Enzymatic reaction catalyst and application thereof in synthesis of di-O-beta-d-glucoside

Abstract

The invention relates to an enzymatic reaction catalyst and application thereof in synthesis of di-O-beta-d-glucoside, and relates to the technical field of biological catalysis. The enzymatic reaction catalyst comprises glycosyl transferase and / or sucrose synthase, the amino acid sequence of the glycosyl transferase is as shown in SEQ ID NO: 2, and the amino acid sequence of the sucrose synthase is as shown in SEQ ID NO: 4. According to the invention, glycosyltransferase and sucrose synthase are used as enzymatic reaction catalysts, and glycosyltransferase can be used for glycosylation of 4'hydroxyl of quercetin-3-O-beta-D-glucoside and also can be used for glycosylation of 7 and 4 'hydroxyl of quercetin; the sucrose synthase can catalyze decomposition of sucrose to generate glucose, and the glucose can be combined with UDP to generate UDPG; the invention discloses a method for preparing quercetin-7, 4 '-di-O-beta-D-glucoside from quercetin-7, 4'-di-O-beta-D-glucoside and quercetin-3, 4 '-di-O-beta-D-glucoside, which are characterized in that quercetin-7, 4'-di-O-beta-D-glucoside and quercetin-3, 4 '-di-O-beta-D-glucoside which are natural products are subjected to enzymatic synthesis at high efficiency and low cost, so that the 7 and 4'-hydroxy glycosylation of quercetin and 3-hydroxy glycosylation reaction of quercetin-4 '-O-beta-D-glucoside are established.

Description

Technical Field

[0001] The invention relates to the technical field of biocatalysis, and in particular to an enzymatic reaction catalyst and application thereof in synthesizing di-O-β-d-glucoside. Background Art

[0002] Quercetin-7,4'-di-O-β-D-glucoside is currently only sold as a standard product. Experimental data show that the anti-inflammatory activity of quercetin-7,4'-di-O-β-D-glucoside is slightly weaker than that of quercetin-3,4'-di-O-β-D-glucoside, but much higher than that of quercetin and dihydroquercetin, and has great development potential. Quercetin-3,4'-di-O-β-D-glucoside has high water solubility and bioavailability. It is the main dietary flavonoid in vegetables, especially onion (Alliumcepa), and can be used as a dietary supplement, functional food ingredient, or valuable phytochemical for the pharmaceutical and food industries. Quercetin-3,4'-di-O-β-D-glucoside has basic neuroprotective effects [Sarchielli, Erica, et al. "Neuroprotective effects of quercetin 4'-O-β-D-diglucoside on humans triatal precursor cells in nutrient deprivation condition. Acta histochemica 2018, 120: 122-128.] In addition, quercetin-3,4'-di-O-β-D-glucoside has antioxidant and anti-aging functions [ D, Colina-Coca C, Char CD, Cano MP, de Ancos B, Sánchez-Moreno C. Hyaluronidase inhibiting activity and radical scavenging potential of flavonols in processed onion. J Agric Food Chem. 2013, 61: 4862-72.]. However, the content of quercetin-3,4'-di-O-β-D-glucoside in fruits and vegetables is very low, for example, about 200 mg per kilogram of fresh weight in onions. Therefore, obtaining a large amount of this substance from fruits and vegetables requires large-scale planting of fruits and vegetables, resulting in a huge waste of land resources and raw materials. In addition, the steps used in the extraction process are complicated, resulting in low economic benefits. Therefore, the use of glycosyltransferases to directly bioconvert substrates into quercetin-3,4'-di-O-β-D-glucoside is the preferred method [Tao Y, Xu J, Shao J, He X, Cai R, Chen K, Li Y, Jia H. Three Glycosyltransferase Mutants in a One-Pot Multi-enzyme System with Enhanced Efficiency for Biosynthesis of Quercetin-3,4'-O-diglucoside. J Agric Food Chem. 2023, 71: 6662-6672.].

