Enzyme, enzyme catalysis system and application of enzyme and enzyme catalysis system in catalysis of tetrahydromagnolol synthesis

Abstract

The invention provides a hydrogenase, an enzyme catalysis system and application of the hydrogenase and the enzyme catalysis system in catalysis of tetrahydromagnolol synthesis. Based on the advantages of strong specificity, high catalytic efficiency, no pollution and the like of a biological method, the characteristics of high conversion rate and good purity are realized after the hydrogenase of SEQ ID NO.1 is applied to a reaction for catalytically synthesizing 5, 5 '-dipropyl-[1, 1'-biphenyl]-2, 2 '-diol by taking 5', 5-di-2-allyl-1, 1 '-biphenyl-2, 2'-diphenol as a substrate.

Description

Technical Field

[0001] The present application relates to the field of synthesis of raw materials for cosmetics, and in particular to a method for synthesizing tetrahydromagnanol, a cosmetic active substance. Background Art

[0002] Tetrahydromagnanol is developed based on the natural ingredients in the bark of Magnolia officinalis (Magnolia officinalis, Magnoliaceae). This ingredient with significant whitening effect was created through natural pathways and artificial synthesis. It can hinder the maturation of tyrosinase before it is activated, inhibit the production of melanin at an early stage; it helps to inhibit the maturation of tyrosinase, the main cause of melanin formation, and enhance the skin's repair function against ultraviolet damage, thereby fading dark spots and sun spots. Therefore, tetrahydromagnanol is widely used in personal care products such as cosmetics, and is used in cosmetics for the purpose of whitening and skin protection.

[0003] At present, tetrahydromagnanol is generally prepared by diazotization reaction of sulfur and cyclohexane; specific preparation methods also include cyclization reaction 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-mentioned defects. Based on the advantages of biological methods such as strong specificity, high catalytic efficiency and no pollution, a hydrogenase that can be used for catalytic hydrogenation is developed, and the hydrogenase is 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'-diphenol, 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 an enzyme used as a hydrogenase as a catalyst in a catalytic hydrogenation reaction, which has the following characteristics: the amino acid sequence of the hydrogenase is as shown in SEQ ID NO.1, or has at least 80% to 99% similarity to 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 comprises the above-mentioned hydrogenase and a 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 catalysis system provided by the present invention 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 catalysis system provided by the present invention has the following characteristics: the addition amount of the above coenzyme is 0.2-2g / L.

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

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

[0015] In addition, the present invention also provides a method for synthesizing tetrahydromagnanol, a cosmetic active ingredient, which has the following characteristics: using the above-mentioned hydrogenase as a catalyst, or using the above-mentioned enzyme catalytic system, the substrate 5',5-di-2-propenyl-1,1'-biphenyl-2,2'-diphenol is catalyzed to be reduced 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 50g / L-150g / L.

[0017] Furthermore, the above-mentioned synthesis method provided by the present invention 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 pH regulation by acid (such as acidic solvents such as hydrochloric acid and acetic acid) or alkali (such as alkaline solvents such as sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, ammonia water, etc.).

[0018] Furthermore, the above-mentioned synthesis method provided by the present invention 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 has the following characteristics: the enzyme catalyzed reduction reaction is carried out under oscillation conditions, and the rotation speed can be 150 rpm-300 rpm, preferably 200 rpm-250 rpm. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 , the hydrogen spectrum of tetrahydromagnanol in Example 1. DETAILED DESCRIPTION

[0021] Example 1. Source of hydrogenase, recombinant expression of hydrogenase encoding gene and acquisition of crude enzyme solution

[0022] Description of the culture medium:

[0023] LB liquid medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl, sterilize in an autoclave at 121°C for 20 min, add kanamycin to a final concentration of 50 ug / mL before use.

[0024] TB liquid medium: peptone 11.8 g / L, yeast extract 23.6 g / L, K 2 HPO 4 9.4 g / L, KH 2 PO 4 2.2 g / L, glycerol 4 ml / L, sterilize in an autoclave at 121 ℃ for 20 min, add kanamycin to a final concentration of 50 ug / mL before use.

[0025] KPi buffer(pH 8.0): K 2 HPO 4 16.284 g / L, KH 2 PO 4 0.888 g / L soluble in pure water.

