Hydrogenase and enzyme catalytic system and its application in catalytic hydrogenation reaction

Abstract

The application provides a hydrogenase and an enzyme catalytic system and application of the hydrogenase in catalytic hydrogenation, the amino acid sequence of the hydrogenase is shown in SEQ ID NO. 1, or the hydrogenase has at least 80%-99% similarity with the sequence shown in SEQ ID NO. 1, and the hydrogenase has the advantages of high catalytic efficiency and no pollution.

Description

Technical Field

[0001] This invention relates to the field of synthesis of cosmetic raw materials, specifically to an enzyme and an enzyme catalytic system and its application in catalytic hydrogenation reactions. In this invention, the enzyme catalytic system was used to successfully prepare cosmetic active ingredients such as dihydrokaempferol, dihydrocapsaicin, dihydromyricetin, dihydroroic acid (10-hydroxydecanoic acid), tetrahydromagnoliol, tetrahydrocurcumin, and octahydrocurcumin. Background Technology

[0002] For active ingredients in skincare products, appropriate hydrogenation modification can alter the spatial configuration and electronic properties of molecules, thereby improving their chemical stability, photostability, and oxidative stability, and reducing the risk of degradation or discoloration during storage and formulation. Simultaneously, hydrogenated derivatives may also exhibit lower irritation, better skin tolerance, and superior transdermal properties, which is beneficial for improving the bioavailability and skincare efficacy of active ingredients. Furthermore, some hydrogenated products show promising application potential in areas such as anti-oxidation, anti-inflammation, soothing, repair, anti-aging, and whitening, thus possessing broad application prospects in functional skincare products.

[0003] As consumers increasingly demand higher standards for the safety, efficacy, and sustainable development of cosmetics, the application of biocatalysis technology in the development of active ingredients in cosmetics has received growing attention. Among these technologies, enzyme-catalyzed hydrogenation is gradually becoming an important alternative to traditional chemical hydrogenation processes due to its advantages such as mild reaction conditions, high catalytic selectivity, fewer byproducts, and environmental friendliness. Enzyme-catalyzed hydrogenation can efficiently and selectively transform unsaturated bonds and carbonyl groups in natural products or functional molecules, improving their physicochemical properties while preserving the core activity of the molecule. Compared to traditional metal-catalyzed hydrogenation, enzyme-catalyzed hydrogenation does not require high temperature and high pressure conditions, avoiding heavy metal residues and complex post-processing. It better aligns with the requirements of green manufacturing and sustainable development, and is more compatible with the safety and quality consistency requirements of cosmetic ingredients.

[0004] However, the application of enzyme-catalyzed hydrogenation technology in the development of active ingredients in cosmetics is still in its developmental stage. Existing research mainly focuses on process optimization, and further research is needed on the structure-activity relationship, efficacy enhancement mechanisms, and industrial applications of hydrogenated products in the skincare field. Therefore, establishing corresponding preparation methods and application systems for enzyme-catalyzed hydrogenated products has significant scientific and industrial value. Summary of the Invention

[0005] Based on the advantages of biological methods, such as high specificity, high catalytic efficiency, and no pollution, this invention has developed a hydrogenase that can be used to catalyze hydrogenation. Using this hydrogenase as a substrate, various compounds have been successfully used to prepare the corresponding hydrogenation products as follows:

[0006] In this invention, the amino acid sequence of the hydrogenase used is as shown in SEQ ID NO.1, or has at least 80%-99% similarity to the sequence shown in SEQ ID NO:1.

[0007] Furthermore, based on the aforementioned hydrogenase, the present invention also provides an enzyme catalytic system for catalyzing hydrogenation, comprising the aforementioned hydrogenase and a coenzyme; wherein the aforementioned coenzyme is selected from at least one of nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP).

[0008] The amount of hydrogenase added is 1 / 5 to 1 / 2 of the total reaction volume.

[0009] The amount of the coenzyme added is 1-4 g / L.

[0010] The above-described enzyme catalytic system may further include a solvent; wherein the solvent is selected from at least one of isopropanol, dimethyl sulfoxide, tetrahydrofuran, water, and ethanol. The volume concentration of the solvent is 20%-60%.

