An enzyme and enzyme catalytic system and their application in the synthesis of tetrahydromagnolol

Abstract

The present invention provides a hydrogenase and an enzyme catalytic system and their application in the catalytic synthesis of tetrahydromagnolol. Based on the advantages of strong specificity, high catalytic efficiency and no pollution shown by the biological method, after using the hydrogenase of SEQ ID NO.1 in the reaction of catalyzing the synthesis of 5,5'-dipropyl-[1,1'-biphenyl]-2,2'-diol with 5',5-di-2-propenyl-1,1'-biphenyl-2,2'-diphenol as the substrate, the characteristics of high conversion rate and good purity are achieved.

Description

Technical Field

[0001] This application relates to the field of synthesis of raw materials for cosmetics, and specifically to a method for synthesizing the cosmetic active ingredient tetrahydromagnolol. Background Art

[0002] Tetrahydromagnolol was developed based on the natural components in the bark of Magnolia denudata (Magnoliaceae Magnolia officinalis), and this ingredient with significant whitening effect was created through natural and artificial synthesis. It can hinder the maturation of tyrosinase before its activation, inhibiting the formation of melanin at an early stage; it helps to inhibit the maturation of tyrosinase, the main cause of melanin formation, and enhances the skin's repair function against ultraviolet damage, thus fading dark spots and sunburn marks. Therefore, tetrahydromagnolol is widely used in personal care products such as cosmetics, and its purpose in cosmetics is as a whitening agent and skin protectant.

[0003] Currently, tetrahydromagnolol is generally prepared by the diazotization reaction of sulfur and cyclohexane; the specific preparation methods also include cyclization reactions of diethyl thiobarbiturate, etc. These chemical methods have problems such as high cost and serious environmental pollution. Summary of the Invention

[0004] The present invention aims to overcome the above defects. Based on the advantages of strong specificity, high catalytic efficiency, and no pollution shown by biological methods, a hydrogenase that can be used for catalytic hydrogenation was developed, and this hydrogenase was used to catalyze the synthesis of 5,5'-dipropyl-[1,1'-biphenyl]-2,2'-diol using 5',5-di-2-propenyl-1,1'-biphenyl-2,2'-diol as a substrate.

[0005] The substrate is 5',5-di-2-propenyl-1,1'-biphenyl-2,2'-diol, and its structure is shown in formula (1):

[0006]

[0007] The product is 5,5'-dipropyl-[1,1'-biphenyl]-2,2'-diol, and its structure is shown in formula (2):

[0008]

[0009] The present invention provides the application of an enzyme as a hydrogenase as a catalyst in a catalytic hydrogenation reaction, which has the following characteristics: the amino acid sequence of the above hydrogenase is as shown in SEQ ID NO.1, or has at least 80%-99% similarity with the sequence shown in SEQ ID NO:1.

[0010] Furthermore, the present invention provides an enzyme catalytic system for catalytic hydrogenation, which also has the following characteristics: it includes the above-mentioned hydrogenase and coenzyme; wherein, the above-mentioned coenzyme is selected from at least one of nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP).

[0011] Furthermore, the enzyme catalytic system provided by the present invention also has the following characteristics: the addition amount of the above-mentioned hydrogenase is 1 / 2 - 1 / 4 of the total reaction volume.

[0012] Furthermore, the enzyme catalytic system provided by the present invention also has the following characteristics: the addition amount of the above-mentioned coenzyme is 0.2 - 2 g / L.

[0013] Furthermore, the enzyme catalytic system provided by the present invention also has the following characteristics: it further includes a solvent; wherein, the above-mentioned solvent is selected from at least one of dimethyl sulfoxide, tetrahydrofuran, acetonitrile, and water.

[0014] Furthermore, the enzyme catalytic system provided by the present invention also has the following characteristics: it further includes a solvent; wherein, the volume concentration of the above-mentioned solvent is 10% - 50%.

