRDFIRAVLILKEKGIYDRYIAWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFERDLQSINPEVTIPYWEWETDAQMQDPSQSKIWSTDFMGGNGNPKKDFIVDTGPFAAGRWTTIDEQGNPAGGLKRNFGATKEAPTLPTRDDVLNALKITQYDTPPWDMTSQNSFRNQLEGFINGPQLHNRVHRWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQIVHRNQNYQPMKNGPFGQNFRDFMYPWDTTPKDVMDHRKLGYVYDIELRKSKRSS

[0038] The gene encoding tyrosinase composed of the amino acid sequences of SEQ ID NOs 1 to 6 may each be a gene having the nucleotide sequences of SEQ ID NOs 7 to 12. SEQ ID NOs 7 to 12 are as shown in Table 2.

[0039] 서열번호 염기 서열 서열번호 7(서열번호 1의 아미노산 서열을 코딩하는 유전자) ATGAGTAACAAGTATAGAGTTAGAAAAAACGTATTACATCTTACAGATACCGAAAAAAGAGATTTTATTCGTGCCGTGCTAATACTAAAGGAAAAAGGAATATATGACCGCTATGTAGCCTGGCATGGTGCAGCAGGTAAATTTCATACTCCTCCGGGCAGCGATCGAAATGCAGCACATATGAGTTCTGCTTTTTTGCCGTGGCATCGTGAATACCTTTTGCGATTCGAACGTGACCTCCAGTCGATCAATCCAGAAGTAACCCTTCCTTATTGGGAATGGGAAACGGATGCACAGCTGCAGGATCCATCACAATCACCAATTTGGAACGCAGATTTTATGGGCGGAAATGGAAATCCCAAAAAGGATTTTATCGTCGATACCGGGCCATTTGCAGCTGGGCGCTGGACGACGATCGATGAACAAGGAAATCCTTCCGGAGGGTTAAAACGTAATTTCGGAGCAACGAAAGAGGCGCCTACACTCCCTACTCGAGATGATGTTCTCAAAGCTTTAAAAATAACTCAATATGATACGCCGCCTTGGGATATGACCAGCCAAAACAGCTTTCGTAATCAGCTTGAAGGATTTATTAACGGACCGCAGCTTCACAATCGTGTACACCGTTGGGTTGGCGGACAGATGGGCGTTGTCCCTACTGCTCCGAATGATCCTGTCTTCTTTTTACACCACGCAAATGTGGATCGTATTTGGGCTGTATGGCAAATTGTTCACCGCAATCAAAACTATCTGCCGATGAAAAACGGGCCGTTTGGTCAAAACTTTAGAGATCCGATGTATCCTTGGAATACAACCCCTGAAGACGTTATGAACCATCGAAAGCTTGGGTACGTATACGATATAGAATTAAGAAAATCAAAACGTTCCTCATAA 서열번호 8(서열번호 2의 아미노산 서열을 코딩하는 유전자) ATGAGTAACAAGTATAGAGTTAGAAAAAACGTATTACATCTTACAGACGCGGAAAAAAGAGATTTTATTCGGGCCGTGCTAATACTAAAGGAAAAAGGAATATATGACCGCTATATAGCCTGGCATGGTGCAGCAGGTAAATTTCATACTCCTCCGGGCAGCGATCGAAATGCAGCACATATGAGTTCTGCTTTTTTGCCGTGGCATCGTGAATACCTTTTGCGATTCGAACGTGACCTCCAGTCGATCAATCCAGAAGTAACCCTTCCTTATTGGGAATGGGAAACGGATGCACAGCTGCAGGATCCATCACAATCACCAATTTGGAGCGCAGATTTTATGGGCGGAAACGGAAATCCCAAAAAGGATTTTATCGTCGATACCGGGCCATTTGCAGCTGGGCGCTGGACGACGATCGATGAACAAGGAAATCCTTCCGGAGGGTTAAAACGTAATTTTGGAGCAACGAAAGAGGCGCCTACACTCCCTACTCGAGATGATGTTCTCAAAGCTTTAAAAATAACTCAATATGATACGCCGCCTTGGGATATGACCAGCCAAAACAGCTTTCGTAATCAGCTTGAAGGATTTATTAACGGGCCGCAGCTTCACAATCGCGTACATCGTTGGGTTGGCGGGCAGATGGGCGTTGTCCCTACTGCTCCGAATGATCCTGTCTTCTTTTTACACCACGCAAATGTGGATCGTATTTGGGCTGTATGGCAAATTGTTCATCGCGATCAAAACTATCGGCCGATGAAAAACGGGCCGTTCGGTCAAAACTTTAGAGATCCGATGTATCCTTGGAATACAACCCCTGAAGACGTTATGAACCATCGAAAGCTTGGGTACGTATACGATATAGAATTAAGAAAATCAAAACGTTCCTCATAA 서열번호 9(서열번호 3의 아미노산 서열을 코딩하는 유전자) ATGAGTAACAAGTATAGAGTTAGAAAAAACGTATTACATCTTACCGACACGGAAAAAAGAGATTTTGTTCGTACCGTGCTAATACTAAAGGAAAAAGGAATATACGACCGCTATATAGCCTGGCATGGCGCAGCAGGTAAATTTCATACGCCTCCGGGCAGCGATCGAAATGCAGCACATATGAGTTCTGCTTTTTTGCCATGGCATCGTGAATACCTTTTACGATTCGAACGTGACCTTCAGTCAATCAATCCAGAAGTAACCCTTCCTTATTGGGAATGGGAAACGGACGCACAGATGCAGGATCCCTCACAATCACAAATTTGGAGTGCAGATTTTATGGGAGGAAACGGAAATCCTAAAAAAGATTTTATCGTCGACACCGGTCCGTTTGCAGCTGGGCGCTGGACGACAATCGATGAACAAGGAAATCCTTCTGGAGGGCTAAAACGTAATTTTGGAGCAACGAAAGAGGCACCTACACTCCCTACTCGAGATGATGTTCTCAATGCTTTAAAAATAACTCAGTATGATACGCCGCCTTGGGATATGACCAGCCAAAACAGCTTTCGTAATCAGCTTGAAGGATTTATTAACGGGCCGCAGCTTCACAATCGCGTACACCGTTGGGTTGGCGGACAGATGGGCGTTGTGCCTACTGCTCCGAATGATCCTGTCTTCTTTTTACACCACGCAAATGTGGATCGTATTTGGGCTGTATGGCAAATTGTTCATCGTAATCAAAACTATCAGCCGATGAAAAATGGGCCATTTGGTCAAAACTTTAGAGATCCGATGTACCCTTGGAACACAACCCCTGAAGACGTTATGAACCATCGAAAGCTTGGGTACGTATACGATATAGAATTAAGAAAATCAAAACGTTCCTCATAA 서열번호 10(서열번호 4의 아미노산 서열을 코딩하는 유전자) ATGAGTAACAAGTATAGAGTTAGAAAAAACGTATTGAATCTTACCGACATGGAAAAAAGAGATTTTGTTCGTACCGTGCTAATACTAAAGGAAAAAGGAATATATGACCGCTATATAGCGTGGCATGGTGCAGCAGGTAAATTTCATACGCCTCCGGGCAGCGATCGAAATGCAGCACATATGAGTTCTGCTTTTTTGCCATGGCATCGTGAATACCTTTTACGATTTGAGCGTGACCTTCAGTCAATCAATCCAGAAGTAACCCTTCCTTATTGGGAATGGGAAACGGACGCACAGATGCAGGATCCCTCACAATCACAAATTTGGAGTGCAGATTTTATGGGAGGAAACGGAAATCCTAAAAAAGATTTTATCGTCGACACCGGTCCGTTTGCAGCTGGGCGCTGGACGACGATTGATGAACAAGGAAATCCTTCCGGAGGGCTAAAACGTAATTTTGGCGCAACGAAAGAGGCACCTACACTCCCTACTCGAGATGATGTTCTCAATGCTTTAAAAATAACTCAGTATGATACGCCGCCTTGGGATATGACCAGCCAAAACAGCTTCCGTAATCAGCTTGAAGGATTTATTAACGGGCCGCAGCTTCACAATCGCGTACACCGTTGGGTTGGCGGACAGATGGGCGTTGTCCCTACTGCTCCGAATGATCCTGTCTTCTTTTTACACCACGCAAATGTGGATCGTATTTGGGCTGTATGGCAAATTGTTCATCGCAATCAAAACTATCAGCCAATGAAAAACGGGCCATTTGGTCAAAACTTTAGAGATCCGATGTATCCTTGGAATACAACTCCTGAAGACGTGATGAACCATCGAAAGCTTGGGTACGTATACGATATAGAATTAAGAAAATCAAAACGTTCCTCATAA 