[0003] Uridine diphosphate glucose (UDPG) is an activated form of glucose and can serve as a glycosyl donor in the glycosylation enzymatic reaction system to form a glycosidic bond through nucleophilic attack of the substrate [Takeshi Hiromoto, Eijiro Honjo, Naonobu Noda, Taro Tamada, Kohei Kazuma, Masahiko Suzuki, Michael Blaber, Ryota Kuroki. Structural basis for acceptor-substraterecognition of UDP-glucose: anthocyanidin 3-O-glucosyltransferase from Clitoria ternatea. Protein Science, 2015, 24: 395-407.]. UDPG-glycosyltransferase catalyzes the glycosylation reaction of the substrate in one step to produce di-O-β-D-glucoside, which has the advantages of simple steps, economic efficiency and the like [Brazier-Hicks Melissa, Offen Wendy, Gershater Markus, Revett Timothy, Lim Eng-Kiat, Bowles Dianna, Davies Gldeon, Edwards Robert. Characterization and engineering of the bifunctional N-and O-glucosyltransferase involved in xenobiotic metabolism in plants. PNAS 2007, 104: 20238-20243].

[0004] At present, it is reported that the glycosyltransferase UGT73G1 from A.cepa can catalyze quercetin or quercetin-3-O-β-D-glucoside to produce quercetin-3,4'-di-O-β-D-glucoside. However, when quercetin and quercetin-3-O-β-D-glucoside are used as substrates, the conversion rate of the product quercetin-3,4'-di-O-β-D-glucoside can only reach about 10% to 50% [Kramer CM, Prata RT, Willits MG, De Luca V, Steffens JC, Graser G. Cloning and regiospecificity studies of two flavonoid glucosyltransferases from Alliumcepa. Phytochemistry. 2003, 64: 1069-1076.]. For quercetin-7,4'-di-O-β-D-glucoside, glycosyltransferase ZjOGT3 can convert quercetin into quercetin-7,4'-di-O-β-D-glucoside, and the substrate loading capacity is only 30.2 mg / L, which cannot meet the requirements of industrial production [Functional characterization, structural basis, and regio-selectivity control of a promiscuous flavonoid 7,4′-di-O-glycosyltransferase from Ziziphus jujuba var.spinosa].

[0005] The existing raw materials for synthesizing two disaccharides, quercetin-3,4'-di-O-β-D-glucoside and quercetin-7,4'-di-O-β-D-glucoside, both use UDPG, which has high synthesis cost. In view of this, the present invention provides an enzymatic reaction catalyst and its application in synthesizing di-O-β-d-glucoside. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide an enzymatic reaction catalyst and its application in the synthesis of di-O-β-d-glucoside. The purpose is to provide a glycosyltransferase (UGT73C1) and a sucrose synthase (GMSUS) to achieve high efficiency and low cost preparation of quercetin-7,4'-di-O-β-d-glucoside and quercetin-3,4'-di-O-β-d-glucoside.

[0007] The technical solution of the present invention to solve the above technical problems is as follows:

[0008] In a first aspect, an enzymatic reaction catalyst comprises a glycosyltransferase and / or a sucrose synthase, wherein the amino acid sequence of the glycosyltransferase is shown in SEQ ID NO: 2, and the amino acid sequence of the sucrose synthase is shown in SEQ ID NO: 4.

[0009] The beneficial effects of the present invention are as follows: the present invention utilizes glycosyltransferase (UGT73C1) and sucrose synthase (GMSUS) as enzymatic reaction catalysts, the glycosyltransferase (UGT73C1) can glycosylate the 4'-hydroxyl group of quercetin-3-O-β-D-glucoside, and can also glycosylate the 7- and 4-hydroxyl groups of quercetin; the sucrose synthase can catalyze the decomposition of sucrose to generate a molecule of fructose and a molecule of glucose, and the glucose can combine with UDP to generate UDPG; thus, the glycosylation reaction of the 7- and 4-hydroxyl groups of quercetin and the glycosylation reaction of the 3-hydroxyl group of quercetin-4'-O-β-D-glucoside are established, and the high-efficiency and low-cost enzymatic synthesis of natural product di-O-β-d-glucoside (quercetin-7,4'-di-O-β-D-glucoside and quercetin-3,4'-di-O-β-D-glucoside) is realized. The enzymatic reaction also has the following catalytic capabilities: it can catalyze the conversion of dihydroquercetin into corresponding monosaccharide and disaccharide glycoside derivatives and catalyze the conversion of quercetin into monosaccharide quercetin and chicory glycoside, and catalyze 10-OH camptothecin to generate corresponding monosaccharide glycoside products.

