A method for synthesizing dihydrochalcone using chalcone reductase

Abstract

The invention discloses a method for synthesizing dihydrochalcone by using chalcone reductase, wherein chalcone is used as a substrate, and its carbon-carbon double bond is selectively reduced by a chalcone reductase catalytic system to synthesize dihydrochalcone, wherein the catalytic system is composed of a chalcone reductase and a cofactor regeneration system. The method for synthesizing dihydrochalcone by using chalcone reductase as a catalyst in the invention has a reasonable design, and has the advantages of simple reaction steps, mild reaction conditions, environmental friendliness, high catalytic efficiency, simple enzyme purification, good stability, etc., and has good application development prospects in the field of industrial synthesis of dihydrochalcone and related drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical engineering, and specifically relates to a method for synthesizing dihydrochalcone by using chalcone reductase, and is a method for obtaining dihydrochalcone by using chalcone as a catalyst to selectively reduce the carbon-carbon double bond of chalcone. Background Art

[0002] Dihydrochalcone and its derivatives not only have important application value as low-energy sweeteners in the fields of beverage, candy and food manufacturing, but also have anti-inflammatory, anti-tumor, antibacterial and hypoglycemic effects, and are a class of biologically active substances with great development prospects. Dihydrochalcone and its derivatives are mainly derived from plant secondary metabolism, and their content in plants is relatively low. At present, chemical synthesis is still their main source, among which the selective reduction of the carbon-carbon double bond of chalcone is an important synthetic step. Therefore, dihydrochalcone is the most important intermediate compound for the preparation of its derivatives. However, the carbon-carbon double bond in the chalcone structure is conjugated with the adjacent carbonyl group to form an α, β-unsaturated carbonyl structural unit. When reduced by chemical methods, the carbonyl and double bond are very likely to compete to generate 1,4- and 1,2-reduced mixtures, and it is challenging to achieve regional selectivity; the chemical method mainly uses transition metals as catalysts, which are expensive, and the reaction conditions are harsh and easy to pollute the environment. Compared with the chemical method, enzyme catalysis has the advantages of high efficiency, environmental protection, mild reaction conditions and good chemical selectivity. Currently, double bond reductases that catalyze carbon-carbon double bonds are limited by factors such as their substrate spectrum, catalytic efficiency, enzyme yield and stability. New and efficient double bond reductases that can be used for chalcone double bond reduction are still urgently needed to be developed.

[0003] Chalcone reductase (CHR), also known as chalcone polyketide reductase (CHR), is a member of the AKR4 family of the Aldo-Keto Reductase (AKR) superfamily. It participates in the biosynthesis of plant flavonoids and isoflavonoids and catalyzes the production of dehydrochalcone in collaboration with chalcone synthase (Bomati EK, Austin MB, Bowman ME, Dixon RA, Noel JP. Structural elucidation of chalcone reductase and implications for deoxychalcone biosynthesis. J Biol Chem 2005, 280, 30496-30503.). However, the natural substrate and substrate spectrum of CHR have not been determined so far. Summary of the invention

[0004] The present invention aims to provide a method for synthesizing dihydrochalcone by using chalcone reductase, which is a method for selectively reducing the carbon-carbon double bond of chalcone to generate dihydrochalcone by using chalcone reductase. The method has the advantages of good chemical selectivity, high conversion efficiency, simple reaction steps, etc.

[0005] The invention provides a method for synthesizing dihydrochalcone by using chalcone reductase, which takes chalcone as substrate and obtains the corresponding dihydrochalcone through catalysis of an enzyme catalysis system, wherein the enzyme catalysis system consists of a chalcone reductase and a coenzyme regeneration system.

[0006] The specific steps are: using chalcone as a raw material and reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor, the carbon-carbon double bond is reduced to generate the corresponding dihydrochalcone under the selective reduction catalysis of chalcone reductase in the enzyme catalytic system, and the reaction formula is as follows:

[0007]

[0008] During the reaction, NADPH is oxidized to nicotinamide adenine dinucleotide phosphate (NADP + ), but due to the high price of NADPH, NADP + Regeneration into NADPH can significantly reduce reaction costs.

[0009] In the present invention, the cofactor regeneration system comprises: glucose dehydrogenase as a cofactor regeneration enzyme, glucose as a cofactor regeneration substrate, NADPH and NADP + Glucose dehydrogenase cofactor regeneration system.