서열번호 11(서열번호 5의 아미노산 서열을 코딩하는 유전자) ATGAGTAACAAGTATAGAGTTAGAAAAAACGTATTACATCTTACAGATACCGAAAAAAGAGATTTTATTCGTGCCGTGCTAATACTAAAGGAAAAAGGAATATATGACCGCTATGTAGCCTGGCATGGTGCAGCAGGTAAATTTCATACTCCTCCGGGCAGCGATCGAAATGCAGCACATATGAGTTCTGCTTTTTTGCCGTGGCATCGTGAATACCTTTTGCGATTCGAACGTGACCTCCAGTCGATCAATCCAGAAGTAACCCTTCCTTATTGGGAATGGGAAACGGATGCACAGCTGCAGGATCCATCACAATCACCAATTTGGAACGCAGATTTTATGGGCGGAAATGGAAATCGCAAAAAGGATTTTATCGTCGATACCGGGCCATTTGCAGCTGGGCGCTGGACGACGATCGATGAACAAGGAAATCCTTCGGGAGGGTTAAAACGTAATTTTGGAGCAACGAAAGAGGCGCCTACACTCCCTACTCGAGATGATGTTCTCAAAGCTTTAAAAATAACTCAATATGATACGCCGCCTTGGGATATGACCAGCCAAAACAGCTTTCGTAATCAGCTTGAAGGATTTATTAACGGGCCGCAGCTTCACAATCGCGTACACCGTTGGGTTGGCGGACAGATGGGCGTTGTCCCTACTGCCCCGAATGATCCTGTCTTCTTTTTACACCACGCAAATGTGGATCGTATTTGGGCTGTATGGCAAATTGTTCATCGCAATCAAAACTATCTGCCGATGAAAAATGGGCCGTTTGGTCAAAACTTTAGAGATCCGATGTATCCTTGGAATACAACCCCTGAAGACGTTATGAACCATCGAAAGCTTGGGTACGTATACGATATAGAATTAAGAAAATCAAAACGTTCCTCATAA 서열번호 12(서열번호 6의 아미노산 서열을 코딩하는 유전자) ATGAGTAACAAGTATAGAGTTAGAAAAAACGTATTACATCTTACCGTCACGGAAAAAAGAGATTTTATTCGTGCTGTGCTCATACTAAAGGAAAAAAGGAATATATGACCGCTATATAGCATGGCATGGCGCAGCAGGTAAATTTCATACGCCTCCGGGCAGCGATCGAAATGCAGCACATATGAGTTCTGCTTTTTTGCCATGGCATCGTGAATACCTTTTAC GATTTGAGCGTGACCTTCAGTCAATCAATCCAGAAGTAACGATTCCTTATTGGGAATGGGAAACGGATGCACAAATGCAAGATCCATCACAATCAAAAATTTGGAGCACAGATTTTATGGGCGGAAACGGAAATCCCAAAAAAGATTTTATCGTTGATACCGGTCCGTTTGCAGCTGGGCGCTGGACGACGATCGATGAGCAAGGAAATCCTGCTGGAGGGTTA AAACGTAATTTTGGCGCAACGAAAGAAGCACCTACACTCCCTACTCGAGATGATGTTCTTAATGCTTTAAAAATAACTCAATATGATACGCCGCCTTGGGATATGACGAGCCAAAACAGCTTTCGTAATCAGCTTGAAGGATTTATTAACGGGCCGCAGCTTCACAATCGTGTACATCGCTGGGGTCGGCGGACAGATGGGCGTTGTTCCTACTGCTCCAAATG ATCCGGTTTTCTTTTTACACCACGCAAATGTAGATCGTATTTGGGCTGTATGGCAAATTGTTCATCGCAATCAAAACTATCAGCCGATGAAAAACGGGCCTTTCGGTCAAAACTTTAGAGACTTCATGTATCCTTGGGATACAACTCCAAAAGATGTTATGGACCATCGAAAGCTTGGGTACGTATACGATATAGAATTAAGAAAATCAAAACGTTCCTCATAA