[0010] Based on the above technical solution, the present invention can also be improved as follows.

[0011] In a second aspect, a gene encoding an enzymatic reaction catalyst, wherein the gene encodes the glycosyltransferase and / or sucrose synthase.

[0012] Furthermore, the nucleotide sequence of the gene encoding the glycosyltransferase is shown in SEQ ID NO: 1; and the nucleotide sequence of the gene encoding the sucrose synthase is shown in SEQ ID NO: 3.

[0013] In a third aspect, a recombinant plasmid of an enzymatic reaction catalyst is provided, wherein the recombinant plasmid comprises a gene encoding the enzymatic reaction catalyst.

[0014] The recombinant plasmid of the enzymatic reaction catalyst was constructed as follows: the base sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 were selected, the 5' end site was NdeI, the 3' end site was XhoI endonuclease, the target vector was pET28a, and Beijing Qingke Biotechnology Co., Ltd. was commissioned to perform gene synthesis.

[0015] Furthermore, the backbone of the recombinant plasmid is at least one of pET28a and pYES2.

[0016] In a fourth aspect, a transformant of an enzymatic reaction catalyst, the transformant comprises a recombinant plasmid of the enzymatic reaction catalyst, wherein the transformant is selected from Escherichia coli or Saccharomyces cerevisiae, preferably Escherichia coli.

[0017] The expression process of glycosyltransferase (UGT73C1) or sucrose synthase (GMSUS) is as follows: the transformant containing the enzymatic reaction catalyst is cultured and expanded. When the cell density reaches OD 600 =0.5, isopropyl thiogalactoside is added for induction culture for a period of time, and then the transformants are collected by centrifugation. The transformant precipitate is resuspended in sodium citrate buffer and homogenized (e.g., 1000 bar, 5 min) until the solution becomes translucent; then centrifuged, and the supernatant obtained by centrifugation after the broken cells are collected. Prepare a Ni-NTE column (Ni SepharoseTM excel purchased from GE Healthcare, 17-3712-01), wash with ultrapure water, wash with Lysisi buffer, pass the supernatant through the Ni-NTE column, flow through twice, collect the protein mixture and pass through the binding column without binding part; rinse the nickel column with Wash buffer to remove the impurities on the nickel column, 3 mL each time, wash at least 3 times; elute the target band with Eluntion buffer to obtain purified sucrose synthase (UGT73C1) or sucrose synthase (GMSUS), and then pass through molecular sieve SD75 (purchased from GE Healthcare) to obtain sucrose synthase (UGT73C1) or sucrose synthase (GMSUS) with higher purity.

[0018] A fifth aspect provides an application of an enzymatic reaction catalyst, wherein the enzymatic reaction catalyst is used to synthesize di-O-β-d-glucoside.

[0019] Furthermore, the di-O-β-d-glucoside includes at least one of quercetin-3,4'-di-O-β-D-glucoside and quercetin-7,4'-di-O-β-d-glucoside.

[0020] In a sixth aspect, a method for preparing di-O-β-d-glucoside comprises the following steps:

[0021] The glycosyltransferase and the sucrose synthase are added to a reaction system containing sodium citrate buffer, uridine diphosphate, sucrose and a reaction substrate, wherein the reaction substrate includes at least one of quercetin-3-O-β-D-glucoside and quercetin, and an enzymatic reaction is performed to prepare di-O-β-d-glucoside.