[0015] In addition, the present invention also provides a method for synthesizing the cosmetic active ingredient tetrahydromagnolol, which has the following characteristics: using the above-mentioned hydrogenase as a catalyst, or adopting the above-mentioned enzyme catalytic system, to catalyze the reduction of the substrate 5',5 - bis - 2 - propenyl - 1,1'-biphenyl - 2,2'-diphenol to 5,5'-dipropyl - [1,1'-biphenyl] - 2,2'-diol.

[0016] Furthermore, the above-mentioned synthesis method provided by the present invention also has the following characteristics: the concentration of the substrate is 50 g / L - 150 g / L.

[0017] Furthermore, the above-mentioned synthesis method provided by the present invention also has the following characteristics: the pH of the enzyme-catalyzed reduction reaction is 6.0 - 9.0. Preferably 7.8 - 8.0. The method of regulation is to achieve the regulation of pH through an acid (such as acidic solvents like hydrochloric acid, acetic acid, etc.) or a base (such as basic solvents like sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, ammonia water, etc.).

[0018] Furthermore, the above-mentioned synthesis method provided by the present invention also has the following characteristics: the temperature of the enzyme-catalyzed reduction reaction is 25°C - 65°C. Preferably 30°C - 45°C.

[0019] Furthermore, the above-mentioned synthesis method provided by the present invention also has the following characteristics: the enzyme-catalyzed reduction reaction is carried out under oscillating conditions. The rotation speed can be 150 rpm - 300 rpm, preferably 200 rpm - 250 rpm. Description of the Drawings

[0020] Figure 1 1H NMR spectrum of tetrahydromagnolol of Example 1 Detailed implementation manners

[0021] Example 1. Source of hydrogenase, recombinant expression of hydrogenase-encoding gene, and acquisition of crude enzyme solution. Instructions for culture media:

[0022] LB liquid medium: 5 g / L of yeast extract, 10 g / L of peptone, 10 g / L of NaCl, sterilized in an autoclave at 121 °C for 20 min, and kanamycin with a final concentration of 50 μg / mL was added before use.

[0023] TB liquid medium: 11.8 g / L of peptone, 23.6 g / L of yeast extract, 9.4 g / L of K2HPO4, 2.2 g / L of KH2PO4, 4 mL / L of glycerol, sterilized in an autoclave at 121 °C for 20 min, and kanamycin with a final concentration of 50 μg / mL was added before use.

[0024] KPi buffer (pH 8.0): 16.284 g / L of K2HPO4 and 0.888 g / L of KH2PO4 were dissolved in pure water.

[0025] Referring to the amino acid sequence of hydrogenase from Galactomyces reessii on NCBI, a synthesis company was commissioned to perform codon optimization and gene sequence synthesis for Escherichia coli, cloned into the pet28a plasmid vector, and transformed into the expression host bacterium Escherichia coli BL21(DE3) for recombinant expression. LB liquid medium and TB liquid medium were used as the growth and induction media respectively. IPTG with a final concentration of 0.5 mM was added as an inducer during induction, and induction was carried out at 20 °C for more than 12 h.

[0026] This example provides a crude enzyme solution of hydrogenase, and the specific method for obtaining it includes the following steps:

[0027] (1) Inoculate the genetically engineered bacterium expressing hydrogenase into LB liquid medium containing kanamycin with a final concentration of 50 μg / mL, culture at 37 °C and 200 r / min until the OD600 is about 0.6 - 0.8 to obtain the seed bacterial solution; inoculate the seed bacterial solution into TB liquid medium containing kanamycin with a final concentration of 50 μg / mL at an inoculation amount of 1%, culture at 37 °C and 200 r / min for 4 - 6 h, when the OD600 is between about 0.8 - 1, add IPTG with a final concentration of 0.5 mM for induction, induce expression at 20 °C and 200 r / min for 12 - 16 h. After the induction is completed, centrifuge at 8000 r / min and 4 °C to collect the bacterial cells, and resuspend the bacterial cells with KPi buffer (pH 8.0).