[0011] Furthermore, this invention also suggests the application of the above-mentioned enzyme catalysts or enzyme catalytic systems in catalytic hydrogenation / reduction reactions, mostly in the reduction reactions of compounds containing double bonds.

[0012] The concentration of the reaction substrate for the above catalytic hydrogenation reaction is 5 g / L-15 g / L; The pH of the reaction is 6.0-9.0, preferably 7.0-8.0. The pH is adjusted by using acids (such as hydrochloric acid, acetic acid, etc.) or bases (such as sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, ammonia, etc.). The reaction temperature is 25℃-55℃, preferably 30℃-40℃; The reaction is carried out under oscillating conditions, with a rotation speed of 150 rpm to 300 rpm, preferably 200 rpm to 250 rpm. Detailed Implementation

[0013] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0014] Example 1. Source of hydrogenase, recombinant expression of hydrogenase encoding gene, and acquisition of crude enzyme solution. 1.1. Preparation of culture medium: LB liquid medium: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L, autoclave at 121℃ for 20 min, add kanamycin to a final concentration of 50 ug / mL before use.

[0015] TB liquid medium: peptone 11.8 g / L, yeast extract 23.6 g / L, K2HPO4 9.4 g / L, KH2PO4 2.2 g / L, glycerol 4 ml / L, autoclaved at 121 ℃ for 20 min, add kanamycin to a final concentration of 50 ug / mL before use.

[0016] KPi buffer (pH 8.0): 16.284 g / L K2HPO4 and 0.888 g / L KH2PO4 dissolved in pure water.

[0017] 1.2. Preparation of crude hydrogenase solution: Referenced from NCBI Geotrichum galactomycetum The amino acid sequence of the hydrogenase was obtained by commissioning a synthetic company to optimize the codons and synthesize the gene sequence of E. coli. The sequence was cloned into the pet28a plasmid vector and transformed into the expression host E. coli BL21(DE3) for recombinant expression. The growth and induction media were LB liquid medium and TB liquid medium, respectively. IPTG with a final concentration of 0.5 mM was added as an inducer during induction and the induction was carried out at 20 ℃ for 12 h.

[0018] The specific method for obtaining the crude enzyme solution of hydrogenase includes the following steps: (1) The recombinant hydrogenase-expressing genetically engineered bacteria were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C and 200 r / min until the OD600 was about 0.4-0.7 to obtain seed culture. The seed culture was inoculated into TB liquid medium containing 50 μg / mL kanamycin at a 1% inoculation rate and cultured at 37°C and 200 r / min for 4-6 h until the OD600 was about 0.8-1. IPTG was added to induce expression at a final concentration of 0.5 mM and the expression was induced at 20°C and 200 r / min for 12 h. After the induction was completed, the cells were collected by centrifugation at 8000 r / min and 4°C and resuspended in KPi buffer (pH 8.0).

[0019] (2) The bacterial suspension was disrupted using ultrasonic disruption. The wet bacterial cells were resuspended in KPi buffer (pH 8.0) to prepare a 10% bacterial suspension. The disruption conditions were as follows: 1 second operation, 2 seconds interval, 180 W, disruption for 8 minutes.

[0020] (3) Remove various impurities in the cell lysate by centrifugation to obtain crude enzyme solution. Centrifugation parameters: 8000 r / min, 4℃, centrifugation for 5~10 min.