[0022] The beneficial effect of adopting the above scheme is that the glycosyltransferase (UGT73C1) of the present invention combined with sucrose synthase (GMSUS) can catalyze 100% complete conversion of the substrate to generate products, the loading amounts of the substrates quercetin and quercetin-3-O-β-D-glucoside are 0.64 g / L and 1 g / L, respectively, and the glycosyltransferase and sucrose synthase are used for the synthesis reaction of quercetin-7,4'-di-O-β-D-glucoside and quercetin-3,4'-di-O-β-D-glucoside. Compared with the chemical method, the chemical reaction steps are relatively simplified and low temperature control is not required.

[0023] Furthermore, the molar ratio of the uridine diphosphate to the reaction substrate is 2-4:8-12; the dosage ratio of the glycosyltransferase to the reaction substrate is 300-350 μg:2-3 mmol; the mass ratio of the glycosyltransferase to the sucrose synthase is 300-350:300-350;

[0024] The temperature of the enzymatic reaction is 35-45°C and the time is 12-24h.

[0025] The sodium citrate buffer only needs to meet the requirements of the above-mentioned reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of the enzymatic reaction of quercetin or quercetin-3-O-β-D-glucoside catalyzed by the glycosyltransferase (UGT73C1) of the present invention in combination with sucrose synthase (GMSUS);

[0027] Figure 2 It is the SDS-PAGE gel electrophoresis diagram of the glycosyltransferase (UGT73C1) and sucrose synthase (GMSUS) of the present invention; wherein A is quercetin-7,4'-di-O-β-D-glucoside; B is quercetin-3,4'-di-O-β-D-glucoside;

[0028] Figure 3The present invention is a schematic diagram of the liquid phase detection results of quercetin-7,4'-di-O-β-D-glucoside and quercetin-3,4'-di-O-β-D-glucoside; a substrate is a quercetin standard; b is a substrate quercetin-3-O-β-D-glucoside standard; c is a product quercetin-3,4'-di-O-β-D-glucoside standard; d is a product quercetin-7,4'-di-O-β-D-glucoside standard; β-D-glucoside standard; Reaction group 1: glycosyltransferase (UGT73C1) and sucrose synthase (GMSUS) jointly catalyze the substrate quercetin to generate quercetin-7,4'-di-O-β-d-glucoside; Reaction group 2: UGT73C1 and sucrose synthase jointly catalyze the substrate quercetin-3-O-β-d-glucoside to generate quercetin-3,4'-di-O-β-d-glucoside;

[0029] Figure 4 It is a schematic diagram of the high-resolution mass spectrometry identification results of quercetin-7,4'-di-O-β-D-glucoside and quercetin-3,4'-di-O-β-D-glucoside of the present invention and a standard substance;

[0030] Figure 5 It is a schematic diagram of mass-to-charge ratio analysis of the product obtained in the present invention; wherein A is quercetin-7,4'-di-O-β-D-glucoside; and B is quercetin-3,4'-di-O-β-D-glucoside. DETAILED DESCRIPTION

[0031] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0032] Example

[0033] 1. Construction of glycosyltransferase (UGT73C1) and sucrose synthase (GMSUS)

[0034] The construction of glycosyltransferase (UGT73C1) or sucrose synthase (GMSUS) comprises the following steps:

[0035] (1) Construction of expression strain: The base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 was selected, the 5' end site was NdeI, the 3' end site was XhoI, the target vector was pET28a (provided by Beijing Qingke Biotechnology Co., Ltd.), and Beijing Qingke Biotechnology Co., Ltd. was entrusted to perform gene synthesis; 4 μg of the synthesized plasmid was added to 100 μL of ddH2O, 3 μL of the plasmid was taken out and added to 50 μL of E coli BL21 (DE3) competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), the EP tube was reset in ice for 30 min, then placed in a 42°C water bath for heat shock for 45 s, and then placed in ice for 2 min. Add 900 μL LB medium (Wuhan Taisheng Biotechnology Co., Ltd.) to each tube and culture at 37°C, 180 rpm shaking for 60 min; then centrifuge at 2000g for 5 min, take 850 μL supernatant, gently mix the precipitate, spread it on an LB plate containing 100 mg / L kanamycin, and culture in a 37°C incubator for 10-12 h.