[0028] (2) The bacterial solution was disrupted by ultrasonic disruption method. The wet bacterial cells were resuspended with KPi buffer (pH 8.0) to prepare a bacterial solution with a bacterial concentration of 10%. The disruption conditions were as follows: working for 1 s, intermittent for 2 s, 180 W, and disrupting for 8 min.

[0029] (3) Various impurities in the cell disruption solution were removed by centrifugation to obtain a crude enzyme solution. The centrifugation parameters were 8000 r / min, 4 °C, and centrifugation for 5 - 10 min.

[0030] Among them, the amino acid sequence of the hydrogenase is SEQ ID NO.1: Met Ser Val Gln Asn Thr Thr His Phe Thr Gly Leu Gly Pro Leu Pro Gln Pro Ala Pro Lys Pro Ala Ser Asn Val Leu Asp Leu Phe Ser Leu Lys Gly Lys Val Ala Ser Val Thr Gly Ser Ser Thr Gly Ile Gly Tyr Ala Val Ala Glu Ala Phe Ala Gln Ala Gly Ala Asp Val Ala Leu Trp Tyr Asn Ser His Asn Ala Glu Ala Lys Ala Lys Ala Leu Ser Glu Lys Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr Asp Ser Ala Ala Val Glu Ala Ala Ile Lys Glu Gln Ile Glu Tyr Phe Gly Lys Ile Asp Ile Phe Val Ala Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu Ile Asp Thr Glu Asp Asp Lys Glu Trp Lys Lys Val Ile Asp Leu Asp Phe Thr Gly Val Tyr Tyr Cys Ala Lys Tyr Ile Gly Arg His Phe Lys Glu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Ser Met Ser Gly His Ile Val Asn Phe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala Gly Val Arg His Phe Cys Thr Ser Leu Ala Val Glu Trp Ala Gly Phe Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Glu Ile Ser Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro Leu Gly Arg Glu Gly Glu AlaGln Glu Leu Val Gly Ala Tyr LeuTyr Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp Ile Arg Val Asp GlyGly Tyr Thr Ala Pro

[0031] Example 2. Catalytic synthesis of tetrahydromagnolol based on hydrogenase

[0032] Construct an enzyme-catalyzed system: the crude hydrogenase solution, coenzyme, and solvent of Example 1. The coenzyme is selected from at least one of nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate. The solvent is selected from at least one of dimethyl sulfoxide, tetrahydrofuran, acetonitrile, and water.

[0033] In the reaction of using the above enzyme-catalyzed system to catalyze the reduction of the substrate 5',5-di-2-propenyl-1,1'-biphenyl-2,2'-diphenol to 5,5'-dipropyl-[1,1'-biphenyl]-2,2'-diol, the pH of the reduction reaction is 6 - 9, preferably pH 7 - 8; the reaction temperature is 25 - 65 °C, preferably 30 - 60 °C. The reduction reaction is carried out under oscillating conditions, and the rotation speed is 150 rpm - 300 rpm, preferably 200 rpm - 250 rpm.

[0034] The concentration of the substrate is 50 g / L - 150 g / L, preferably 100 g / L - 150 g / L. The addition amount of hydrogenase is 1 / 2 - 1 / 4 of the total volume of the reaction; the addition amount of coenzyme is 0.2 - 2 g / L, preferably 0.5 - 1.5 g / L. Based on the total volume of the reaction system, the concentration of the organic solvent is 20% (v / v) - 50% (v / v), preferably 30% (v / v) - 40% (v / v).

[0035] Example 2.1. Optimal reaction example

[0036] The specific experimental method includes the following steps: (1) Add 50 mL of acetonitrile to a 500 mL reaction flask, add 10 g of the substrate, and stir to dissolve at 45 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 7.5 with 1M HCl, add 50 mL of the crude hydrogenase solution, and simultaneously add 50 mg of coenzyme NAD+; (3) Continuously react for 24 h under the oscillating conditions of a shaker at 37 °C and 250 rpm, and after sampling, detect the product conversion rate by HPLC.