[0021] The amino acid sequence of the hydrogenase is SEQ ID NO.1: Met Val Asn Thr His Phe Thr Gly LeuGly Pro Pro Gln Pro Ala Pro Lys Pro Ser Asn Val Leu Asp Leu Ser Leu Lys LysVal Ala Ser Val Thr Gly Ser Ser Thr Gly Gly Tyr Ala Val Ala Glu Ala Phe AlaGln Ala Gly Ala Asp Val Ala Leu Tyr Asn Ser His Asn Ala Glu Ala Lys Ala LysAla Leu Ser Glu Lys Tyr Gly Ile Lys Ala Lys Ala Tyr Lys Val Leu Val Thr AspSer Ala Ala Val Glu Ala Ala Ile Lys Glu Tyr Phe Gly Lys Ile Asp Ile Phe ValAla Asn Ala Gly Val Pro Trp Thr Ala Gly Pro Leu Ile Asp Thr Glu Asp Asp LysGlu Trp Lys Lys Val Ile Asp Leu Asp Phe Thr Gly Val Tyr Tyr Tyr Ile Phe LysGlu Arg Gly Ser Gly Ser Phe Ile Ala Thr Ser Ser Met Ser Gly His Ile Val AsnPhe Pro Gln Leu Gln Ala Ala Tyr Asn Gly Ala Lys Ala Gly Val Arg His Ser LeuAla Val Glu Trp Ala Gly Phe Ala Arg Val Asn Thr Val Ser Pro Gly Tyr Ile IleSer Ala Phe Val Pro Asp Glu Thr Lys Ser Lys Trp Trp Ser Phe Thr Pro Leu GlyArg Glu Gly Glu Ala Gln Glu Leu Val Gly Ala Tyr Leu Tyr Leu Ala Ser Asp AlaSer Thr Tyr Thr Thr Ile Arg Val Asp Gly GlyTyr Gln Thr Leu Ala Phe Gly IleTrp Ala Lys Gly Arg His Phe Cys Thr Ala Thr Glu Gly Ala Asp Glu Gln Ile CysThr Ala Pro .

[0022] Example 2. Catalytic hydrogenation reaction Construction of the enzyme catalytic system: The crude hydrogenase solution from Example 1, coenzyme, and solvent were used. The coenzyme was selected from at least one of nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate. The solvent was selected from at least one of isopropanol, dimethyl sulfoxide, tetrahydrofuran, and water.

[0023] In the reaction catalyzed by the above-described enzyme catalysis system for the corresponding double-bonded substrate, the pH of the reduction reaction is 6-9, preferably 7-8; the reaction temperature is 25-55℃, preferably 30-40℃. The reduction reaction is carried out under oscillation conditions at a rotation speed of 150-300 rpm, preferably 200-250 rpm.

[0024] The substrate concentration is 5 g / L-15 g / L, preferably 10 g / L-15 g / L. The amount of hydrogenase added is 20-70% of the total reaction volume; the amount of coenzyme added is 1-4 g / L. The concentration of organic solvent, based on the total volume of the reaction system, is 20% (v / v)-60% (v / v), preferably 40% (v / v)-50% (v / v).

[0025] Example 2.1. Dihydroroyalic acid (10-hydroxydecanoic acid) (Example of optimal reaction conditions) The specific experimental method includes the following steps: (1) Add 50 mL of isopropanol to a 500 mL reaction flask, add 1 g of the substrate royal icing, stir and dissolve at 45 °C to obtain a mixture; (2) Adjust the pH of the reaction solution to 7.5 with 1 M NaOH, add 50 mL of crude hydrogenase solution, and add 200 mg of coenzyme NAD+ at the same time; (3) Continue the reaction for 24 h under the shaking conditions of 37 °C and 250 rpm, and then take a sample to detect the product conversion rate by HPLC.

[0026] The reaction system contains 50% (v / v) isopropanol, 10 g / L substrate, and the total reaction volume is 1:1 of hydrogenase solution / organic solvent (v / v). The pH is controlled at 7.5 during the reaction process.

[0027] Experimental results: The dihydroroic acid produced by the hydrogenase of SEQ ID NO: 1 after 24h was purified by column chromatography with a purity of 99% and a yield of 78%.

[0028] Example 2.2. The reaction temperature was 45℃. The specific experimental method includes the following steps: (1) Add 50 mL of isopropanol to a 500 mL reaction flask, add 1 g of the substrate royal icing, stir and dissolve at 45 °C to obtain a mixture; (2) Adjust the pH of the reaction solution to 7.5 with 1 M NaOH, add 50 mL of crude hydrogenase solution, and add 200 mg of coenzyme NAD+ at the same time; (3) Continue the reaction for 24 h under the shaking conditions of 45 °C and 250 rpm, and then take a sample to detect the product conversion rate by HPLC.