[0026] Reference NCBI from Galactomyces reessii The amino acid sequence of hydrogenase was optimized and a synthesis company was commissioned to perform codon optimization and gene sequence synthesis in Escherichia coli. The gene was 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 medium, respectively. IPTG with a final concentration of 0.5 mM was added as the inducer during induction, and the induction was carried out at 20°C for more than 12 h.

[0027] This embodiment provides a crude enzyme solution of hydrogenase, and the specific method for obtaining the crude enzyme solution comprises the following steps:

[0028] (1) The genetically engineered bacteria expressing recombinant hydrogenase were inoculated into LB liquid medium containing kanamycin at a final concentration of 50 ug / mL, and cultured at 37°C, 200 r / min until OD600 was about 0.6-0.8 to obtain a seed bacterial solution; the seed bacterial solution was inoculated into TB liquid medium containing kanamycin at a final concentration of 50 ug / mL at a 1% inoculum amount, and cultured at 37°C, 200 r / min for 4-6 h, until OD600 was about 0.8-1, IPTG with a final concentration of 0.5 mM was added for induction, and expression was induced at 20°C, 200 r / min for 12-16 h. After the induction, the bacteria were collected by centrifugation at 8000 r / min and 4°C, and the bacteria were resuspended in KPi buffer (pH 8.0).

[0029] (2) Ultrasonic disruption was used to disrupt the bacterial solution. The wet bacterial cells were resuspended in KPi buffer (pH 8.0) to prepare a 10% bacterial solution. The disruption conditions were as follows: working for 1 s, resting for 2 s, 180 W, and disruption for 8 min.

[0030] (3) Various impurities in the cell disruption solution were removed by centrifugation to obtain crude enzyme solution. The centrifugation parameters were 8000 r / min, 4°C, and centrifugation for 5-10 min.

[0031] 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 85 90 95 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 le 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 145 150 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 ArgGlu Gly Glu Ala Gln Glu Leu ValGly Ala Tyr Leu Tyr Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp IleArg Val Asp Gly Gly Tyr Thr Ala Pro

[0032] Example 2. Hydrogenase-based catalytic synthesis of tetrahydromagnanol

[0033] Construct an enzyme catalytic system: the crude hydrogenase solution of Example 1, a coenzyme, and a solvent. 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.

[0034] In the reaction of catalyzing 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 by using the above enzyme catalytic system, 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 shaking conditions, with a rotation speed of 150 rpm-300 rpm, preferably 200 rpm-250 rpm.

[0035] The concentration of the substrate is 50 g / L-150 g / L, preferably 100 g / L-150 g / L. The amount of hydrogenase added is 1 / 2-1 / 4 of the total reaction volume; the amount of coenzyme added is 0.2-2 g / L, preferably 0.5-1.5 g / L. The concentration of the organic solvent is 20% (v / v)-50% (v / v), preferably 30% (v / v)-40% (v / v) based on the total volume of the reaction system.

[0036] Example 2.1. Best response example

[0037] The specific experimental method includes the following steps: (1) adding 50 mL of acetonitrile to a 500 mL reaction bottle, adding 10 g of substrate, stirring at 45°C to dissolve, and obtaining a mixed solution; (2) adjusting the pH of the reaction solution to 7.5 with 1 M HCl, adding 50 mL of crude hydrogenase solution, and adding 50 mg of coenzyme NAD+; (3) continuing the reaction for 24 h under shaking conditions at 37°C and 250 rpm, and detecting the product conversion rate by HPLC after sampling.

[0038] The reaction system contained 50% (v / v) acetonitrile, 100 g / L substrate, total reaction solution: hydrogenase solution (v / v) = 2:1, and the pH was controlled at 7.5 during the reaction process.

[0039] Experimental results: The purity of the hydrogenase product of SEQ ID NO: 1 was 99% and the conversion rate was 67% within 24 hours.

[0040] 1 H NMR (500 MHz, DMSO- d 6 ) 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- d 6 ) 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] +

[0041] Example 2.2. Reaction temperature is 45°C

[0042] The specific experimental method includes the following steps: (1) adding 50 mL of acetonitrile to a 500 mL reaction bottle, adding 10 g of substrate, stirring and dissolving at 45°C to obtain a mixed solution; (2) adjusting the pH of the reaction solution to 7.5 with 1 M HCl, adding 50 mL of crude hydrogenase solution, and adding 50 mg of coenzyme NAD+; (3) continuing the reaction for 24 hours under shaking conditions at 45°C and 250 rpm, and detecting the product conversion rate by HPLC after sampling.

