Curcumin / dihydrocurcumin reductase mutant and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a curcumin / dihydrocurcumin reductase mutant and application thereof. According to the curcumin / dihydrocurcumin reductase mutant obtained through screening, in the reaction of catalyzing 1, 7-bis (4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3, 5-diketone to prepare tetrahydrocurcumin, the highest catalytic efficiency can reach 1641 mM <-1 > S <-1 >, the highest catalytic efficiency is 8.16 times that of initial curcumin / dihydrocurcumin reductase, the catalytic efficiency is greatly improved, and the curcumin can be used for preparing tetrahydrocurcumin by catalyzing 1, 7-bis (4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3, 5-diketone. The production efficiency of the tetrahydrocurcumin is remarkably improved, and a novel method is provided for preparing the tetrahydrocurcumin.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to a curcumin / dihydrocurcumin reductase mutant and application thereof. Background Art

[0002] Curcumin is a well-known yellow food pigment that has the functions of protecting blood vessel health and regulating blood sugar levels. Tetrahydrocurcumin is one of the main reduced metabolites of curcumin. Since the solubility, stability and bioavailability of tetrahydrocurcumin are better than those of curcumin, and its pharmacological activity is similar, the market demand for tetrahydrocurcumin has been increasing in recent years.

[0003] At present, the synthesis of tetrahydrocurcumin mainly relies on chemical methods, and there are two common routes: 1. Using heavy metal catalyst Pd / C to hydrogenate curcumin to tetrahydrocurcumin; 2. Acylation of curcumin to diacetylcurcumin, using hydrogen or hydrogen donor in the presence of catalyst and solvent to reduce it to diacetyltetrahydrocurcumin intermediate, and then deacetylation to obtain tetrahydrocurcumin. The above chemical methods often have problems such as the use of heavy metal catalysts, large environmental pollution, and difficulty in product purification; compared with chemical methods, the bioenzymatic synthesis of tetrahydrocurcumin has the advantages of safety, high efficiency, low cost and environmental friendliness, but there are few relevant literature disclosures.

[0004] Therefore, if a biological enzyme with stronger catalytic ability can be developed for the production of tetrahydrocurcumin, it will greatly improve the industrial preparation efficiency of tetrahydrocurcumin and have important significance for the market development of tetrahydrocurcumin. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a curcumin / dihydrocurcumin reductase mutant and its application. Taking EcCurA from Escherichia coli as the starting gene, curcumin / dihydrocurcumin reductase KcCurA is determined, and a curcumin / dihydrocurcumin reductase mutant with high activity is further obtained, which shows good catalytic effect in the catalytic reaction of preparing tetrahydrocurcumin with 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (curcumin) as a substrate.

[0006] The technical solution of the present invention is as follows: The present invention provides a curcumin / dihydrocurcumin reductase mutant capable of efficiently catalyzing the reaction of 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione to prepare tetrahydrocurcumin; in addition, the amino acid sequence, nucleotide sequence, recombinant expression plasmid, genetic engineering strain and preparation method thereof corresponding to the curcumin / dihydrocurcumin reductase mutant, and the application of the curcumin / dihydrocurcumin reductase mutant in synthesizing tetrahydrocurcumin are also within the protection scope of the present invention.

[0007] The curcumin / dihydrocurcumin reductase mutant provided by the present invention is based on the EcCurA gene from Escherichia coli first reported in the literature in 2011 as the starting gene. Through BLAST analysis of the NCBI database, the enzyme activity and solubility test confirmed that it is from Cossackia Kosakoniacowanii The curcumin / dihydrocurcumin reductase KcCurA was isolated and its mutants were screened to identify the key sites affecting the enzymatic properties of the initial curcumin / dihydrocurcumin reductase, and curcumin / dihydrocurcumin reductase mutants with high activity were obtained.