[0029] Experimental results: The yield of dihydroroic acid from the hydrogenase of SEQ ID NO: 1 was 25%.

[0030] Example 2.3. The reaction temperature was 25°C. The specific experimental method includes the following steps: (1) Add 50 mL of isopropanol to a 500 mL reaction flask, add 1 g of the substrate royal icing, stir and dissolve at 45 °C to obtain a mixture; (2) Adjust the pH of the reaction solution to 7.5 with 1 M NaOH, add 50 mL of crude hydrogenase solution, and add 200 mg of coenzyme NAD+ at the same time; (3) Continue the reaction for 24 h under the shaking conditions of 25 °C and 250 rpm, and then take a sample to detect the product conversion rate by HPLC.

[0031] Experimental results: The yield of dihydroroic acid obtained by hydrogenase of SEQ ID NO: 1 after 24 hours was 31%.

[0032] Example 2.4. The reaction temperature was 65℃. The specific experimental method includes the following steps: (1) Add 50 mL of isopropanol to a 500 mL reaction flask, add 1 g of the substrate royal icing, stir and dissolve at 45 °C to obtain a mixture; (2) Adjust the pH of the reaction solution to 7.5 with 1 M NaOH, add 50 mL of crude hydrogenase solution, and add 200 mg of coenzyme NAD+ at the same time; (3) Continue the reaction for 24 h under the shaking conditions of 65 °C and 250 rpm, and then take a sample to detect the product conversion rate by HPLC.

[0033] Experimental results: The hydrogenase of SEQ ID NO: 1 yielded 21% dihydroroic acid after 24 hours.

[0034] Example 2.5. The organic solvent is dimethyl sulfoxide. The specific experimental method includes the following steps: (1) Add 50 mL of dimethyl sulfoxide (DMSO) to a 500 mL reaction flask, add 1 g of the substrate royal icing, stir and dissolve at 37 °C to obtain a mixture; (2) Adjust the pH of the reaction solution to 7.5 with 1 M NaOH, add 50 mL of crude hydrogenase solution, and add 200 mg of coenzyme NAD+ at the same time; (3) Continue the reaction for 24 h under the shaking conditions of 37 °C and 250 rpm, and then take a sample to detect the product conversion rate by HPLC.

[0035] Experimental results: The hydrogenase of SEQ ID NO: 1 yielded dihydroroic acid with a yield of 6% after 24 hours.

[0036] Example 2.6. The organic solvent is tetrahydrofuran. The specific experimental method includes the following steps: (1) Add 50 mL of tetrahydrofuran to a 500 mL reaction flask, add 1 g of the substrate royal icing, stir and dissolve at 37 °C to obtain a mixture; (2) Adjust the pH of the reaction solution to 7.5 with 1 M NaOH, add 50 mL of crude hydrogenase solution, and add 200 mg of coenzyme NAD+ at the same time; (3) Continue the reaction for 24 h under the shaking conditions of 37 °C and 250 rpm, and then take a sample to detect the product conversion rate by HPLC.

[0037] Experimental results: The hydrogenase of SEQ ID NO: 1 yielded 35% dihydroroic acid after 24 hours.

[0038] Example 2.7. No organic solvent The specific experimental method includes the following steps: (1) Add 50 mL of water to a 500 mL reaction flask, add 1 g of the substrate royal icing, stir and dissolve at 37 °C (the dissolution is not good, it is a suspension) to obtain a mixture; (2) Adjust the pH of the reaction solution to 7.5 with 1 M NaOH, add 50 mL of crude hydrogenase solution, and add 200 mg of coenzyme NAD+ at the same time; (3) Continue the reaction for 24 h under the shaking conditions of 37 °C and 250 rpm, and then take a sample to detect the product conversion rate by HPLC.

[0039] Experimental results: The hydrogenase of SEQ ID NO: 1 yielded 11% dihydroroic acid after 24 hours.