[0040] A microorganism expressing tyrosinase may express a tyrosinase consisting of any one of the amino acid sequences of SEQ ID NOs 1 to 6, either intrinsically or extrinsically. In the case of extrinsically expressing the tyrosinase, the microorganism may be transformed into a gene encoding a tyrosinase consisting of any one of the amino acid sequences of SEQ ID NOs 1 to 6.

[0041] Microorganisms may be used without limitation as long as they are suitable for the production of fisetanol. The microorganisms may be bacteria, fungi, or a combination thereof, and the bacteria may be Gram-positive bacteria, Gram-negative bacteria, or a combination thereof. The microorganisms may be Bacillus megaterium ( Bacillus megaterium It can be.

[0042] A culture of microorganisms may include a medium containing microorganisms after culture, a medium from which microorganisms have been isolated after culture, or substances secreted by microorganisms during culture. The medium may be a solid medium or a liquid medium.

[0043] The pulverized microorganism may be a pulverized microorganism obtained through sonication, etc., and may contain a tyrosinase composed of the amino acid sequences of SEQ ID NOs 1 to 6 within the microorganism.

[0044] The method for producing ficetanol according to the present invention includes the step of reacting resveratrol with the composition for producing ficetanol.

[0045] Resveratrol may be represented by the following chemical formula 2.

[0046]

[0047] The present invention relates to a method for producing ficetanol comprising the step of reacting a tyrosinase having an amino acid sequence of any one of SEQ ID NOs 1 to 6 with a substrate, resveratrol.

[0048] The method for producing ficetanol according to the present invention may further include a step of dissolving resveratrol in ethanol or an aqueous solution thereof. The step of dissolving resveratrol in ethanol or an aqueous solution thereof may be performed before, after, or during the step of reacting resveratrol with a tyrosinase having an amino acid sequence of any one of SEQ ID NOs 1 to 6. The tyrosinase may be added to a reactor after dissolving resveratrol in ethanol or an aqueous solution thereof, or the ethanol or aqueous solution thereof may be added after adding the tyrosinase in the presence of the substrate resveratrol. The addition of ethanol or an aqueous solution thereof may be performed during the intermediate process of the reaction between resveratrol and tyrosinase. That is, the step of dissolving resveratrol or the product ficetanol in ethanol or an aqueous solution thereof may be performed at any step of the method for producing ficetanol according to the present invention and is not limited to a specific step.

[0049] In the method for producing picetanol according to the present invention, aqueous ethanol solutions of, for example, 0%, 2%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% may be used, but the concentration of the aqueous ethanol solution is not limited thereto. A tyrosinase composed of any one of the amino acid sequences of SEQ ID NOs 1 to 6 of the present invention has ethanol resistance, so it is possible to react even in a high-concentration aqueous ethanol solution. Ethanol resistance means that the enzyme maintains reactivity even in a high-concentration ethanol environment.

[0050] In one example, when the reaction was carried out by adding a 40% aqueous ethanol solution, a large amount of picetanol reaching 11.8 mM was produced.

[0051] The method for producing fisetanol according to the present invention may further include a step of supplying oxygen to the reaction. Tyrosinase is an enzyme that requires oxygen for the reaction, and for the mass production of fisetanol, oxygen must be supplied to the reaction process. The oxygen may be supplied in the form of dissolved oxygen. The oxygen may be supplied by shaking the reactor in a circular motion, or by generating horizontal circular shaking from low to high speed.