[0043] Experimental results: The purity of the hydrogenase product of SEQ ID NO: 1 was 99% and the conversion rate was 42% within 24 hours.

[0044] Example 2.3. Reaction temperature is 25°C

[0045] The specific experimental method includes the following steps: (1) adding 50 mL of acetonitrile to a 500 mL reaction bottle, adding 10 g of substrate, stirring and dissolving at 45°C to obtain a mixed solution; (2) adjusting the pH of the reaction solution to 7.5 with 1 M HCl, adding 50 mL of crude hydrogenase solution, and adding 50 mg of coenzyme NAD+; (3) continuing the reaction for 24 hours under shaking conditions at 25°C and 250 rpm, and detecting the product conversion rate by HPLC after sampling.

[0046] Experimental results: The purity of the hydrogenase product of SEQ ID NO: 1 was 99% and the conversion rate was 27% within 24 hours.

[0047] Example 2.4. Reaction temperature is 65°C

[0048] The specific experimental method includes the following steps: (1) adding 50 mL of acetonitrile to a 500 mL reaction bottle, adding 10 g of substrate, stirring and dissolving at 25°C to obtain a mixed solution; (2) adjusting the pH of the reaction solution to 7.5 with 1 M HCl, adding 50 mL of crude hydrogenase solution, and adding 50 mg of coenzyme NAD+; (3) continuing the reaction for 24 hours under shaking conditions at 65°C and 250 rpm, and detecting the product conversion rate by HPLC after sampling.

[0049] Experimental results: The purity of the hydrogenase product of SEQ ID NO: 1 was 99% and the conversion rate was 21% within 24 hours.

[0050] Example 2.5. The organic solvent is dimethyl sulfoxide

[0051] The specific experimental method includes the following steps: (1) adding 50 mL of dimethyl sulfoxide (DMSO) to a 500 mL reaction bottle, adding 10 g of substrate, stirring and dissolving at 37°C to obtain a mixed solution; (2) adjusting the pH of the reaction solution to 7.5 with 1 M HCl, adding 50 mL of crude hydrogenase solution, and adding 50 mg of coenzyme NAD+; (3) continuing the reaction for 24 h under shaking conditions at 37°C and 250 rpm, and detecting the product conversion rate by HPLC after sampling.

[0052] Experimental results: The purity of the hydrogenase product of SEQ ID NO: 1 was 99% and the conversion rate was 43% within 24 hours.

[0053] Example 2.6. The organic solvent is tetrahydrofuran

[0054] The specific experimental method includes the following steps: (1) adding 50 mL of tetrahydrofuran to a 500 mL reaction bottle, adding 10 g of substrate, stirring and dissolving at 37°C to obtain a mixed solution; (2) adjusting the pH of the reaction solution to 7.5 with 1 M HCl, adding 50 mL of crude hydrogenase solution, and adding 50 mg of coenzyme NAD+; (3) continuing the reaction for 24 hours under shaking conditions at 37°C and 250 rpm, and detecting the product conversion rate by HPLC after sampling.

[0055] Experimental results: The purity of the hydrogenase product of SEQ ID NO: 1 was 99% and the conversion rate was 60% within 24 hours.

[0056] Example 2.7. The organic solvent is DMF

[0057] The specific experimental method includes the following steps: (1) adding 50 mL of DMF to a 500 mL reaction bottle, adding 10 g of substrate, stirring and dissolving at 37°C to obtain a mixed solution; (2) adjusting the pH of the reaction solution to 7.5 with 1 M HCl, adding 50 mL of crude hydrogenase solution, and adding 50 mg of coenzyme NAD+; (3) continuing the reaction for 24 hours under shaking conditions at 37°C and 250 rpm, and detecting the product conversion rate by HPLC after sampling.

[0058] Experimental results: The purity of the hydrogenase product of SEQ ID NO: 1 was 99% and the conversion rate was 47% within 24 hours.

[0059] Example 2.8. The organic solvent is ethanol

[0060] The specific experimental method includes the following steps: (1) adding 50 mL of ethanol to a 500 mL reaction bottle, adding 10 g of substrate, stirring and dissolving at 37°C to obtain a mixed solution; (2) adjusting the pH of the reaction solution to 7.5 with 1 M HCl, adding 50 mL of crude hydrogenase solution, and adding 50 mg of coenzyme NAD+; (3) continuing the reaction for 24 hours under shaking conditions at 37°C and 250 rpm, and detecting the product conversion rate by HPLC after sampling.