[0040] Example 2.8. The organic solvent is ethanol. The specific experimental method includes the following steps: (1) Add 50 mL of ethanol to a 500 mL reaction flask, add 1 g of substrate, stir and dissolve at 37 °C to obtain a mixture; (2) Adjust the pH of the reaction solution to 7.5 with 1 M NaOH, add 50 mL of crude hydrogenase solution, and add 200 mg of coenzyme NAD+ at the same time; (3) Continue the reaction for 24 h under the shaking conditions of 37 °C and 250 rpm, and then take a sample to detect the product conversion rate by HPLC.

[0041] Experimental results: The hydrogenase of SEQ ID NO: 1 yielded 49% dihydroroic acid after 24 hours.

[0042] Example 2.9. pH is 6 The specific experimental method includes the following steps: (1) Add 50 mL of isopropanol to a 500 mL reaction flask, add 1 g of the substrate royal icing, stir and dissolve at 45 °C to obtain a mixture; (2) Adjust the pH of the reaction solution to 6 with 1 M NaOH, add 50 mL of crude hydrogenase solution, and add 200 mg of coenzyme NAD+ at the same time; (3) Continue the reaction for 24 h under the shaking conditions of 37 °C and 250 rpm, and then take a sample to detect the product conversion rate by HPLC.

[0043] Experimental results: The hydrogenase of SEQ ID NO: 1 yielded 24% dihydroroic acid after 24 hours.

[0044] Example 2.10. pH is 9 The specific experimental method includes the following steps: (1) Add 50 mL of isopropanol to a 500 mL reaction flask, add 1 g of substrate, stir and dissolve at 45 °C to obtain a mixture; (2) Adjust the pH of the reaction solution to 9 with 1 M NaOH, add 50 mL of crude hydrogenase solution, and add 200 mg of coenzyme NAD+ at the same time; (3) Continue the reaction for 24 h under the shaking conditions of 37 °C and 250 rpm, and then take a sample to detect the product conversion rate by HPLC.

[0045] Experimental results: The hydrogenase of SEQ ID NO: 1 yielded dihydroroic acid with a yield of 4% after 24 hours.

[0046] Example 2.11. Rotation speed 150 rpm The specific experimental method includes the following steps: (1) Add 50 mL of isopropanol to a 500 mL reaction flask, add 1 g of the substrate royal icing, stir and dissolve at 45 °C to obtain a mixture; (2) Adjust the pH of the reaction solution to 7.5 with 1 M NaOH, add 50 mL of crude hydrogenase solution, and add 200 mg of coenzyme NAD+ at the same time; (3) Continue the reaction for 24 h under the shaking conditions of 37 °C and 150 rpm, and then take a sample to detect the product conversion rate by HPLC.

[0047] Experimental results: The hydrogenase of SEQ ID NO: 1 yielded 65% dihydroroic acid after 24 hours.

[0048] Example 2.12. Rotation speed 300 rpm The specific experimental method includes the following steps: (1) Add 50 mL of isopropanol to a 500 mL reaction flask, add 1 g of the substrate royal icing, stir and dissolve at 45 °C to obtain a mixture; (2) Adjust the pH of the reaction solution to 7.5 with 1 M NaOH, add 50 mL of crude hydrogenase solution, and add 200 mg of coenzyme NAD+ at the same time; (3) Continue the reaction for 24 h under the shaking conditions of 37 °C and 300 rpm, and then take a sample to detect the product conversion rate by HPLC.

[0049] Experimental results: The yield of dihydroroic acid from the hydrogenase product of SEQ ID NO: 1 after 24 hours was 71%.

[0050] Example 2.13. The substrate concentration was 15 g / L. The specific experimental method includes the following steps: (1) Add 50 mL of isopropanol to a 500 mL reaction flask, add 1.5 g of the substrate royal ic acid, stir and dissolve at 45 °C (it is not completely dissolved and is a suspension) to obtain a mixture; (2) Adjust the pH of the reaction solution to 7.5 with 1 M NaOH, add 50 mL of crude hydrogenase solution, and add 200 mg of coenzyme NAD+ at the same time; (3) Continue the reaction for 24 h under the shaking conditions of 37 °C and 250 rpm, and then take a sample to detect the product conversion rate by HPLC.

[0051] Experimental results: The hydrogenase of SEQ ID NO: 1 yielded 44% dihydroroic acid after 24 hours.