[0052] The above oxygen supply may be supplied at a stirring speed of 50 rpm to 400 rpm, for example, at a stirring speed of 50 rpm to 400 rpm, 60 rpm to 350 rpm, 70 rpm to 300 rpm, 80 rpm to 300 rpm, 90 rpm to 250 rpm, 100 rpm to 250 rpm, 150 rpm to 250 rpm, or 170 rpm to 230 rpm.

[0053] However, the manufacturing method of the present invention is not limited to whether or not oxygen is supplied and the method thereof.

[0054] The method for producing picetanol according to the present invention may be carried out at a reaction temperature of 30 to 50 ℃. Although picetanol can be produced at a temperature range other than 30 to 50 ℃, a higher concentration of picetanol can be obtained in the above range.

[0055] The method for producing picetanol according to the present invention may be carried out at a reaction pH of pH 3 to 9, for example, at pH 4 to 9, pH 4 to 8, pH 3 to 8, pH 4 to 7, pH 5 to 9, or pH 6 to 9. Although picetanol can be produced in pH ranges other than pH 3 to 9, a higher concentration of picetanol can be obtained in the above ranges.

[0056] The method for producing picetanol according to the present invention may further include a step of reducing a quinone-type structural substance ((E)-4-(3,5-dihydroxystyryl)cyclohexa-3,5-diene-1,2-dione) produced by the peroxidation reaction of resveratrol to picetanol.

[0057] Tyrosinase carries out a primary hydroxylation reaction (monophenolase activity) in which monophenol-type structural materials (e.g., L-tyrosine) are hydroxylated into catechol-type structural materials, and a secondary oxidation reaction (diphenolase activity) in which catechol-type structural materials bioconverted from monophenol-type structural materials are oxidized into quinone-type materials (quinonic compounds), which are melanin pigment precursors. Therefore, secondary oxidation reactions can occur from resveratrol to produce quinone-type structural materials of resveratrol, and a step of reducing these can be added to produce fisetanol at a high concentration.

[0058] The method for producing fisetanol according to the present invention may be carried out in the presence of any one reducing agent selected from the group consisting of NADH, NADPH, ascorbic acid, cysteine, p-coumaric acid, hydroxylamine, catechol, pyrogallol, and caffeic acid. By reacting in the presence of a reducing agent, the quinone-type structural material produced by the peroxidation reaction of tyrosinase can be reduced to maximize the production yield of fisetanol. In one embodiment of the present invention, L-ascorbic acid (LAA) was used, but it is not limited thereto.

[0059] The method for producing fisetanol according to the present invention may further include a step of separating or purifying fisetanol from a reaction solution. The method of separation or purification may use any method commonly used in the art without limitation. For example, for separation, centrifugation or filtration may be used, and for purification, salting out (e.g., ammonium sulfate precipitation, sodium phosphate precipitation), solvent precipitation (protein fractionation and precipitation using acetone, ethanol, etc.), dialysis, gel filtration, ion exchange, column chromatography, ultrafiltration, or a combination of two or more of these may be used.

[0061] Hereinafter, the present invention will be described in detail with reference to examples to specifically explain the invention.

[0063] Example 1. Confirmation of Pisetanol production according to ethanol concentration

[0064] Since resveratrol is insoluble in water, ethanol, to which tyrosinase is resistant, was used as a cosolvent to determine the production of ficetanol. A composition of 20 μg / mL tyrosinase (DY801), 10 mM resveratrol, 0.1 M L-ascorbic acid, and 50 mM KPi (pH 6) was reacted at 40°C for 1 hour at varying ethanol concentrations. As a result, the production of ficetanol was maximum at an ethanol concentration of 20% (v / v), confirming that this represents more than three times the production compared to that at an ethanol concentration of 2% (Fig. 1, left graph). In the above composition, when the amount of resveratrol was increased to 20 mM and reacted for 2 hours, it was confirmed that a maximum amount of ficetanol reaching 11.8 mM could be produced at an ethanol concentration of 40% (Fig. 1, right graph).