[0036] (2) Screening of positive clones: A single colony grown on the plate was inoculated on an LB plate containing 50 mg / L kanamycin and cultured at 37°C for 5 h. A single colony was picked for sequencing. The sequencing results showed that the length of the loaded glycosyltransferase (UGT73C1) fragment was 1443 bp, and the length of the sucrose synthase (UGT73C1) fragment was 2427 bp;

[0037] (3) Glycosyltransferase (UGT73C1) or sucrose synthase (GMSUS): Select the kanamycin-resistant strain from step (2) and expand it to 1 L. When the cell density reaches OD 600 =0.5, 0.5 mM isopropylthiogalactoside (IPTG, purchased from BioFroxx Biotechnology Co., Ltd., purity ≥99%) was added for induction culture. After 24 h, the bacteria were collected by centrifugation, and the bacterial pellet was resuspended in 100 mL Tris-HCl buffer and homogenized (1000 bar, 5 min) until the solution became translucent; then centrifuged at 4 ° C, 12000 rpm for 1 h, and the supernatant obtained by centrifugation after the broken cells were collected.

[0038] Prepare a Ni-NTE column (Ni SepharoseTM excel purchased from GE Healthcare, 17-3712-01), wash it 3 times with ultrapure water, wash it 3 times with Lysisi buffer, pass the supernatant through the Ni-NTE column, flow through it twice, collect the protein mixture that has not bound to the binding column; rinse the nickel column with Wash buffer to remove the impurities on the nickel column, 3 mL each time, wash 3 times; elute the target band with Eluntion buffer to obtain purified glycosyltransferase (UGT73C1) or sucrose synthase (GMSUS), and then pass it through molecular sieve SD75 (purchased from GE Healthcare) to obtain glycosyltransferase (UGT73C1) or sucrose synthase (GMSUS) with higher purity.

[0039] Buffer used for protein purification:

[0040] Lysis buffer components: 20 mM sodium citrate (pH = 6.0), 300 mM KCl, 10% glycerol, 1 mM PMSF;

[0041] Wash Buffer components: 20 mM sodium citrate (pH = 6.0), 300 mM KCl, 10 mM, 20 mM, 30 mM, 50 mM, 100 mM, 200 mM imidazole, 10% glycerol, 1 mM PMSF;

[0042] Elution Buffer components: 20 mM sodium citrate (pH = 6.0), 300 mM KCl, 500 mM imidazole, 10% glycerol, 1 mM PMSF;

[0043] Molecular sieve buffer: 20 mM sodium citrate (pH=6.0), 10% glycerol.

[0044] After the obtained glycosyltransferase (UGT73C1) or sucrose synthase (GMSUS) was subjected to nickel column affinity chromatography, a portion of the collected product was mixed with loading buffer and analyzed by SDS-PAGE electrophoresis. The results were as follows: Figure 2 As shown by Figure 2 It can be seen that glycosyltransferase (UGT73C1) or sucrose synthase (GMSUS) was produced.

[0045] 2. Determination of enzyme activity

[0046] Determination of enzyme activity: The concentration of glycosyltransferase (UGT73C1) and sucrose synthase (GMSUS) was determined by BCA method:

[0047] 0μL, 1μL, 2μL, 4μL, 8μL, 12μL, 16μL, 20μL, 2mg / mL BSA solution (Wuhan Taisheng Biotechnology Co., Ltd.) was respectively added to the first column of the 96-well plate, and 20μL, 19μL, 18μL, 16μL, 12μL, 8μL, 4μL, 0μL PBS buffer solution was added to the corresponding wells. 19μL PBS buffer and 1μL unknown protein solution were added to the first row of the second, third, and fourth columns, and 200μL of mixed BCA reagent A and BCA reagent B solution (volume ratio of 50:1) was added to each well, and incubated at 37°C for 30min using a microplate incubator and an oscillator. The 96-well plate was cooled to room temperature, and the absorbance of the sample at 562 nm was measured by an ELISA instrument. A standard curve y=0.039x+0.243 (y is absorbance, x is protein concentration) was drawn to calculate the concentration of unknown protein.