[0010] The enzyme catalysis system of the present invention comprises chalcone reductase, glucose dehydrogenase, glucose and trace NADP + The chalcone reductase and glucose dehydrogenase in the enzyme catalysis system are purified to be free pure enzymes. When chalcone is added to the enzyme reaction system, glucose dehydrogenase oxidizes glucose to generate gluconic acid and NADPH under the conditions of controlling pH and temperature. The generated NADPH can act as a cofactor to participate in the chalcone reductase-catalyzed chalcone double bond reduction reaction to generate dihydrochalcone and NADP. + , while NADP + It further participates in the glucose oxidation reaction, so it can continuously obtain NADPH in a cycle, participate in the chalcone reduction reaction, and obtain dihydrochalcone. The reaction formula is as follows:

[0011]

[0012] Specifically, the chalcone reductase is derived from the plant alfalfa (Medicago sativa), and the nucleotide sequence encoding the chalcone reductase is derived from GenBank, with the serial number U13925.1. After codon optimization, it is shown as CHR-DNA (SEQ ID No. 1) in the sequence table.

[0013] Specifically, the amino acid sequence of chalcone reductase is shown as CHR-AA (SEQ ID No. 2) in the sequence listing.

[0014] As known to those skilled in the art, the nucleotide sequence of the chalcone reductase gene of the present invention may also be any other nucleotide sequence encoding the amino acid sequence shown in CHR-AA (SEQ ID No. 2) in the sequence table.

[0015] Any nucleotide sequence obtained by substituting, confirming or inserting one or more nucleotides into the nucleotide sequence shown by CHR-DNA, as long as it has more than 90% homology with the nucleotide sequence, belongs to the protection scope of the present invention.

[0016] Any deletion, insertion or substitution of one or more amino acids in the amino acid sequence represented by CHR-AA and having chalcone reductase activity still falls within the protection scope of the present invention.

[0017] Specifically, the glucose dehydrogenase sequence is derived from Bacillus megaterium IAM1030, and the nucleotide sequence encoding the glucose dehydrogenase is derived from GenBank, numbered D10626.1, as shown in GDH-DNA (SEQ ID No. 3) in the sequence table, and is obtained by whole gene synthesis.

[0018] Specifically, the amino acid sequence of glucose dehydrogenase is shown as GDH-AA (SEQ ID No. 4) in the sequence listing.

[0019] Preferably, in the catalytic system, the added amounts of the chalcone reductase and glucose dehydrogenase are 0.1-5 mg / ml and 0.1-3 mg / ml respectively.

[0020] In the catalytic system, the addition amount of substrate chalcone is 0.1-15 mM; the addition amount of coenzyme regeneration substrate is 0.15-20 mM.

[0021] Preferably, in the enzyme catalysis system, the reaction temperature is 20-45°C, and the reaction time is 0.5-24h; more preferably, the temperature is 30-40°C, and the reaction time is 2-10h.

[0022] Preferably, the pH value of the reaction is controlled to be 6-9, sodium hydroxide is used to control the pH drop, and formic acid is used to control the pH rise.

[0023] The beneficial effects of the present invention are mainly reflected in: providing a method for synthesizing dihydrochalcone by selectively reducing the carbon-carbon double bond of chalcone using chalcone reductase as a biocatalyst, which has not been reported yet; the conversion rate of the chalcone reductase catalytic system described in the present invention can reach more than 90%, the purified free enzyme can be stored at 4°C for more than 7 days, the heterologous expression amount of Escherichia coli can reach 30 mg / liter of culture medium, and the free enzyme with a purity of more than 95% can be obtained by one-step purification by nickel affinity chromatography; at the same time, the reaction is carried out at a pH value close to neutral and at room temperature, and no transition metal and organic solvent are required to participate in the reaction, so it has the characteristics of high catalytic efficiency, good thermal stability, high heterologous expression amount, easy purification, etc., and also has the advantages of biocatalysis such as mild reaction conditions and environmental friendliness, and has good industrial application development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the SDS-PAGE picture of purified chalcone reductase.

[0025] Figure 2 This is the HPLC chromatogram of chalcone blank control.