[0008] Wherein, the curcumin / dihydrocurcumin reductase mutant comprises: (a) a protein having the amino acid sequence shown in SEQ ID NO: 1; or (b) a protein having at least one of the following sites of the amino acid sequence in (a): R264, V265, P266, L267, L268, M269, A270, T271, I272, L273, K274, K275 or Q286, which has undergone amino acid mutation and has the function of curcumin / dihydrocurcumin reductase; or (c) a protein having more than 80% homology with the above amino acid sequence defined in any one of (a) and (b) and having the function of curcumin / dihydrocurcumin reductase.

[0009] Furthermore, the amino acid mutation in (b) is selected from at least one of the following: R264A, R264L, R264F, R264V, R264C, R264I, R264M, V265A, V265L, V265F, V265G, P266A, P266L, P266 F, P266G, P266Y, P266W, L267A, L267F, L268A, L268F, M269A, M269L, M269F, A270L, A270F, T271A, T27 1L, T271F, I272A, I272L, I272F, L273A, L273F, K274A, K274L, K274F, K274V, K274C, K274I, K274M, K 275A, K275L, K275F, K275V, K275C, K275I, K275M, K275Y, K275W, Q286A, Q286L, Q286F, Q286Y, Q286W.

[0010] Furthermore, the amino acid mutation in (b) is selected from any one of the following: R264A, R264L, R264F, R264V, R264C, R264I, R264M, V265A, V265L, V265F, V265G, P266A, P266L, P266F, P266G, P266Y, P2 66W, L267A, L267F, L268A, L268F, M269A, M269L, M269F, A270L, A270F, T271A, T271L, T271F, I272A, I272L, I272F, L273 A. L273F, K274A, K274L, K274F, K274V, K274C, K274I, K274M, K275A, K275L, K275F, K275V, K275C, K275I, K275M, K275Y, K275W, Q286A, Q286L, Q286F, Q286Y, Q286W, K274L+R264I, K274L+R264L, K274L+V265A, K274L+V265L, K274L+P266A, K27 4L+P266L, K274L+P266F, K274L+P266Y, K274L+P266G, K274L+P266I, K274L+K275L, K274L+K275F, K274L+Q286F, K274L +Q286Y、K274L+P266F+R264I、K274L+P266F+R264L、K274L+P266F+V265A、K274L+P266F+V265L、K274L+P266F+K275L、K 274L+P266F+K275F, K274L+P266F+Q286F, K274L+P266F+Q286Y, K274L+P266F+Q286Y+R264I, K274L+P266F+Q286Y+R26 4L, K274L+P266F+Q286Y+V265A, K274L+P266F+Q286Y+V265L, K274L+P266F+Q286Y+K275L or K274L+P266F+Q286Y+K275F.

[0011] The second aspect of the present invention provides a method for preparing the above-mentioned curcumin / dihydrocurcumin reductase mutant, the preparation method comprising the following steps: site-directed mutagenesis of the initial curcumin / dihydrocurcumin reductase encoding gene, construction of an expression vector, transformation of a protein expression host bacterium, and induction of protein expression.

[0012] Furthermore, the present invention uses molecular cloning technology to obtain the coding gene of the mutant, and constructs a 6×His fusion expression vector of the mutant gene and introduces it into the genetic engineering bacteria BL21 (DE3) for induced expression to obtain the mutant enzyme protein, and uses the mutant as a catalyst and 1,7-di(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione as a substrate to carry out an enzyme catalytic reaction under appropriate conditions. The results show that the catalytic efficiency of the mutant provided by the present invention in synthesizing tetrahydrocurcumin is 8.13 times higher than that of the wild-type curcumin / dihydrocurcumin reductase, and has great application potential and value in the industrial production of tetrahydrocurcumin.

[0013] All mutation sites and any combination of mutations encoding the curcumin / dihydrocurcumin reductase mutants mentioned above in the present invention are the key protections of the present invention. All modifications made on the genes are key protections of the present invention. In addition, expression cassettes, vectors or recombinant bacteria containing the genes also fall within the technical scope protected by the present invention.