[0061] Experimental results: The purity of the hydrogenase product of SEQ ID NO: 1 was 99% and the conversion rate was 53% within 24 hours.

[0062] Example 2.9. pH 6

[0063] The specific experimental method includes the following steps: (1) adding 50 mL of acetonitrile to a 500 mL reaction bottle, adding 10 g of substrate, stirring and dissolving at 37°C to obtain a mixed solution; (2) adjusting the pH of the reaction solution to 6 with 1 M HCl, adding 50 mL of crude hydrogenase solution, and adding 50 mg of coenzyme NAD+; (3) continuing the reaction for 24 hours under shaking conditions at 37°C and 250 rpm, and detecting the product conversion rate by HPLC after sampling.

[0064] Experimental results: The purity of the hydrogenase product of SEQ ID NO: 1 was 99% and the conversion rate was 36% within 24 hours.

[0065] Example 2.10. pH 9

[0066] The specific experimental method includes the following steps: (1) adding 50 mL of acetonitrile to a 500 mL reaction bottle, adding 10 g of substrate, stirring and dissolving at 37°C to obtain a mixed solution; (2) adjusting the pH of the reaction solution to 9 with 1 M NaOH, adding 50 mL of crude hydrogenase solution, and adding 50 mg of coenzyme NAD+; (3) continuing the reaction for 24 hours under shaking conditions at 37°C and 250 rpm, and detecting the product conversion rate by HPLC after sampling.

[0067] Experimental results: The purity of the hydrogenase product of SEQ ID NO: 1 was 99% and the conversion rate was 45% within 24 hours.

[0068] Example 2.11. Rotation speed 150 rpm

[0069] The specific experimental method includes the following steps: (1) adding 50 mL of acetonitrile to a 500 mL reaction bottle, adding 10 g of substrate, stirring and dissolving at 37°C to obtain a mixed solution; (2) adjusting the pH of the reaction solution to 7.5 with 1 M HCl, adding 50 mL of crude hydrogenase solution, and adding 50 mg of coenzyme NAD+; (3) continuing the reaction for 24 hours under shaking conditions at 37°C and 150 rpm, and detecting the product conversion rate by HPLC after sampling.

[0070] Experimental results: The purity of the hydrogenase product of SEQ ID NO: 1 was 99% and the conversion rate was 56% within 24 hours.

[0071] Example 2.12. Rotation speed 300 rpm

[0072] The specific experimental method includes the following steps: (1) adding 50 mL of acetonitrile to a 500 mL reaction bottle, adding 10 g of substrate, stirring and dissolving at 37°C to obtain a mixed solution; (2) adjusting the pH of the reaction solution to 7.5 with 1 M HCl, adding 50 mL of crude hydrogenase solution, and adding 50 mg of coenzyme NAD+; (3) continuing the reaction at 37°C and 300 rpm under shaking conditions for 24 h, and detecting the product conversion rate by HPLC after sampling.

[0073] Experimental results: The purity of the hydrogenase product of SEQ ID NO: 1 was 99% and the conversion rate was 62% within 24 hours.

[0074] Example 2.13. The concentration of the substrate is 50 g / L

[0075] The specific experimental method includes the following steps: (1) adding 50 mL of acetonitrile to a 500 mL reaction bottle, adding 5 g of substrate, stirring and dissolving at 37°C to obtain a mixed solution; (2) adjusting the pH of the reaction solution to 7.5 with 1 M HCl, adding 50 mL of crude hydrogenase solution, and adding 50 mg of coenzyme NAD+; (3) continuing the reaction for 24 hours under shaking conditions at 37°C and 250 rpm, and detecting the product conversion rate by HPLC after sampling.

[0076] Experimental results: The purity of the hydrogenase product of SEQ ID NO: 1 was 99% and the conversion rate was 65% within 24 hours.

[0077] Example 2.14. The concentration of the substrate is 150 g / L

[0078] The specific experimental method includes the following steps: (1) adding 50 mL of acetonitrile to a 500 mL reaction bottle, adding 15 g of substrate, stirring and dissolving at 37°C to obtain a mixed solution; (2) adjusting the pH of the reaction solution to 7.5 with 1 M HCl, adding 50 mL of crude hydrogenase solution, and adding 50 mg of coenzyme NAD+; (3) continuing the reaction for 24 hours under shaking conditions at 37°C and 250 rpm, and detecting the product conversion rate by HPLC after sampling.