[0026] Figure 3 This is the HPLC chromatogram of the reaction solution after 10 hours of reaction in the chalcone reductase catalytic system. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0028] The experimental methods in the present invention are all conventional methods unless otherwise specified.

[0029] The plasmid extraction kit in the present invention was purchased from Axygen Hangzhou Co., Ltd.; E. coli DH5α, E. coli BL21 (DE3) and the like were purchased from Novagen; and the prestained protein Maker was purchased from Fermantas.

[0030] NADPH and NADP used in the present invention + All were purchased from Shanghai Yishen Biological Co., Ltd.; other commonly used reagents, including substrates, were purchased from Aladdin Chemical Reagent Co., Ltd. and Shanghai Yuanye Biotechnology Co., Ltd.

[0031] Example 1 Expression of chalcone reductase

[0032] The sequence of the gene encoding alfalfa (Medicago sativa) chalcone reductase (GenBank: U13925.1) was fully synthesized by Sangon Biotech (Shanghai) Co., Ltd. after codon optimization (sequence as SEQ ID NO.1) and then connected to the pET-28a (+) vector to construct a CHR-pET-28a (+) plasmid. After sequencing and verifying the sequence, it was heat-shock transformed into E. coli BL21 (DE3) competent cells to obtain chalcone reductase-expressing engineered bacteria. A single colony was picked from an LB plate medium containing 50 μg / ml kanamycin and inoculated into a TB liquid medium containing 50 μg / ml kanamycin. After shaking and culturing at 37°C and 200 rpm for 12 hours, it was transferred to 3L liquid TB medium for expansion culture. Shaking and culturing at 37°C and 200 rpm was continued for 12 hours. When the optical density OD of the culture solution reached 0.04, the expression of the gene was detected by Western blotting. 600 When it reached 0.6, the temperature was lowered to 16°C, and an IPTG solution with a final concentration of 1.0 mM was added to induce expression for 12 h. The culture solution was centrifuged at 4000 rpm for 25 min, the supernatant culture solution was discarded, and the bacteria were stored at -20°C for later use.

[0033] Example 2 Expression of glucose dehydrogenase

[0034] In the previous research work, the applicant's laboratory has completed the construction of Bacillus megaterium glucose dehydrogenase plasmid (Sun Lianli, Chen Yuanyuan, Shao Nana, Zhou Yun, A method for synthesizing chiral alcohols using perakine reductase, Chinese patent, ZL201811521466.0; Cai S, Shao N, Chen Y, Li A, Pan J, Zhu H, Zou H, Zeng S, Sun L, Zhao J. Enantioselective reduction of α, β-unsaturated ketones and aryl ketones by perakine reductase. Org. Lett. 2019, 21, 4411-4414.) The nucleotide sequence is shown in the sequence table SEQ ID NO.3, and the amino acid sequence is shown in the sequence table SEQ ID NO.4. Heat shock transformation to E. coli BL21 (DE3) competent cells to obtain glucose dehydrogenase expression engineering bacteria. The engineered bacteria expressing glucose dehydrogenase were inoculated into LB medium containing 50 μg / ml kanamycin and cultured at 37°C for 12 h. The culture was transferred to 1 L LB medium containing the same concentration of kanamycin. When the optical density of the culture solution was OD 600When it reached 0.6, IPTG was added at a final concentration of 0.1 mM and induced at 20°C for 16 h. The culture solution was centrifuged at 8000 rpm for 10 min, the supernatant medium was discarded, and the bacteria were stored at -20°C for later use.

[0035] Example 3 Purification of chalcone reductase and glucose dehydrogenase

[0036] 3 g of chalcone reductase-expressing engineered bacteria or glucose dehydrogenase-expressing engineered bacteria were resuspended in 20 ml of lysis buffer (20 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, Tris-HCl pH 8.0). After shaking, lysozyme (1 mg / ml) was added, ice-bathed for 40 min, and ultrasonically disrupted for 3 times, 3 min / time, with an interval of 15 min each time, and centrifuged at 22000×g for 50 min. The resulting supernatant was the crude enzyme solution. Ni-NTA was used as the purification material, the column volume was 3 ml, 15 ml of lysis buffer was used to balance the Ni-NTA column, the crude enzyme solution was loaded at a rate of 1 ml / min, the non-adsorbed protein was eluted with lysis buffer (20 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0), and the target protein was eluted with elution buffer (250 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 8.0) to collect the target protein, and the target protein was dialyzed with 5 L Kpi buffer (50 mM KH2PO4, 50 mM K2HPO4, pH 7.0) to remove salt and imidazole. The results of SDS-PAGE analysis showed that the purified yields of chalcone reductase and glucose dehydrogenase could reach 30 mg / L culture medium and 57 mg / L culture medium, respectively, and the purity was above 95% ( Figure 1 ), and both enzymes were relatively stable. Compared with the freshly prepared enzymes, the relative enzyme activity could still be maintained above 90% after being stored at 4°C for 7 days.