[0066] Example 2. Confirmation of Pisetanol production volume according to temperature

[0067] As confirmed in the above example, we aimed to optimize the reaction temperature under conditions of an ethanol concentration of 40% to maximize the production of ficetanol. When reacted for 30 minutes under a composition of 20 μg / mL tyrosinase (DY801), 5 mM resveratrol, 10 mM L-ascorbic acid, 50 mM KPi (pH 6), and a 40% ethanol aqueous solution, the maximum ficetanol production was observed at 50°C (Fig. 2). Furthermore, when the amount of the substrate resveratrol was increased to 10 mM, 20 mM, or 30 mM while keeping the other compositions the same, and the reaction was carried out for 1 hour, it was confirmed that as the substrate concentration increased, the optimal temperature for maximum production was around 40°C (Fig. 3).

[0069] Example 3. Confirmation of Pisetanol production volume according to reaction pH

[0070] The reaction was carried out for 1 hour with varying pH under a composition of 20 μg / mL tyrosinase (DY801), 10 mM resveratrol, 10 mM L-ascorbic acid, 50 mM KPi, and a 40% ethanol aqueous solution, and the production of fisetanol was confirmed. As a result, it was confirmed that activity was maintained at all pH levels from 3 to 9, and that a large amount of fisetanol was produced, particularly at pH 6 to 9 (Fig. 4).

[0072] Example 4. Optimization of Pisetanol Production According to Reaction Time and Substrate Concentration

[0073] The production of ficetanol was determined according to reaction time and aeration under a composition of 20 μg / mL tyrosinase (DY801), 30 mM resveratrol, 0.1 M L-ascorbic acid, 50 mM KPi (pH 6), a reaction temperature of 40 ℃, and a 40% ethanol aqueous solution. It was confirmed that maximum production of ficetanol was possible under aeration of 200 rpm for 2 hours (Fig. 5).

[0074] As a result of analyzing the production of ficetanol according to the substrate concentration under the above optimal production conditions (reaction conditions; 20 μg / mL tyrosinase (DY801), 10 to 100 mM resveratrol, 0.1 M L-ascorbic acid, 50 mM KPi (pH 6), reaction temperature 40 ℃, 40% ethanol aqueous solution, reaction 2 hours), a maximum of 27 mM ficetanol was produced by reacting with 50 mM substrate for 2 hours under aeration at 200 rpm, which corresponds to a production rate of 3.3 g / L / h (Fig. 6).

[0076] Example 5. HPLC Analysis

[0077] HPLC (High Performance Liquid Chromatography) chromatograms were analyzed to compare the reactions of various tyrosinase variants. The reaction for analysis was carried out under the conditions of 20 μg / mL tyrosinase, 50 mM resveratrol, 0.1 M L-ascorbic acid, 50 mM KPi (pH 6), reaction temperature 40 ℃, 40% ethanol aqueous solution, and 200 rpm aeration.

[0078] As a pretreatment step for HPLC analysis, ethyl acetate was first added to the reaction mixture and vigorously vortexed, and the ethyl acetate in the upper layer was extracted by centrifugation at 3000 rpm, 10 minutes, and 4 ℃. After removing the organic solvent using a nitrogen evaporator, the residue was dissolved in a solution of HPLC mobile phase buffer A:B = 6:4.

[0079] After pretreatment, a Gemini 5 μm C18 110 Å column was used. Mobile phase A was a solution prepared by adding 0.5% acetic acid to 3 times the volume of distilled water (3DW) and mixing it with acetonitrile in a volume ratio of 95:5 (v / v), and mobile phase B was a solution prepared by adding 0.5% acetic acid to 3 times the volume of distilled water (3DW) and mixing it with acetonitrile in a volume ratio of 5:95 (v / v). The mobile phases were flowed at a rate of 1 mL / min, and UV absorbance was measured under conditions of B concentration of 15% and 320 nm. The HPLC analysis results are shown in Figure 7.