[0014] Similarly, the catalyst comprising the above-mentioned curcumin / dihydrocurcumin reductase mutant is also the technical content protected by the present invention.

[0015] Furthermore, the present invention also provides the use of the above-mentioned curcumin / dihydrocurcumin reductase mutant or a catalyst comprising the above-mentioned curcumin / dihydrocurcumin reductase mutant in the preparation of tetrahydrocurcumin, specifically, using 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione as a substrate for catalytic reaction to synthesize tetrahydrocurcumin.

[0016] The application is specifically: using the above-mentioned curcumin / dihydrocurcumin reductase mutant or a catalyst containing the above-mentioned curcumin / dihydrocurcumin reductase mutant, taking 1,7-di(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione as a substrate, carrying out a catalytic reaction under the conditions of 25-40° C. and pH=5.0-8.0, to synthesize tetrahydrocurcumin.

[0017] Furthermore, the concentration of 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione is 0.025 mM.

[0018] The beneficial effects of the present invention are: (1) The highly active curcumin / dihydrocurcumin reductase mutant screened and obtained by the present invention realizes the efficient catalysis of producing tetrahydrocurcumin using 1,7-di(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione as raw material, significantly improves the production efficiency of tetrahydrocurcumin, and provides a new method for preparing tetrahydrocurcumin; (2) The curcumin / dihydrocurcumin reductase mutant screened by the present invention has a catalytic efficiency of up to 1641 mM in the reaction of catalyzing 1,7-di(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione to prepare tetrahydrocurcumin. -1 S -1 , and its highest catalytic efficiency was 8.16 times that of the initial curcumin / dihydrocurcumin reductase, which greatly improved the catalytic efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the reaction principle of the present invention using curcumin / dihydrocurcumin reductase mutant to catalyze 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione to synthesize tetrahydrocurcumin; Figure 2 High performance liquid chromatography was used to detect the production of tetrahydrocurcumin from 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione catalyzed by KcCurA. DETAILED DESCRIPTION

[0020] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0021] The main reagents and consumables used in the present invention are: Phanta Max Super-Fidelity DNA polymerase P505 was purchased from Nanjing Novogen Biotechnology Co., Ltd.; FastDigest DpnI DNA endonuclease was purchased from ThermoFisher Scientific; pET28(a+) plasmid is a known Escherichia coli expression vector with a vector size of 5369 bp, T7 promoter, vector tag N-6×Hi, and vector resistance Kanamycin (kanamycin), purchased from Novogen; E. coli DH5α competent cells and BL21 (DE3) competent cells were purchased from Ang Yu Biotechnology Co., Ltd.; agar powder was purchased from Solarbio; tryptophan and yeast extract were purchased from OXOID.

[0022] LB medium Each 100 mL of LB medium contains: 1 g tryptone, 0.5 g yeast extract and 1 g NaCl, pH 7.0-7.2.

[0023] Preparation method: Dissolve 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl in 1 L of distilled water, do not adjust the pH value, and sterilize with high pressure steam at 121°C for 20 min. If preparing solid culture medium, add 1.8 g of agar powder to 100 mL of culture medium.

[0024] Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, which can be obtained commercially or prepared according to conventional methods in the art, and the specifications are laboratory pure grade. Unless otherwise specified, the methods used in the following examples are all conventional methods in the art, and the experimental conditions used are all conventional experimental conditions in the art, and reference can be made to the relevant experimental manuals or manufacturer's instructions.

[0025] Example 1 Preparation of curcumin / dihydrocurcumin reductase mutants 1. Design of curcumin / dihydrocurcumin reductase mutants Through gene mining, the gene of curcumin / dihydrocurcumin reductase KcCurA was screened from the NCBI database. The open reading frame of the gene is 1038 bp in length. The curcumin / dihydrocurcumin reductase encoded by it consists of 345 amino acids, and its amino acid sequence (345 aa) is shown in SEQ ID NO:1.

