A cyclodextrin glucosyltransferase mutant and its application

Abstract

The present invention provides a cyclodextrin glucosyltransferase mutant, belonging to the technical fields of genetic engineering and enzyme mutants. The mutant is obtained by performing single-point mutation or multi-point mutation on amino acids at positions 197, 345, 407, and 412 of the amino acid sequence shown in SEQ ID No. 1; the mutation includes mutating position 197 of the amino acid sequence shown in SEQ ID No. 1 to H, position 345 to N, position 407 to M, and position 412 to D. The enzyme mutant provided by the present invention can be used to prepare polyglycosylated flavonoids, has a high conversion rate, a simple and convenient purification method, and a low amount of residual glycosyl donors, making it suitable for industrial production and application.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering and enzyme mutants, in particular to a cyclodextrin glucosyltransferase mutant and application thereof. Background Art

[0002] Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) is a versatile enzyme capable of catalyzing hydrolysis, cyclization, disproportionation, and conjugation reactions, with broad potential applications in the food and pharmaceutical industries. Disproportionation is an intermolecular transglycosylation reaction catalyzed by CGTase. This reaction breaks the glycosidic bond of the donor molecule and transfers a glucose group to the acceptor molecule, thereby altering the physicochemical properties of the acceptor molecule. This reaction has potential applications in drug modification and the production of food additives. For example, it can catalyze the glycosylation of drug molecules, thereby improving their stability and bioavailability.

[0003] Flavonoids are important products of plant secondary metabolism. Pharmacological studies have shown that flavonoids possess a variety of biological activities, including antioxidant, antiviral, anti-inflammatory, and anti-tumor activities. They are widely used in clinical medicine for liver protection and cardiovascular disease treatment. However, the dense molecular arrangement and strong attractive forces between flavonoids, flavonols, and chalcones make them poorly soluble in water, resulting in low bioavailability. Structural modification to enhance the solubility of flavonoids is crucial for improving their bioavailability.

[0004] Currently, common structural modifications of flavonoids include glycosylation, methylation, and acylation. Glycosylation has become a research hotspot and focus both domestically and internationally in recent years. To address the inherent deficiencies of natural flavonoids, structural modifications are being made through glycosylation. This not only alters the structure of flavonoids but also enhances their biological activity, endowing them with diverse functions.

[0005] For example, glucose is connected to rutin through an enzymatic reaction to prepare glucosylrutin, which has a solubility about 12,000 times higher than that of rutin and has good application prospects. Zheng Jianyong’s research team at Zhejiang University of Technology expressed Bacillus stearothermophilus cyclodextrin glycosyltransferase (CGTase) extracellularly in Bacillus subtilis SCK6 and catalyzed the transglycosylation reaction of rutin. The optimal conversion rate reached 80.13%, the yield of glycosylated rutin was 56.1%, and the purity was 74.3%. However, the products of its catalytic transglycosylation of rutin are mainly monoglycosylated products, while the proportion of polyglycosylated products is low. In actual industrial production, obtaining more polyglycosylated rutin will help develop more application scenarios for rutin, which is one of the problems that need to be solved urgently.

[0006] Furthermore, current methods for the separation and purification of polyglycosylated flavonoids have significant shortcomings. Existing methods are generally only suitable for the separation of monoglycosylated flavonoids, and research on the separation of polyglycosylated flavonoids is severely underrepresented. When isolating monoglycosylated flavonoids, the substrate is typically hydrolyzed with amylase prior to separation. This method hydrolyzes the glycosyl donors (such as soluble starch and dextrin) into monosaccharides, which are easily removed during the subsequent purification process. However, when isolating polyglycosylated flavonoids, some of these products are also degraded by amylase, making it difficult to remove the residual glycosyl donors by treating the substrate with amylase prior to separation. Therefore, to fill the technological gap in the separation of polyglycosylated flavonoids and further promote the research and application of polyglycosylated flavonoids, it is crucial and urgent to develop an effective method for the separation and purification of polyglycosylated flavonoids. Summary of the Invention

[0007] To address the aforementioned issues in the prior art, the present invention provides a cyclodextrin glucosyltransferase mutant and its application. The enzyme mutant provided by the present invention can produce polyglycosylated flavonoids with high conversion rates, simple and convenient purification methods, and low levels of residual glycosyl donors, making it suitable for industrial production and application.

[0008] The technical solutions of the present invention are as follows:

[0009] A cyclodextrin glucosyltransferase mutant is obtained by performing single-point mutation or multi-point mutation on the 197th, 345th, 407th and 412th amino acids of the amino acid sequence shown in SEQ ID No. 1.