[0037] Example 4 Synthesis of dihydrochalcone by chalcone reductase

[0038] The pure chalcone reductase and glucose dehydrogenase obtained in Example 3 were added to the reaction system at a concentration of 2 mg / ml, and 50 mM pH 7.0 Kpi was used as a buffer, and 0.8 mM chalcone, 1.2 mM glucose, and 0.02 mM NADP were added respectively. +After the reaction was carried out at 30°C with constant temperature shaking (666rpm) for 10 hours, an equal volume of methanol was added to terminate the reaction. The reaction solution was centrifuged at 12000rpm for 30 minutes, and the supernatant was sampled for high performance liquid chromatography (HPLC) to analyze the amount of substrate and product. The HPLC analysis method is: Agilent high performance liquid chromatography 1260; chromatographic column Agilent 5HC-C18 250*4.6mm; column temperature 30°C; flow rate 1ml / min; detection wavelength 254nm (substrate chalcone conversion detection), 214nm (product dihydrochalcone yield detection); mobile phase: water 55%, acetonitrile 45%. The conversion rate and yield of chalcone reductase-catalyzed reduction of chalcone carbon-carbon double bonds were calculated using the concentration curves of chalcone and dihydrochalcone standards. It was calculated that the conversion rate can reach 90%. The corresponding high performance liquid chromatogram above is shown in Figure 2 and Figure 3 .