[0037] The reaction system contains 50% (v / v) acetonitrile, 100 g / L substrate, and the total volume of the reaction solution: hydrogenase solution (v / v) = 2:1. The pH during the reaction process is controlled at 7.5.

[0038] Experimental results: The purity of the product of hydrogenase with SEQ ID NO: 1 after 24 h is 99%, and the conversion rate is 67%.

[0039] 1 H NMR (500 MHz, DMSO-d6) δ 8.99 (s, 2H), 7.01–6.94 (m, 4H), 6.84 (d, J = 8.1 Hz, 2H), 2.49 (t, J = 7.6 Hz, 4H), 1.58 (h, J = 7.4 Hz, 4H), 0.91 (t, J = 7.4 Hz, 6H). 13 C NMR (126 MHz, DMSO-d6) δ 152.66, 132.90, 131.75, 128.26, 126.33, 116.20, 37.05, 24.90, 14.13. LC-MS: 271.39 [M+H] +

[0040] Example 2.2. The reaction temperature is 45 °C

[0041] The specific experimental method includes the following steps: (1) Add 50 mL of acetonitrile to a 500 mL reaction flask, add 10 g of the substrate, and stir to dissolve at 45 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 7.5 with 1 M HCl, add 50 mL of crude hydrogenase solution, and simultaneously add 50 mg of coenzyme NAD+; (3) Continuously react for 24 h under the conditions of shaking in a shaker at 45 °C and 250 rpm. After sampling, the product conversion rate is detected by HPLC.

[0042] Experimental results: The purity of the product of hydrogenase with SEQ ID NO: 1 after 24 h is 99%, and the conversion rate is 42%.

[0043] Example 2.3. The reaction temperature is 25 °C

[0044] The specific experimental method includes the following steps: (1) Add 50 mL of acetonitrile to a 500 mL reaction flask, add 10 g of the substrate, and stir to dissolve at 45 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 7.5 with 1 M HCl, add 50 mL of crude hydrogenase solution, and simultaneously add 50 mg of coenzyme NAD+; (3) Continuously react for 24 h under the conditions of shaking in a shaker at 25 °C and 250 rpm. After sampling, the product conversion rate is detected by HPLC.

[0045] Experimental results: The purity of the product of hydrogenase with SEQ ID NO: 1 after 24 h is 99%, and the conversion rate is 27%.

[0046] Example 2.4. The reaction temperature is 65 °C

[0047] The specific experimental method includes the following steps: (1) Add 50 mL of acetonitrile to a 500 mL reaction flask, add 10 g of the substrate, and stir to dissolve at 25 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 7.5 with 1 M HCl, add 50 mL of crude hydrogenase solution, and simultaneously add 50 mg of coenzyme NAD+; (3) Continuously react under the conditions of shaking in a shaker at 65 °C and 250 rpm for 24 h. After sampling, detect the product conversion rate by HPLC.

[0048] Experimental results: The product purity of the hydrogenase of SEQ ID NO: 1 after 24 h is 99%, and the conversion rate is 21%.

[0049] Example 2.5. The organic solvent is dimethyl sulfoxide

[0050] The specific experimental method includes the following steps: (1) Add 50 mL of dimethyl sulfoxide (DMSO) to a 500 mL reaction flask, add 10 g of the substrate, and stir to dissolve at 37 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 7.5 with 1 M HCl, add 50 mL of crude hydrogenase solution, and simultaneously add 50 mg of coenzyme NAD+; (3) Continuously react under the conditions of shaking in a shaker at 37 °C and 250 rpm for 24 h. After sampling, detect the product conversion rate by HPLC.

[0051] Experimental results: The product purity of the hydrogenase of SEQ ID NO: 1 after 24 h is 99%, and the conversion rate is 43%.

[0052] Example 2.6. The organic solvent is tetrahydrofuran

[0053] The specific experimental method includes the following steps: (1) Add 50 mL of tetrahydrofuran to a 500 mL reaction flask, add 10 g of the substrate, and stir to dissolve at 37 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 7.5 with 1 M HCl, add 50 mL of crude hydrogenase solution, and simultaneously add 50 mg of coenzyme NAD+; (3) Continuously react under the conditions of shaking in a shaker at 37 °C and 250 rpm for 24 h. After sampling, detect the product conversion rate by HPLC.