[0079] Experimental results: The purity of the hydrogenase product of SEQ ID NO: 1 was 99% and the conversion rate was 54% within 24 hours.

[0080] Example 2.15. The amount of hydrogenase added is about 25% of the total amount

[0081] The specific experimental method includes the following steps: (1) adding 50 mL of acetonitrile to a 500 mL reaction bottle, adding 10 g of substrate, stirring and dissolving at 37°C to obtain a mixed solution; (2) adjusting the pH of the reaction solution to 7.5 with 1 M HCl, adding 20 mL of crude hydrogenase solution, and adding 50 mg of coenzyme NAD+; (3) continuing the reaction for 24 hours under shaking conditions at 37°C and 250 rpm, and detecting the product conversion rate by HPLC after sampling.

[0082] Experimental results: The purity of the hydrogenase product of SEQ ID NO: 1 was 99% and the conversion rate was 26% within 24 hours.

[0083] Example 2.16. The amount of hydrogenase added is about 65% of the total amount

[0084] The specific experimental method includes the following steps: (1) adding 50 mL of acetonitrile to a 500 mL reaction bottle, adding 10 g of substrate, stirring and dissolving at 37°C to obtain a mixed solution; (2) adjusting the pH of the reaction solution to 7.5 with 1 M HCl, adding 100 mL of crude hydrogenase solution, and adding 50 mg of coenzyme NAD+; (3) continuing the reaction for 24 hours under shaking conditions at 37°C and 250 rpm, and detecting the product conversion rate by HPLC after sampling.

[0085] Experimental results: The purity of the hydrogenase product of SEQ ID NO: 1 was 99% and the conversion rate was 43% within 24 hours.

[0086] Example 2.17. The amount of coenzyme NAD added is 20 mg

[0087] The specific experimental method includes the following steps: (1) adding 50 mL of acetonitrile to a 500 mL reaction bottle, adding 10 g of substrate, stirring and dissolving at 37°C to obtain a mixed solution; (2) adjusting the pH of the reaction solution to 7.5 with 1 M HCl, adding 50 mL of crude hydrogenase solution, and adding 20 mg of coenzyme NAD+; (3) continuing the reaction for 24 hours under shaking conditions at 37°C and 250 rpm, and detecting the product conversion rate by HPLC after sampling.

[0088] Experimental results: The purity of the hydrogenase product of SEQ ID NO: 1 was 99% and the conversion rate was 52% within 24 hours.

[0089] Example 2.18. The amount of coenzyme NAD added is 200 mg

[0090] The specific experimental method includes the following steps: (1) adding 50 mL of acetonitrile to a 500 mL reaction bottle, adding 10 g of substrate, stirring and dissolving at 37°C to obtain a mixed solution; (2) adjusting the pH of the reaction solution to 7.5 with 1 M HCl, adding 50 mL of crude hydrogenase solution, and adding 200 mg of coenzyme NAD+; (3) continuing the reaction for 24 hours under shaking conditions at 37°C and 250 rpm, and detecting the product conversion rate by HPLC after sampling.

[0091] Experimental results: The purity of the hydrogenase product of SEQ ID NO: 1 was 99% and the conversion rate was 62% within 24 hours.

[0092] Example 2.19. Coenzyme is NADP

[0093] The specific experimental method includes the following steps: (1) adding 50 mL of acetonitrile to a 500 mL reaction bottle, adding 10 g of substrate, stirring and dissolving at 37°C to obtain a mixed solution; (2) adjusting the pH of the reaction solution to 7.5 with 1 M HCl, adding 50 mL of crude hydrogenase solution, and adding 50 mg of coenzyme NADP; (3) continuing the reaction for 24 hours under shaking conditions at 37°C and 250 rpm, and detecting the product conversion rate by HPLC after sampling.

[0094] Experimental results: The purity of the hydrogenase product of SEQ ID NO: 1 was 99% and the conversion rate was 63% within 24 hours.

[0095] Example 2.20. Coenzyme is FAD

[0096] The specific experimental method includes the following steps: (1) adding 50 mL of acetonitrile to a 500 mL reaction bottle, adding 10 g of substrate, stirring and dissolving at 37°C to obtain a mixed solution; (2) adjusting the pH of the reaction solution to 7.5 with 1 M HCl, adding 50 mL of crude hydrogenase solution, and adding 50 mg of coenzyme FAD; (3) continuing the reaction for 24 hours under shaking conditions at 37°C and 250 rpm, and detecting the product conversion rate by HPLC after sampling.