[0026] The nucleotide sequence encoding the curcumin / dihydrocurcumin reductase is shown in SEQ ID NO: 2.

[0027] In this example, molecular docking of the substrate 1,7-di(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione was performed according to the structure of curcumin / dihydrocurcumin reductase KcCurA by homology modeling. The docking results were analyzed, and 13 residues near the active neutral that may affect the catalytic properties of the protein were selected. These residues included R264, V265, P266, L267, L268, M269, A270, T271, I272, L273, K274, K275 or Q286. These residues were subjected to site-directed mutagenesis to obtain mutants with higher enzyme activity, i.e., the mutants provided by the present invention, which exhibited better catalytic performance and enzyme activity.

[0028] 2. Obtaining the Curcumin / Dihydrocurcumin Reductase Mutant Gene The gene of the curcumin / dihydrocurcumin reductase mutant obtained above can be obtained by a total gene synthesis method or a molecular cloning method.

[0029] The curcumin / dihydrocurcumin reductase KcCurA gene obtained by the whole gene synthesis method in our laboratory was used to obtain the curcumin / dihydrocurcumin reductase mutant gene by PCR. The specific steps are as follows: 1. Obtaining the complete gene of initial curcumin / dihydrocurcumin reductase Curcumin / dihydrocurcumin reductase KcCurA has been synthesized in our laboratory, and the above gene was synthesized by Nanjing GenScript Biotechnology Co., Ltd. and ligated into plasmid pET28a(+).

[0030] 2. Site-directed mutagenesis of curcumin / dihydrocurcumin reductase (1) Design of primers for site-directed mutagenesis (2) Site-directed mutagenesis of curcumin / dihydrocurcumin reductase Using the pET28a(+) plasmid containing the original curcumin / dihydrocurcumin reductase gene as a template, the curcumin / dihydrocurcumin reductase mutant gene was amplified according to the following PCR system and procedure using upstream primers and downstream primers containing mutation sites designed according to the template.

[0031] PCR system: Phanta Max Super-Fidelity DNA polymerase P520 20 μL, template plasmid 1 μL, upstream primer-F (10 μM) 1 μL, downstream primer-R (10 μM) 1 μL, dd H 2 O 2 μL.

[0032] The PCR program was as follows: a. pre-denaturation at 98°C for 30 s; b. denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C for 40 s, and 32 cycles; c. extension at 72°C for 5 min, cooling to 4°C, and digestion of the template plasmid containing the initial curcumin / dihydrocurcumin reductase gene with DpnI enzyme at 37°C for 30 min.

[0033] (3) Transformation of DH5α competent cells The digestion product of the pET28a(+) plasmid containing the curcumin / dihydrocurcumin reductase mutant obtained in (2) was transformed into DH5α competent cells.

[0034] Place DH5α competent cells on ice, add 10 μL of plasmid solution after the cells thaw, place on ice for 30 minutes, heat shock at 42°C for 45 seconds, then place on ice for 2 minutes, add 700 μL of sterile LB liquid culture medium, culture in a shaker at 37°C and 200 rpm for 1 hour, centrifuge at 4000 rpm for 1 min, discard 600 μL of supernatant, resuspend the precipitated bacteria with the remaining supernatant and spread all on LB plate culture medium containing Kana resistance (50 μg / mL), and culture inverted at 37°C overnight.

[0035] (4) Positive clone screening A single colony was picked from the LB plate and inoculated into LB liquid medium containing Kana resistance (50 μg / mL), cultured overnight at 37°C with a shaker at 200 rpm, and then sent to Anshengda (Suzhou, China) for sequencing to identify whether the initial curcumin / dihydrocurcumin reductase was successfully mutated.