[0054] Experimental results: The product purity of the hydrogenase of SEQ ID NO: 1 after 24 h is 99%, and the conversion rate is 60%.

[0055] Example 2.7. The organic solvent is DMF

[0056] The specific experimental method includes the following steps: (1) Add 50 mL of DMF to a 500 mL reaction flask, add 10 g of the substrate, and stir to dissolve at 37 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 7.5 with 1 M HCl, add 50 mL of crude hydrogenase solution, and simultaneously add 50 mg of coenzyme NAD+; (3) Continuously react under the condition of shaking in a shaker at 37 °C and 250 rpm for 24 h. After sampling, detect the product conversion rate by HPLC.

[0057] Experimental results: The purity of the product of the hydrogenase of SEQ ID NO: 1 after 24 h is 99%, and the conversion rate is 47%.

[0058] Example 2.8. The organic solvent is ethanol

[0059] The specific experimental method includes the following steps: (1) Add 50 mL of ethanol to a 500 mL reaction flask, add 10 g of the substrate, and stir to dissolve at 37 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 7.5 with 1 M HCl, add 50 mL of crude hydrogenase solution, and simultaneously add 50 mg of coenzyme NAD+; (3) Continuously react under the condition of shaking in a shaker at 37 °C and 250 rpm for 24 h. After sampling, detect the product conversion rate by HPLC.

[0060] Experimental results: The purity of the product of the hydrogenase of SEQ ID NO: 1 after 24 h is 99%, and the conversion rate is 53%.

[0061] Example 2.9. The pH is 6

[0062] The specific experimental method includes the following steps: (1) Add 50 mL of acetonitrile to a 500 mL reaction flask, add 10 g of the substrate, and stir to dissolve at 37 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 6 with 1 M HCl, add 50 mL of crude hydrogenase solution, and simultaneously add 50 mg of coenzyme NAD+; (3) Continuously react under the condition of shaking in a shaker at 37 °C and 250 rpm for 24 h. After sampling, detect the product conversion rate by HPLC.

[0063] Experimental results: The purity of the product of the hydrogenase of SEQ ID NO: 1 after 24 h is 99%, and the conversion rate is 36%.

[0064] Example 2.10. The pH is 9

[0065] The specific experimental method includes the following steps: (1) Add 50 mL of acetonitrile to a 500 mL reaction flask, add 10 g of the substrate, and stir to dissolve at 37 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 9 with 1 M NaOH, add 50 mL of crude hydrogenase solution, and simultaneously add 50 mg of coenzyme NAD+; (3) Continuously react for 24 h under the shaking condition of a shaker at 37 °C and 250 rpm. After sampling, detect the product conversion rate by HPLC.

[0066] Experimental results: The purity of the product of the hydrogenase of SEQ ID NO: 1 after 24 h is 99%, and the conversion rate is 45%.

[0067] Example 2.11. Rotation speed 150 rpm

[0068] The specific experimental method includes the following steps: (1) Add 50 mL of acetonitrile to a 500 mL reaction flask, add 10 g of the substrate, and stir to dissolve at 37 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 7.5 with 1 M HCl, add 50 mL of crude hydrogenase solution, and simultaneously add 50 mg of coenzyme NAD+; (3) Continuously react for 24 h under the shaking condition of a shaker at 37 °C and 150 rpm. After sampling, detect the product conversion rate by HPLC.

[0069] Experimental results: The purity of the product of the hydrogenase of SEQ ID NO: 1 after 24 h is 99%, and the conversion rate is 56%.

[0070] Example 2.12. Rotation speed 300 rpm

[0071] The specific experimental method includes the following steps: (1) Add 50 mL of acetonitrile to a 500 mL reaction flask, add 10 g of the substrate, and stir to dissolve at 37 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 7.5 with 1 M HCl, add 50 mL of crude hydrogenase solution, and simultaneously add 50 mg of coenzyme NAD+; (3) Continuously react for 24 h under the shaking condition of a shaker at 37 °C and 300 rpm. After sampling, detect the product conversion rate by HPLC.