[0039] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims. Sequence Listing <110> Zhejiang University <120> A method for synthesizing dihydrochalcone using chalcone reductase <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 939 <212> DNA <213> Artificial sequence (Unknown) <400> 1 atgggcagcg ttgaaatccc gaccaaagtt ctgaccaaca cctctagcca gctgaaaatg 60 ccggttgttg gtatgggttc tgctccggat ttcacctgta aaaaagatac caaagatgcg 120 atcatcgaag cgatcaaaca gggctaccgt cacttcgata ccgcggcggc ttatggtagc 180 gaacaggcgc tgggcgaagc gctgaaagaa gcgatcgaac tgggcctggt tacccgtgat 240 gatctcttcg ttacctctaa actgtgggtt accgaaaacc acccgcacct ggttatcccg 300 gcgctgcaga aatctctgaa aaccctgcag ctggattacc tggatctgta cctgatccac 360 tggccgctga gcagccagcc gggtaaattc agcttcccga tcgatgttgc ggatctgctg 420 ccgtttgatg ttaaaggcgt gtgggaatcc atggaagaaa gcctgaaact gggtctgacc 480 aaagcgatcg gtgtttctaa cttcagcgtt aaaaaactgg aaaacctgct gagcgttgcg 540 accgttctgc cggcagttaa ccaggttgaa atgaacctgg cgtggcagca gaaaaaactg 600 cgtgaattct gtaacgcgca cggtatcgtt ctgaccgcgt tcagcccggt tcgtaaaggt 660 gcgagccgtg gtccgaacga agttatggaa aacgatatgc tgaaagaaat tgcggatgcg 720 cacggtaaaa gcgttgcaca gatctctctg cgttggctgt acgaacaggg tgttaccttc 780 gttccgaaat cctacgataa agaacgtatg aaccagaacc tgcgtatctt cgattggagc 840 ctgaccaaag aagatcacga aaaaatcgcg cagatcaaac agaaccgtct gattccgggc 900 ccgaccaaac cgggtctgaa cgatctgtac gatgattaa 939 <210> 2 <211> 312 <212> PRT <213> Medicago sativa <400> 2 Met Gly Ser Val Glu Ile Pro Thr Lys Val Leu Thr Asn Thr Ser Ser 1 5 10 15 Gln Leu Lys Met Pro Val Val Gly Met Gly Ser Ala Pro Asp Phe Thr 20 25 30 Cys Lys Lys Asp Thr Lys Asp Ala Ile Ile Glu Ala Ile Lys Gln Gly 35 40 45 Tyr Arg His Phe Asp Thr Ala Ala Ala Tyr Gly Ser Glu Gln Ala Leu 50 55 60 Gly Glu Ala Leu Lys Glu Ala Ile Glu Leu Gly Leu Val Thr Arg Asp 65 70 75 80 Glu Leu Phe Val Thr Ser Lys Leu Trp Val Thr Glu Asn His Pro His 85 90 95 Leu Val Ile Pro Ala Leu Gln Lys Ser Leu Lys Thr Leu Gln Leu Asp 100 105 110 Tyr Leu Asp Leu Tyr Leu Ile His Trp Pro Leu Ser Ser Gln Pro Gly 115 120 125 Lys Phe Thr Phe Pro Ile Asp Val Ala Asp Leu Leu Pro Phe Asp Val 130 135 140 Lys Gly Val Trp Glu Ser Met Glu Glu Ser Leu Lys Leu Gly Leu Thr 145 150 155 160 Lys Ala Ile Gly Val Ser Asn Phe Ser Val Lys Lys Leu Glu Asn Leu 165 170 175 Leu Ser Val Ala Thr Val Leu Pro Ala Val Asn Gln Val Glu Met Asn 180 185 190 Leu Ala Trp Gln Gln Lys Lys Leu Arg Glu Phe Cys Asn Ala His Gly 195 200 205 Ile Val Leu Thr Ala Phe Ser Pro Leu Arg Lys Gly Ala Ser Arg Gly 210 215 220 Pro Asn Glu Val Met Glu Asn Asp Met Leu Lys Glu Ile Ala Asp Ala 225 230 235 240 His Gly Lys Ser Val Ala Gln Ile Ser Leu Arg Trp Leu Tyr Glu Gln 245 250 255 Gly Val Thr Phe Val Pro Lys Ser Tyr Asp Lys Glu Arg Met Asn Gln 260 265 270 Asn Leu Arg Ile Phe Asp Trp Ser Leu Thr Lys Glu Asp His Glu Lys 275 280 285 Ile Asp Gln Ile Lys Gln Asn Arg Leu Ile Pro Gly Pro Thr Lys Pro 290 295 300 Gly Leu Asn Asp Leu Tyr Asp Asp 305 310 <210> 3 <211> 786 <212> DNA <213> Artificial sequence (Unknown) <400> 3 atgtatacag atttaaaaga taaagtagtt gtaattacag gtggatcaac aggtttagga 60 cgtgcaatgg ctgttcgttt cggtcaagaa gaagcaaaag ttgttattaa ctattacaac 120 aatgaagaag aagctttaga tgcgaaaaaa gaagtagaag aagcaggcgg acaagcaatc 180 atcgttcaag gcgacgtaac aaaagaagaa gacgttgtaa accttgttca aacagctatt 240 aaagaattcg gaacattaga cgttatgatt aataacgctg gtgttgaaaa cccagttcct 300 tctcatgagc tatctttaga caactggaac aaagttattg atacaaactt aacaggtgca 360 ttcttaggaa gccgtgaagc aattaaatat ttcgttgaaa atgacattaa aggaaacgtt 420 attaacatgt ccagcgttca cgaaatgatt ccttggccat tatttgttca ctacgcagca 480 agtaaaggcg gtatgaaact aatgacggaa acattggctc ttgaatatgc gccaaaaggt 540 atccgagtaa atacattgg accaggtgcg atgacacac cattaacgc tgaaaaatttc 600 gctgatcctg tacaacgtgc agacgtagaa agcatgattc caatgggtta catcggtaag 660 ccagaagaag tagcagcagt tgcagcattc ttagcatcat cacaagcaag ctatgtaaca 720 ggtattacat tatttgctga tggtggtatg acgaatacc cttctttcca agcaggaaga 780 ggcta 786 <210> 4 <211> 261 <212> PRT <213> Bacillus megaterium <400> 4 Met Tyr Thr Asp Leu Lys Asp Lys Val Val Val Ile Thr Gly Gly Ser 1 5 10 15 Thr Gly Leu Gly Arg Ala Met Ala Val Arg Phe Gly Gln Glu Glu Ala 20 25 30 Lys Val Val Ile Asn Tyr Tyr Asn Asn Glu Glu Glu Ala Leu Asp Ala 35 40 45 Lys Lys Glu Val Glu Glu Ala Gly Gly Gln Ala Ile Ile Val Gln Gly 50 55 60 Asp Val Thr Lys Glu Glu Asp Val Val Asn Leu Val Gln Thr Ala Ile 65 70 75 80 Lys Glu Phe Gly Thr Leu Asp Val Met Ile Asn Asn Ala Gly Val Glu 85 90 95 Asn Pro Val Pro Ser His Glu Leu Ser Leu Asp Asn Trp Asn Lys Val 100 105 110 Ile Asp Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile 115 120 125 Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser 130 135 140 Ser Val His Glu Met Ile Pro Trp Pro Leu Phe Val His Tyr Ala Ala 145 150 155 160 Ser Lys Gly Gly Met Lys Leu Met Thr Glu Thr Leu Ala Leu Glu Tyr 165 170 175 Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Met Asn 180 185 190 Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Val Gln Arg Ala Asp 195 200 205 Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Lys Pro Glu Glu Val 210 215 220 Ala Ala Val Ala Ala Phe Leu Ala Ser Ser Gln Ala Ser Tyr Val Thr 225 230 235 240 Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Lys Tyr Pro Ser Phe 245 250 255 Gln Ala Gly Arg Gly 260