[0036] 3. Heterologous expression of curcumin / dihydrocurcumin reductase mutant 6×His fusion protein (1) Transformation of competent cells for protein expression BL21 (DE3) competent cells were taken out from the -80°C ultra-low temperature refrigerator and placed on ice. After the cells thawed, 1 μL of pET28a (+) plasmid containing curcumin / dihydrocurcumin reductase mutant was added to each competent cell, placed on ice for 30 min, heat shocked at 42°C for 45 s, then placed on ice for 2 min, added 700 μL of sterile LB liquid culture medium, and cultured at 37°C and 200 rpm for 1 h. 200 μL of the culture solution was aspirated and spread on LB plate culture medium containing Kana resistance (50 μg / mL), and inverted cultured at 37°C overnight.

[0037] (2) Preservation of curcumin / dihydrocurcumin reductase mutant expression strains A single colony was picked from the LB plate and inoculated into a test tube containing LB liquid medium containing Kana resistance (50 μg / mL), and cultured overnight at 37°C and 200 rpm in a shaking incubator to be used as an expression strain for the curcumin / dihydrocurcumin reductase mutant. Sterile glycerol was added to the bacterial solution at a final volume concentration of 15%, and the bacterial solution was stored in a -80°C ultra-low temperature refrigerator for a long time.

[0038] (3) Protein expression Inoculate the overnight cultured test tube bacterial liquid into 50 mL sterile LB liquid culture medium with an inoculum size of 1% and a final kanamycin concentration of 50 μg / mL. Culture at 37°C and 200 rpm for about 2.5 h. When OD600 = 0.6-0.8, add sterile IPTG with a final concentration of 0.1 mM. Induce the culture at 28°C and 200 rpm for 12 h, and then collect the bacteria by centrifugation at 8000 rpm for 8 min.

[0039] Example 2 Synthesis of Tetrahydrocurcumin Using the Curcumin / Dihydrocurcumin Reductase Mutant Provided by the Present Invention In this embodiment, a reaction principle diagram of the synthesis of tetrahydrocurcumin by 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione catalyzed by curcumin / dihydrocurcumin reductase mutant (NADPH) is shown in the attached figure. Figure 1 The specific operations are as follows: 10 μL of purified enzyme solution, 10 μL of substrate 1,7-di(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, and 20 μL of coenzyme NADPH were added to a 96-well plate in sequence, and the system was filled with 200 μL of buffer, and the reaction was carried out at 30°C.

[0040] Enzyme activity assay: The assay system for curcumin / dihydrocurcumin reductase activity is as follows: 160 μL phosphate buffer (100 mM, pH 6.0), 20 μL NADPH (1.5 mM), 10 μL 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (0.5 mM), 10 μL purified mutant enzyme solution, the mixed system was added to a 96-well plate, and the decrease in absorbance at 430 nm within 6 min at 30 °C was detected by a microplate reader, and the amount of curcumin / dihydrocurcumin reductase consumed in the reaction was calculated based on the standard curve.

[0041] One enzyme activity unit U involved in the present invention is defined as: the amount of enzyme required to consume 1 μmol of 1,7-di(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione within 1 minute.

[0042] The enzymatic activities of the original curcumin / dihydrocurcumin reductase and the mutants are shown in Table 1.

[0043] Table 1 Enzyme activities of initial curcumin / dihydrocurcumin reductase and mutants Types of enzymes Specific activity (U / mg) <![CDATA[Conversion number (S -1 )]]> Michaelis constant (Mm) <![CDATA[Catalytic efficiency (mM -1 S -1 )]]> Relative catalytic efficiency Curcumin / dihydrocurcumin reductase 2.29±0.04 5.18±0.46 0.03±0.004 201.89±50.42 1.00 K274L 5.95±0.31 14.31±0.63 0.03±0.002 507.38±59.86 2.51 K274L / P266F 7.44±0.09 15.53±1.24 0.01±0.002 1141.07±265.65 5.65 K274L / P266F / Q286Y 6.31±0.19 12.12±0.42 0.01±0.000 1641.28±214.81 8.12 K274L / P266F / Q286Y / V265L 6.14±0.00 12.75±1.04 0.01±0.001 1115.08±267.35 5.52