[0072] Experimental results: The purity of the product of the hydrogenase of SEQ ID NO: 1 after 24 h is 99%, and the conversion rate is 62%.

[0073] Example 2.13. The concentration of the substrate is 50 g / L

[0074] The specific experimental method includes the following steps: (1) Add 50 mL of acetonitrile to a 500 mL reaction flask, add 5 g of the substrate, and stir to dissolve at 37 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 7.5 with 1 M HCl, add 50 mL of crude hydrogenase solution, and simultaneously add 50 mg of coenzyme NAD+; (3) Continuously react under the shaking conditions of 37 °C and 250 rpm for 24 h. After sampling, detect the product conversion rate by HPLC.

[0075] Experimental results: The purity of the product of hydrogenase of SEQ ID NO: 1 after 24 h is 99%, and the conversion rate is 65%.

[0076] Example 2.14. The concentration of the substrate is 150 g / L

[0077] The specific experimental method includes the following steps: (1) Add 50 mL of acetonitrile to a 500 mL reaction flask, add 15 g of the substrate, and stir to dissolve at 37 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 7.5 with 1 M HCl, add 50 mL of crude hydrogenase solution, and simultaneously add 50 mg of coenzyme NAD+; (3) Continuously react under the shaking conditions of 37 °C and 250 rpm for 24 h. After sampling, detect the product conversion rate by HPLC.

[0078] Experimental results: The purity of the product of hydrogenase of SEQ ID NO: 1 after 24 h is 99%, and the conversion rate is 54%.

[0079] Example 2.15. The addition amount of hydrogenase is about 25% of the total amount

[0080] The specific experimental method includes the following steps: (1) Add 50 mL of acetonitrile to a 500 mL reaction flask, add 10 g of the substrate, and stir to dissolve at 37 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 7.5 with 1 M HCl, add 20 mL of crude hydrogenase solution, and simultaneously add 50 mg of coenzyme NAD+; (3) Continuously react under the shaking conditions of 37 °C and 250 rpm for 24 h. After sampling, detect the product conversion rate by HPLC.

[0081] Experimental results: The purity of the product of hydrogenase of SEQ ID NO: 1 after 24 h is 99%, and the conversion rate is 26%.

[0082] Example 2.16. The addition amount of hydrogenase is about 65% of the total amount

[0083] The specific experimental method includes the following steps: (1) Add 50 mL of acetonitrile to a 500 mL reaction flask, add 10 g of the substrate, and stir to dissolve at 37 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 7.5 with 1 M HCl, add 100 mL of crude hydrogenase solution, and simultaneously add 50 mg of coenzyme NAD+; (3) Continuously react under the conditions of shaking in a shaker at 37 °C and 250 rpm for 24 h. After sampling, detect the product conversion rate by HPLC.

[0084] Experimental results: The purity of the product of the hydrogenase of SEQ ID NO: 1 after 24 h is 99%, and the conversion rate is 43%.

[0085] Example 2.17. The addition amount of coenzyme NAD is 20 mg

[0086] The specific experimental method includes the following steps: (1) Add 50 mL of acetonitrile to a 500 mL reaction flask, add 10 g of the substrate, and stir to dissolve at 37 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 7.5 with 1 M HCl, add 50 mL of crude hydrogenase solution, and simultaneously add 20 mg of coenzyme NAD+; (3) Continuously react under the conditions of shaking in a shaker at 37 °C and 250 rpm for 24 h. After sampling, detect the product conversion rate by HPLC.

[0087] Experimental results: The purity of the product of the hydrogenase of SEQ ID NO: 1 after 24 h is 99%, and the conversion rate is 52%.