[0048] 3. Enzymatic reaction

[0049] Using bovine serum albumin (BCA protein concentration assay kit (enhanced), solution concentration of 2 mg / mL, purchased from Yeasen Biotechnology Co., Ltd.) as the standard, the BCA method was used to determine the concentration of glycosyltransferase (UGT73C1) or sucrose synthase (GMSUS): 161 μg of glycosyltransferase (UGT73C1) and 161 μg of sucrose synthase (GMSUS) were added to 1 mL of the reaction system.

[0050] 1mL enzymatic reaction system is as follows: 50mM sodium citrate buffer (pH=6.0), UDP (≥98%, Shanghai Yuanye Biotechnology Co., Ltd.) 0.75mM, quercetin-3-O-β-D-glucoside (95%, Shanghai Yuanye Biotechnology Co., Ltd.) or quercetin (95%, Shanghai Yuanye Biotechnology Co., Ltd.) 2.5mM, reaction temperature 40°C, reaction time 18h. Glycosyltransferase (UGT73C1) and sucrose synthase (GMSUS) jointly catalyze the enzymatic reaction of quercetin-3-O-β-D-glucoside or quercetin. Figure 1 shown.

[0051] The precise molecular weight of the obtained products was identified by high-resolution mass spectrometry (Thermo Fisher Q Exactive), wherein the products obtained by the enzymatic reaction were detected by mass detectors as follows: Figure 4 As shown, from Figure 4 It can be seen that the retention times of the sample and the standard (Toranto research chemicals Inc, purity 98%) on the mass detector are close (the mobile phase is methanol: 0.1% formic acid water = 50:50).

[0052] The products obtained by the enzymatic reaction were further subjected to mass-to-charge ratio analysis, such as Figure 5 shown; from Figure 5 It can be seen that the precise molecular weights of the products obtained are 627.14563 and 627.15289, and the isotope spectra of the products are consistent with those of the standard products, indicating that quercetin-7,4'-di-O-β-D-glucoside or quercetin-3,4'-di-O-β-D-glucoside is synthesized in each system. The retention time of the products obtained by the enzymatic reaction is close to that of the standard products.

[0053] 0mM, 0.01mM, 0.03mM, 0.0625mM, 0.125mM, 0.25mM, 0.40mM, 0.50mM concentrations of quercetin-3-O-β-D-glucoside or quercetin were selected, and high-resolution liquid chromatography detection was performed, and the standard curve of quercetin-3-O-β-D-glucoside or quercetin was fitted according to the peak area: y1=160.04x1, where y1 represents the integrated peak area of ​​quercetin-3-O-β-D-glucoside , x1 represents the concentration of quercetin-3-O-β-D-glucoside; y2=224.26x2, wherein y2 represents the integrated peak area of ​​quercetin, and x2 represents the concentration of quercetin; the quercetin-3-O-β-D-glucoside or quercetin after the enzymatic reaction is substituted into the standard curve, and after detection, it is obtained that there is no residue of quercetin-3-O-β-D-glucoside or quercetin in the enzymatic reaction system, so the conversion rate of quercetin-3-O-β-D-glucoside or quercetin is 100%.

[0054] Compared with the reference document that when quercetin and quercetin-3-O-β-D-glucoside are used as substrates, the conversion rate of the product quercetin-3,4'-di-O-β-D-glucoside using glycosyltransferase UGT73G1 can only reach about 10% and 50%, the conversion rate of the present invention is significantly higher than that of the reference document; the reference document is: Kramer CM, Prata RT, Willits MG, DeLuca V, Steffens JC, Graser G. Cloning and regiospecificity studies of twoflavonoid glucosyltransferases from Allium cepa. Phytochemistry. 2003, 64: 1069-1076.