[0081] Example 6. Comparison of Pisetanol Productivity of Tyrosinase Variants

[0082] The productivity of fisetanol from various tyrosinase variants derived from Bacillus megaterium other than DY801 (GJ803, GJ804, DY802, DY804, DY805) was confirmed. The reaction was carried out for 2 hours under 20 μg / mL tyrosinase, 50 mM resveratrol, 0.1 M L-ascorbic acid, 50 mM KPi (pH 6), a reaction temperature of 40 ℃, a 40% ethanol aqueous solution, and 200 rpm aeration. As a result, unlike mushroom-derived tyrosinase (MT), fisetanol was produced in the remaining 5 variants derived from Bacillus megaterium, and it was confirmed that all variants produced more than 20 mM fisetanol, corresponding to a level of at least 5.0 g / L productivity (Fig. 8).

[0084] Example 7. Material identification using GC-MS analysis

[0085] For sample preparation for GC-MS (Gas Chromatography-Mass Spectrometry) analysis, the reaction was carried out for 30 minutes under a composition of 20 μg / mL tyrosinase (DY801), 5 mM resveratrol, 10 mM L-ascorbic acid, 50 mM KPi (pH 6), a reaction temperature of 40 °C, and a 40% ethanol aqueous solution. The reaction was stopped by adding 600 μL of EA (ethyl acetate), and after separating the water and organic solvent layers by centrifugation, 100 μL of the upper organic solvent layer containing the sample was extracted. The extracted organic solvent was removed using nitrogen gas, and the sample was derivatized using 70 μL of BSTFA-TMS (99:1) at 75 °C for 20 minutes.

[0086] For GC-MS analysis, a DB-5MS UI column was used, and the mobile phase flow rate was set to 1 ml / min, the injector temperature to 250°C, the ionization source temperature to 250°C, and the interface temperature to 300°C. The initial temperature of the GC oven was maintained at 150°C for 1 minute, and the temperature was increased at a rate of 10°C per minute to a final temperature of 300°C, which was maintained for 5 minutes. The MS scan mode was set from 2.5 minutes to 20 minutes, and the mass-to-charge ratio (m / z) was set between 50 and 650 for scanning.

[0087] As a result, peaks were observed at 15.25 min (resveratrol) and 16.32 min (picetanol), and the mass spectra of derivatized resveratrol and picetanol were observed at 444 and 532, respectively (Figs. 9 to 11). The production of picetanol was confirmed by GC-MS analysis of the reaction mixture.

Claims

Claim 1 A composition for producing picetanol comprising: a tyrosinase having an amino acid sequence of any one of SEQ ID NOs 1 to 6, a microorganism expressing the same, or a culture or pulverized product of said microorganism; and ethanol or an aqueous solution thereof. Claim 2 In claim 1, the tyrosinase is Bacillus megaterium ( Bacillus megaterium Composition for manufacturing pisetanol derived from ). Claim 3 A method for producing picetanol comprising the step of reacting resveratrol with the composition of claim 1. Claim 4 A method for producing ficetanol according to claim 3, wherein the step of reacting the resveratrol with the composition of claim 1 is performed by dissolving the resveratrol in ethanol or an aqueous solution thereof, and reacting the dissolved resveratrol with a tyrosinase having an amino acid sequence of any one of SEQ ID NOs 1 to 6, a microorganism expressing the same, or a culture or pulverized product of said microorganism. Claim 5 A method for producing picetanol according to claim 3, further comprising the step of supplying oxygen. Claim 6 A method for producing picetanol according to claim 3, wherein the reaction is carried out at 30 to 50 ℃. Claim 7 A method for producing picetanol according to claim 3, wherein the reaction is carried out at a pH of 3 to 9. Claim 8 A method for producing picetanol according to claim 3, further comprising the step of reducing (E)-4-(3,5-dihydroxystyryl)cyclohexa-3,5-diene-1,2-dione generated by the peroxidation reaction of resveratrol to picetanol. Claim 9 A method for producing picetanol according to claim 3, wherein the reaction is carried out in the presence of any one reducing agent selected from the group consisting of NADH, NADPH, ascorbic acid, cysteine, p-coumaric acid, hydroxylamine, catechol, pyrogallol, and caffeic acid. Claim 10 A method for producing picetanol according to claim 3, further comprising the step of separating or purifying picetanol from a reaction solution.

Images