[0088] Example 2.18. The addition amount of coenzyme NAD is 200 mg

[0089] The specific experimental method includes the following steps: (1) Add 50 mL of acetonitrile to a 500 mL reaction flask, add 10 g of the substrate, and stir to dissolve at 37 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 7.5 with 1 M HCl, add 50 mL of crude hydrogenase solution, and simultaneously add 200 mg of coenzyme NAD+; (3) Continuously react under the conditions of shaking in a shaker at 37 °C and 250 rpm for 24 h. After sampling, detect the product conversion rate by HPLC.

[0090] Experimental results: The purity of the product of the hydrogenase of SEQ ID NO: 1 after 24 h is 99%, and the conversion rate is 62%.

[0091] Example 2.19. The coenzyme is NADP

[0092] The specific experimental method includes the following steps: (1) Add 50 mL of acetonitrile to a 500 mL reaction flask, add 10 g of the substrate, and stir to dissolve at 37 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 7.5 with 1 M HCl, add 50 mL of crude hydrogenase solution, and simultaneously add 50 mg of coenzyme NADP; (3) Continuously react under the shaking conditions of 37 °C and 250 rpm for 24 h, and after sampling, detect the product conversion rate by HPLC.

[0093] Experimental results: The purity of the product of the hydrogenase of SEQ ID NO: 1 after 24 h is 99%, and the conversion rate is 63%.

[0094] Example 2.20. The coenzyme is FAD

[0095] The specific experimental method includes the following steps: (1) Add 50 mL of acetonitrile to a 500 mL reaction flask, add 10 g of the substrate, and stir to dissolve at 37 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 7.5 with 1 M HCl, add 50 mL of crude hydrogenase solution, and simultaneously add 50 mg of coenzyme FAD; (3) Continuously react under the shaking conditions of 37 °C and 250 rpm for 24 h, and after sampling, detect the product conversion rate by HPLC.

[0096] Experimental results: The conversion rate of the product of the hydrogenase of SEQ ID NO: 1 after 24 h is 0%.

[0097] Example 2.21. Without coenzyme

[0098] The specific experimental method includes the following steps: (1) Add 50 mL of acetonitrile to a 500 mL reaction flask, add 10 g of the substrate, and stir to dissolve at 37 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 7.5 with 1 M HCl, and add 50 mL of crude hydrogenase solution; (3) Continuously react under the shaking conditions of 37 °C and 250 rpm for 24 h, and after sampling, detect the product conversion rate by HPLC.

[0099] Experimental results: The conversion rate of the product of the hydrogenase of SEQ ID NO: 1 after 24 h is 0%.

[0100] Example 2.22. Without solvent

[0101] The specific experimental method includes the following steps: (1) Add 100 mL of crude hydrogenase solution to a 500 mL reaction flask, add 10 g of the substrate, and it is difficult to stir and dissolve at 37 °C to obtain a turbid solution; (2) Adjust the pH of the reaction solution to 7.5 with 1 M HCl, and continuously react under the shaking conditions of 37 °C and 250 rpm for 24 h, and after sampling, detect the product conversion rate by HPLC.

[0102] Experimental results: The purity of the product of hydrogenase with SEQ ID NO: 1 after 24 hours is 99%, and the conversion rate is 5%.

[0103] Example 2.23. The hydrogenase is the F30T mutant of SEQ ID NO: 1

[0104] The specific experimental method includes the following steps: (1) Add 50 mL of acetonitrile to a 500 mL reaction flask, add 15 g of the substrate, and stir and dissolve at 37 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 7.5 with 1 M HCl, and add 50 mL of the crude enzyme solution of the F30T mutant hydrogenase; (3) Continuously react for 24 h under the shaking condition of a shaker at 37 °C and 250 rpm. After sampling, the product conversion rate is detected by HPLC.

[0105] Experimental results: The purity of the product of the F30T mutant of the hydrogenase with SEQ ID NO: 1 after 24 hours is 99%, and the conversion rate is 55%.

[0106] Example 2.24. Other hydrogenation reduction conditions

[0107] The specific experimental method includes the following steps: Dissolve 500 mg of magnolol in 20 mL of ethanol, add 50 mg of palladium-carbon, place the reactor in a hydrogen environment of 3 atm, and stir at 30 °C for 12 hours.