[0055] For quercetin-7,4'-di-O-β-D-glucoside, glycosyltransferase ZjOGT3 can convert quercetin into quercetin-7,4'-di-O-β-D-glucoside, and the substrate quercetin loading is only 30.2 mg / L, which cannot meet the production requirements. The present invention provides a glycosyltransferase (UGT73C1) and a sucrose synthase (GMSUS), which can glycosylate the 7 and 4' hydroxyl groups of quercetin, and can also glycosylate the 4' hydroxyl group of quercetin 3-O-β-D-glucoside, and establish the glycosylation reaction of the 7 and 4' hydroxyl groups of quercetin and the glycosylation reaction of the 4' hydroxyl group of quercetin-3-O-β-D-glucoside. The enzymatic synthesis of natural products quercetin-7,4'-di-O-β-D-glucoside and quercetin-3,4'-di-O-β-D-glucoside was achieved. In the synthesis process, the loading amount of substrate quercetin and substrate quercetin-3-O-β-D-glucoside can reach 0.6g / L and 1g / L respectively, and the conversion rate of both is 100%.

[0056] In summary, the present invention utilizes glycosyltransferase (UGT73C1) and sucrose synthase (GMSUS) as enzyme catalysts, and according to the functions of glycosyltransferase (UGT73C1) and sucrose synthase (GMSUS), realizes the enzymatic synthesis of natural products quercetin-7,4'-di-O-β-D-glucoside and quercetin-3,4'-di-O-β-D-glucoside.

[0057] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An enzymatic reaction catalyst, characterized in that The enzymatic reaction catalyst comprises glycosyltransferase and / or sucrose synthase. The amino acid sequence of the glycosyltransferase is shown in SEQ ID NO: 2, and the amino acid sequence of the sucrose synthase is shown in SEQ ID NO:

4.

2. A gene encoding an enzymatic reaction catalyst, characterized in that: The coding gene encodes the glycosyltransferase and / or sucrose synthase according to claim 1.

3. The gene encoding the enzymatic reaction catalyst according to claim 2, characterized in that: The nucleotide sequence of the gene encoding the glycosyltransferase is shown in SEQ ID NO: 1; the nucleotide sequence of the gene encoding the sucrose synthase is shown in SEQ ID NO:

3.

4. A recombinant plasmid of an enzymatic reaction catalyst, characterized in that: The recombinant plasmid comprises a gene encoding an enzymatic reaction catalyst as claimed in claim 2 or 3.

5. The recombinant plasmid of an enzymatic reaction catalyst according to claim 4, characterized in that: The backbone of the recombinant plasmid is at least one of pET28a and pYES2.

6. A transformant of an enzymatic reaction catalyst, characterized in that: The transformant comprises a recombinant plasmid of an enzymatic reaction catalyst according to claim 4 or 5.

7. An application of an enzymatic reaction catalyst, characterized in that: The enzymatic reaction catalyst according to claim 1 is used to synthesize di-O-β-d-glucoside.

8. The use of an enzymatic reaction catalyst according to claim 7, characterized in that: The di-O-β-d-glucoside includes at least one of quercetin-3,4'-di-O-β-D-glucoside and quercetin-7,4'-di-O-β-d-glucoside.

9. A method for preparing di-O-β-d-glucoside, characterized in that: The steps include: The glycosyltransferase and the sucrose synthase described in claim 1 are added to a reaction system containing sodium citrate buffer, uridine diphosphate (UDP), sucrose, and a reaction substrate, wherein the reaction substrate includes at least one of quercetin-3-O-β-D-glucoside and quercetin, and an enzymatic reaction is performed to prepare di-O-β-d-glucoside.

10. The method for preparing di-O-β-d-glucoside according to claim 9, characterized in that: The molar ratio of the uridine diphosphate to the reaction substrate is 2-4:8-12; the dosage ratio of the glycosyltransferase to the reaction substrate is 300-350 μg:2-3 mmol; the mass ratio of the glycosyltransferase to the sucrose synthase is 300-350:300-350; The temperature of the enzymatic reaction is 35-45°C and the time is 12-24h.

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