[0052] Example 2.14. The substrate concentration was 5 g / L. The specific experimental method includes the following steps: (1) Add 50 mL of isopropanol to a 500 mL reaction flask, add 0.5 g of the substrate royal ic acid, stir and dissolve at 45 °C to obtain a mixed solution; (2) Adjust the pH of the reaction solution to 7.5 with 1 M NaOH, add 50 mL of crude hydrogenase solution, and add 200 mg of coenzyme NAD+ at the same time; (3) Continue the reaction for 24 h under the shaking conditions of 37 °C and 250 rpm, and then take a sample to detect the product conversion rate by HPLC.

[0053] Experimental results: The hydrogenase of SEQ ID NO: 1 yielded 77% dihydroroic acid after 24 hours.

[0054] Example 2.15. The amount of hydrogenase added was approximately 25% of the total amount. The specific experimental method includes the following steps: (1) Add 50 mL of isopropanol to a 500 mL reaction flask, add 1 g of the substrate royal icing, stir and dissolve at 45 °C to obtain a mixture; (2) Adjust the pH of the reaction solution to 7.5 with 1 M NaOH, add 20 mL of crude hydrogenase solution, and add 140 mg of coenzyme NAD+ at the same time; (3) Continue the reaction for 24 h under the shaking conditions of 37 °C and 250 rpm, and then take a sample to detect the product conversion rate by HPLC.

[0055] Experimental results: The yield of dihydroroic acid, the 24-hour product of the hydrogenase of SEQ ID NO: 1, was 33%.

[0056] Example 2.16. The amount of hydrogenase added was approximately 65% ​​of the total amount. The specific experimental method includes the following steps: (1) Add 50 mL of isopropanol to a 500 mL reaction flask, add 1 g of the substrate royal icing, stir and dissolve at 37 °C to obtain a mixture; (2) Adjust the pH of the reaction solution to 7.5 with 1 M HCl, add 100 mL of crude hydrogenase solution, and add 300 mg of coenzyme NAD+ at the same time; (3) Continue the reaction for 24 h under the shaking conditions of 37 °C and 250 rpm, and then take a sample to detect the product conversion rate by HPLC.

[0057] Experimental results: The yield of dihydroroic acid, the 24-hour product of the hydrogenase of SEQ ID NO: 1, was 77%.

[0058] Example 2.17. The amount of coenzyme NAD added was 100 mg. The specific experimental method includes the following steps: (1) Add 50 mL of isopropanol to a 500 mL reaction flask, add 1 g of the substrate royal icing, stir and dissolve at 45 °C to obtain a mixture; (2) Adjust the pH of the reaction solution to 7.5 with 1 M NaOH, add 50 mL of crude hydrogenase solution, and add 100 mg of coenzyme NAD+ at the same time; (3) Continue the reaction for 24 h under the shaking conditions of 37 °C and 250 rpm, and then take a sample to detect the product conversion rate by HPLC.

[0059] Experimental results: The yield of dihydroroic acid, the 24-hour product of the hydrogenase of SEQ ID NO: 1, was 26%.

[0060] Example 2.18. The amount of coenzyme NAD added was 400 mg. The specific experimental method includes the following steps: (1) Add 50 mL of isopropanol to a 500 mL reaction flask, add 1 g of the substrate royal icing, stir and dissolve at 45 °C to obtain a mixture; (2) Adjust the pH of the reaction solution to 7.5 with 1 M NaOH, add 50 mL of crude hydrogenase solution, and add 400 mg of coenzyme NAD+ at the same time; (3) Continue the reaction for 24 h under the shaking conditions of 37 °C and 250 rpm, and then take a sample to detect the product conversion rate by HPLC.

[0061] Experimental results: The yield of pure dihydroroic acid, the 24-hour product of the hydrogenase of SEQ ID NO: 1, was 78%.

[0062] Example 2.19. The coenzyme is NADP. The specific experimental method includes the following steps: (1) Add 50 mL of isopropanol to a 500 mL reaction flask, add 1 g of the substrate royal icing, stir and dissolve at 45 °C to obtain a mixture; (2) Adjust the pH of the reaction solution to 7.5 with 1 M NaOH, add 50 mL of crude hydrogenase solution, and add 200 mg of coenzyme NADP at the same time; (3) Continue the reaction for 24 h under the shaking conditions of 37 °C and 250 rpm, and then take a sample to detect the product conversion rate by HPLC.