[0108] Experimental results: No product is generated as monitored by TLC.

[0109] The function and effect of this example:

[0110] Example Conversion rate Example Conversion rate Example Conversion rate Example 2.1 67％ Example 2.9 36％ Example 2.17 52％ Example 2.2 42％ Example 2.10 45％ Example 2.18 62％ Example 2.3 27％ Example 2.11 56％ Example 2.19 63％ Example 2.4 21％ Example 2.12 62％ Example 2.20 0％ Example 2.5 43％ Example 2.13 65％ Example 2.21 0％ Example 2.6 60％ Example 2.14 54％ Example 2.22 5％ Example 2.7 47％ Example 2.15 26％ Example 2.23 55％ Example 2.8 53％ Example 2.16 43％ Example 2.24 0

[0111] It can be found from the above experimental examples and comparison results that the present invention adopts a hydrogenase and a corresponding enzyme catalytic system, which can efficiently catalyze the substrate 5',5-di-2-propenyl-1,1'-biphenyl-2,2'-diphenol to be reduced to 5,5'-dipropyl-[1,1'-biphenyl]-2,2'-diol. This hydrogenase has good tolerance and stability in acetonitrile, its cost as a catalyst is low, the reaction conditions are mild, and it is environmentally friendly.

[0112] The above has introduced in detail an enzyme catalytic system, a hydrogenase and its application provided by the embodiments of the present application. Specific examples are used in this article to elaborate on the principle and implementation manner of the present application. The description of the above embodiments is only used to help understand the method and its core idea of the present application. At the same time, for those skilled in the art, according to the idea of the present application, there will be changes in the specific implementation manner and application scope. In summary, the content of this specification should not be construed as a limitation to the present application.

Claims

1. Application of a hydrogenase in the synthesis of tetrahydromagnolol, characterized in that: The amino acid sequence of the hydrogenase is shown in SEQ ID NO.1, and the hydrogenase is used to catalyze the reduction of the substrate 5’,5-di-2-propenyl-1,1’-biphenyl-2,2’-diphenol to 5,5'-dipropyl-[1,1'-biphenyl]-2,2'-diol, i.e., tetrahydromagnolol.

2. An enzyme-catalyzed system for synthesizing tetrahydromagnolol, characterized in that: It contains a hydrogenase and a coenzyme; wherein, the coenzyme is selected from at least one of nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate, and the amino acid sequence of the hydrogenase is shown in SEQ ID NO:

1.

3. The enzyme catalysis system according to claim 2, wherein: The addition amount of the hydrogenase is 1 / 2 - 1 / 4 of the total reaction volume.

4. The enzyme-catalyzed system according to claim 2, wherein: The addition amount of the coenzyme is 0.2 - 2 g / L.

5. The enzyme-catalyzed system according to claim 2, wherein: It also contains a solvent; wherein, the solvent is selected from at least one of alcohol, dimethyl sulfoxide, tetrahydrofuran, and acetonitrile.

6. A method for synthesizing tetrahydromagnolol, characterized in that: Using the hydrogenase as a catalyst, the amino acid sequence of the hydrogenase is shown in SEQ ID NO:1, or using the enzyme catalytic system described in any one of claims 2 - 5, catalyze the reduction of the substrate 5’,5-di-2-propenyl-1,1’-biphenyl-2,2’-diphenol to 5,5'-dipropyl-[1,1'-biphenyl]-2,2'-diol, i.e., tetrahydromagnolol.

7. The method according to claim 6, wherein: The concentration of the substrate is 50 g / L - 150 g / L.

8. The method according to claim 6, wherein: The pH of the enzyme-catalyzed reduction reaction is 6.0 - 9.

0.

9. The method according to claim 6, wherein: The temperature of the enzyme-catalyzed reduction reaction is 25°C - 65°C.

10. The method according to claim 6, wherein: The enzyme-catalyzed reduction reaction is carried out under oscillating conditions.

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