[0097] Experimental results: The product conversion rate of the hydrogenase of SEQ ID NO: 1 was 0% in 24 hours.

[0098] Example 2.21. No coenzyme

[0099] The specific experimental method includes the following steps: (1) adding 50 mL of acetonitrile to a 500 mL reaction bottle, adding 10 g of substrate, stirring at 37°C to dissolve, and obtaining a mixed solution; (2) adjusting the pH of the reaction solution to 7.5 with 1 M HCl, and adding 50 mL of crude hydrogenase enzyme solution; (3) continuing the reaction for 24 h under shaking conditions at 37°C and 250 rpm, and detecting the product conversion rate by HPLC after sampling.

[0100] Experimental results: The product conversion rate of the hydrogenase of SEQ ID NO: 1 was 0% in 24 hours.

[0101] Example 2.22. Solvent-free

[0102] The specific experimental method includes the following steps: (1) adding 100 mL of crude hydrogenase solution to a 500 mL reaction bottle, adding 10 g of substrate, and stirring at 37°C until the solution is difficult to dissolve, thereby obtaining a turbid solution; (2) adjusting the pH of the reaction solution to 7.5 with 1 M HCl, continuing the reaction at 37°C and 250 rpm on a shaker for 24 h, and sampling and detecting the product conversion rate by HPLC.

[0103] Experimental results: The purity of the hydrogenase product of SEQ ID NO: 1 was 99% and the conversion rate was 5% within 24 hours.

[0104] Example 2.23. Hydrogenase is the F30T mutant of SEQ ID NO: 1

[0105] The specific experimental method includes the following steps: (1) adding 50 mL of acetonitrile to a 500 mL reaction bottle, adding 15 g of substrate, stirring and dissolving at 37°C to obtain a mixed solution; (2) adjusting the pH of the reaction solution to 7.5 with 1 M HCl, and adding 50 mL of F30T mutant hydrogenase crude enzyme solution; (3) continuing the reaction for 24 hours under shaking conditions at 37°C and 250 rpm, and detecting the product conversion rate by HPLC after sampling.

[0106] Experimental results: The product purity of the hydrogenase F30T mutant of SEQ ID NO: 1 was 99% and the conversion rate was 55% within 24 hours.

[0107] Example 2.24. Other hydrogenation reduction conditions

[0108] The specific experimental method includes the following steps: dissolving 500 mg of magnolol in 20 mL of ethanol, adding 50 mg of palladium carbon, placing the reactor in a 3 atm hydrogen environment, and stirring at 30 degrees Celsius for 12 hours.

[0109] Experimental results: TLC monitoring showed no product was generated.

[0110] Functions and effects of this embodiment: 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] From the above experimental examples and comparative results, it can be found that the present invention uses hydrogenase and a corresponding enzyme catalytic system to efficiently catalyze the reduction of the substrate 5',5-di-2-propenyl-1,1'-biphenyl-2,2'-diphenol to obtain 5,5'-dipropyl-[1,1'-biphenyl]-2,2'-diol. The hydrogenase has good tolerance and stability in acetonitrile, and its cost as a catalyst is low, the reaction conditions are mild, and it is environmentally friendly.

[0112] The above is a detailed introduction to an enzyme catalytic system, hydrogenase and application provided in the examples of the present application. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above examples is only used to help understand the method and core ideas of the present application. At the same time, for those skilled in the art, according to the ideas of the present application, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. Use of an enzyme as a hydrogenase as a catalyst in a catalytic hydrogenation reaction, characterized in that: The amino acid sequence of the hydrogenase is shown in SEQ ID NO.1, or has at least 80% to 99% similarity to the sequence shown in SEQ ID NO:

1.

2. An enzyme catalytic system for catalytic hydrogenation, characterized in that: The method comprises the hydrogenase according to claim 1, and a coenzyme; wherein the coenzyme is selected from at least one of nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate.

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

4. The enzyme catalysis system according to claim 2, characterized in that: The added amount of the coenzyme is 0.2-2g / L.

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

6. A method for synthesizing tetrahydromagnanol, a cosmetic active ingredient, characterized in that: The hydrogenase as claimed in claim 1 is used as a catalyst, or the enzyme catalytic system as claimed in any one of claims 2 to 5 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.

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

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

0.

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

10. The synthesis method according to claim 6, characterized in that: The enzyme-catalyzed reduction reaction was carried out under shaking conditions.

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