[0063] Experimental results: The yield of dihydroroic acid, the 24h product of hydrogenase of SEQ ID NO:1, was 7%.

[0064] Example 2.20. Coenzyme-free The specific experimental method includes the following steps: (1) Add 50 mL of isopropanol to a 500 mL reaction flask, add 1 g of substrate, stir and dissolve at 45 °C to obtain a mixture; (2) Adjust the pH of the reaction solution to 7.5 with 1 M NaOH, and add 50 mL of crude hydrogenase solution; (3) Continue the reaction for 24 h under the shaking conditions of 37 °C and 250 rpm, and then take a sample to detect the product conversion rate by HPLC.

[0065] Experimental results: The conversion rate of dihydroroic acid, the 24h product of the hydrogenase of SEQ ID NO: 1, was 0%.

[0066] Example 2.21. The hydrogenase is the T33F mutant of SEQ ID NO: 1. The specific experimental method includes the following steps: (1) Add 50 mL of isopropanol to a 500 mL reaction flask, add 1 g of the substrate royal icing, stir and dissolve at 45 °C to obtain a mixture; (2) Adjust the pH of the reaction solution to 7.5 with 1 M NaOH, and add 50 mL of crude enzyme solution of LT33F mutant hydrogenase; (3) Continue the reaction for 24 h under the shaking conditions of 37 °C and 250 rpm, and then take a sample to detect the product conversion rate by HPLC.

[0067] Experimental results: The yield of dihydroroic acid, the product of the hydrogenase T33F mutant of SEQ ID NO:1, was 23% after 24 hours.

[0068] Example 2.22. The following are the results of hydrogenation reactions of other substrates under optimal experimental conditions.

[0069] Note: The spectra of the compounds in the above examples are consistent with those of publicly reported spectra.

[0070] The purity levels are as follows: dihydroroic acid 98%; dihydrokaempferol 95%; R-dihydrokaempferol 93%; S-dihydrokaempferol 95%; tetrahydrokaempferol 90%; dihydromyricetin 98%; tetrahydromagnolin 98%; tetrahydrocurcumin 96%; and octahydrocurcumin 91%.

Claims

1. A hydrogenase, characterized in that: The amino acid sequence of the hydrogenase is shown in SEQ ID NO.1, or has at least 80%-99% similarity to the sequence shown in SEQ ID NO:

1.

2. An enzyme catalytic system, characterized in that: It contains hydrogenase and coenzyme; The amino acid sequence of the hydrogenase is shown in SEQ ID NO.1, or has at least 80%-99% similarity to the sequence shown in SEQ ID NO:1; The coenzyme is selected from nicotinamide adenine dinucleotide (NAD).

3. The application of the hydrogenase as described in claim 1 or the enzyme catalytic system as described in claim 2 in catalyzing hydrogenation / reduction reactions.

4. The application as described in claim 3, characterized in that: The substrate for the catalytic hydrogenation / reduction reaction is a compound containing a double bond.

5. The application as described in claim 3, characterized in that: In the catalytic hydrogenation / reduction reaction, the amount of hydrogenase added is 20-70% of the total reaction volume.

6. The application as described in claim 3, characterized in that: In the catalytic hydrogenation / reduction reaction, the amount of the coenzyme added is 1-4 g / L.

7. The application as described in claim 3, characterized in that: In catalytic hydrogenation / reduction reactions, the concentration of the reaction substrate is 5 g / L-15 g / L.

8. The application as described in claim 3, characterized in that: In catalytic hydrogenation / reduction reactions, the pH of the reaction is 6.0-8.

0.

9. The application as described in claim 3, characterized in that: In the catalytic hydrogenation / reduction reaction, the reaction temperature is 25℃-55℃.

10. The application as described in claim 3, characterized in that: The catalytic hydrogenation / reduction reaction is carried out in an organic solvent.

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