Claims

1. A method for synthesizing dihydrochalcone using chalcone reductase, characterized in that: The corresponding dihydrochalcone is obtained by using chalcone as a substrate through catalysis of an enzyme catalytic system. The enzyme catalytic system is composed of a chalcone reductase and a cofactor regeneration system. The method is specifically implemented by the following steps: using chalcone as a raw material and NADPH as a cofactor, reducing its carbon-carbon double bond under the selective reduction catalysis of the chalcone reductase in the enzyme catalytic system to generate the corresponding dihydrochalcone, and the reaction formula is: During the reaction, NADPH is oxidized to NADP + , through the cofactor regeneration system to NADP + Regenerated into NADPH; The chalcone reductase is derived from the plant alfalfa ( Medicago sativa ), the nucleotide sequence encoding chalcone reductase is shown in SEQ ID No.1, and the amino acid sequence thereof is shown in SEQ ID No.2; The cofactor regeneration system comprises: glucose dehydrogenase as a cofactor regeneration enzyme, glucose as a cofactor regeneration substrate, and NADP + , in NADP + In the presence of glucose, glucose dehydrogenase oxidizes glucose to produce gluconic acid and NADPH, and the generated NADPH acts as a cofactor to participate in the chalcone reductase-catalyzed chalcone double bond reduction reaction to produce dihydrochalcone and NADP + , while NADP + Further participate in the glucose oxidation reaction and further circulate to obtain NADPH. The reaction formula is as follows: 。 2. A method for synthesizing dihydrochalcone using chalcone reductase according to claim 1, characterized in that: The enzyme catalysis system comprises chalcone reductase, glucose dehydrogenase, glucose and trace NADP + In the enzyme catalysis system, the chalcone reductase and the cofactor regeneration enzyme are purified to be free pure enzymes.

3. A method for synthesizing dihydrochalcone using chalcone reductase according to claim 1, characterized in that: The glucose dehydrogenase is derived from Bacillus megaterium ( Bacillus megaterium ) Glucose dehydrogenase from IAM1030.

4. A method for synthesizing dihydrochalcone using chalcone reductase according to claim 1, characterized in that: In the enzyme catalysis system, the added amounts of the chalcone reductase and glucose dehydrogenase are both 0.1-5 mg / ml.

5. A method for synthesizing dihydrochalcone using chalcone reductase according to claim 1, characterized in that: In the enzyme catalytic system, the addition amount of substrate chalcone is 0.1~15 mM; the addition amount of cofactor regeneration substrate is 0.15~20 mM, the reaction temperature is 20~45℃, and the reaction time is 0.5~24 h.

6. A method for synthesizing dihydrochalcone using chalcone reductase according to claim 1, characterized in that: The pH value of the reaction was controlled to be between 6 and 9.

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