[0044] Example 3: Use of crude enzyme solutions of curcumin / dihydrocurcumin reductase KcCurA and alcohol dehydrogenase ADH for enzymatic preparation of tetrahydrocurcumin The catalytic reaction system is as follows: 20 g / L KcCurA crude enzyme solution, 20 g / L ADH crude enzyme solution, 20 g / L curcumin, 0.2 g / L NADP + , 2.5% (v / v) isopropanol, 1% Tween 80. The above 20 mL of the reaction solution was placed in a 50 mL round-bottom flask, and stirred with magnetic stirring in a constant temperature water bath at 38° C. for 24 h.

[0045] The catalytic reaction liquid was used for HPLC analysis to obtain the yield of tetrahydrocurcumin. The production of tetrahydrocurcumin in the obtained product was detected by high performance liquid chromatography (HPLC). The results of HPLC are shown in the attached Figure 2 As shown, Figure 2 It can be seen that the retention time of tetrahydrocurcumin is 8.03 min, which is basically completely converted into tetrahydrocurcumin.

[0046] The present invention verifies the specific activity of curcumin / dihydrocurcumin reductase and mutants with other amino acid sequences obtained by the method of the present invention, among which the mutant with the highest catalytic efficiency is 8.16 times that of the initial curcumin / dihydrocurcumin reductase. The experimental results show that the catalytic efficiency of the remaining positive mutant enzymes is higher than 201mM - 1 S -1 , which shows that the mutant has a good catalytic effect in the reaction of catalyzing 1,7-di(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione to synthesize tetrahydrocurcumin.

[0047] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field within the technical scope disclosed by the present invention and the technical solution and inventive concept thereof, which are equivalently replaced or changed, should be covered by the protection scope of the present invention.

Claims

1. A curcumin / dihydrocurcumin reductase mutant, characterized in that: Includes any of the following (a) to (c): (a) a protein having the amino acid sequence shown in SEQ ID NO: 1; (b) a protein having at least one of the following positions: R264, V265, P266, L267, L268, M269, A270, T271, I272, L273, K274, K275, Q286 of the amino acid sequence described in (a) with amino acid mutation and having the function of curcumin / dihydrocurcumin reductase; (c) A protein having 80% or more homology with the amino acid sequence defined in any one of (a) and (b) and having the function of curcumin / dihydrocurcumin reductase.

2. The coding gene of the curcumin / dihydrocurcumin reductase mutant according to claim 1.

3. The gene encoding the curcumin / dihydrocurcumin reductase mutant according to claim 2, characterized in that: The nucleotide sequence of the encoding gene is shown in SEQ ID NO:

2.

4. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the coding gene according to claim 2 or 3.

5. A recombinant genetically engineered bacterium, characterized in that: The recombinant genetically engineered bacteria is obtained by transforming the recombinant expression vector described in claim 4 into a host microorganism.

6. A catalyst, characterized in that The active ingredient of the catalyst comprises the curcumin / dihydrocurcumin reductase mutant according to claim 1.

7. Use of the catalyst according to claim 6 in the preparation of tetrahydrocurcumin.

8. The use of a catalyst as claimed in claim 7 in the preparation of tetrahydrocurcumin, characterized in that: Tetrahydrocurcumin was prepared from 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione.

9. Use of the curcumin / dihydrocurcumin reductase mutant according to claim 1 in the preparation of tetrahydrocurcumin.

10. The use of the curcumin / dihydrocurcumin reductase mutant in the preparation of tetrahydrocurcumin as claimed in claim 9, characterized in that: Tetrahydrocurcumin was prepared from 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione.

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