[0010] Furthermore, the mutation includes mutating the amino acid sequence shown in SEQ ID No. 1 at position 197 to H, mutating the amino acid sequence at position 345 to N, mutating the amino acid sequence at position 407 to M, and mutating the amino acid sequence at position 412 to D.

[0011] The present invention also provides a genetically engineered bacterium of the cyclodextrin glucosyltransferase mutant, which is obtained by cloning a gene encoding the mutant into the NcoI and XhoI sites of a pET-28a vector and then transforming the gene into Escherichia coli.

[0012] Furthermore, the strain of Escherichia coli is W3110 (DE3).

[0013] The present invention further provides a method for preparing polysaccharidized flavonoids, the method comprising the following steps:

[0014] S1. preparing the cyclodextrin glucosyltransferase mutant, or using the cyclodextrin glucosyltransferase mutant obtained by fermentation using the genetically engineered bacteria;

[0015] S2, adding glycosyl donor, flavonoid and cyclodextrin glucosyltransferase mutant to carry out catalytic reaction;

[0016] S3. Remove the glycosyl donor through separation and purification to obtain high-purity polyglycosylated flavonoids.

[0017] The flavonoids include rutin or dihydroflavonols having the structure shown in the following formula I, or flavonols having the structure shown in the following formula II:

[0018]

[0019] (Formula I)

[0020] wherein R1-R6 groups are -H or -OH;

[0021]

[0022] (Formula II)

[0023] Wherein R1-R6 groups are -H or -OH.

[0024] Preferably, the dihydroflavonol has the structure described in Formula III below:

[0025]

[0026] (Formula III)

[0027] Preferably, the flavonol has the structure described in Formula IV below:

[0028]

[0029] (Formula IV)

[0030] Preferably, the glycosyl donor in step S2 is one or more of β-cyclodextrin, maltodextrin, dextrin, and soluble starch.

[0031] Preferably, in step S2, the amount of the glycosyl donor added is 1-300 g / L, the amount of flavonoids added is 1-100 g / L, and the amount of enzyme added is 0.1-10 g / L.

[0032] Preferably, the temperature of the catalytic reaction in step S2 is 40-75° C., the reaction pH is 5.5-8.5, and the reaction time is 1-24 h.

[0033] Preferably, the purification conditions described in step S3 are: styrene-type medium-polarity resin is used for purification, the sample load is 1BV; water is used as the impurity remover, the amount is 3-5 BV; 30% ethanol is used as the desorbent, 1.5-2.5 BV of desorption liquid is collected, and the desorption liquid is then dried by rotary evaporation to obtain high-purity polysaccharidized flavonoids.

[0034] The beneficial technical effects of the present invention are:

[0035] 1. The present invention finds the optimal mutation site and mutation mode through a large number of experiments and molecular docking prediction analysis. The obtained mutant can use cheap glycosyl donors to catalyze the glycosylation reaction of flavonoids to obtain polyglycosylated flavonoids, significantly improving the conversion rate.

[0036] 2. The prominent difference between the polyglycosylated flavonoids prepared by the present invention and the prior art is that the glycosylation products catalyzed by the present invention are mainly polyglycosylated products rather than monoglycosylated products.

[0037] 3. The purification method of polyglycosylated flavonoids provided by the present invention is simple to operate and can effectively remove glycosyl donors. The residual amount of glycosyl donors is less than 0.1%, thereby obtaining high-purity polyglycosylated flavonoids, which has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is an HPLC analysis chart of the catalytic reaction of different types of cyclodextrins as glycosyl donors and wild-type enzyme WT in Example 2;

[0039] Figure 2 This is the relative activity analysis of different types of cyclodextrin glucosyltransferases in Example 2;

[0040] Figure 3 This is the optimal temperature analysis of the enzyme mutant HNMD in Example 3;

[0041] Figure 4 This is the optimal pH analysis of the enzyme mutant HNMD in Example 3;

[0042] Figure 5 This is an HPLC analysis chart of the catalytic reaction of different glycosyl donors and enzyme mutant HNMD in Example 3;

[0043] Figure 6 Screening of different resins for purification of polysaccharidized rutin for Example 4;

[0044] Figure 7 This is the effect of removing glycosyl donors by desorption with different gradients of ethanol in Example 5;

[0045] Figure 8 The effect of the 30% ethanol desorption dosage on the composition of polysaccharidized rutin in Example 5;

[0046] Figure 9 This is the HPLC analysis chart of the high-purity polysaccharide rutin in Example 6;

[0047] Figure 10This is the HPLC analysis chart of the high-purity polysaccharide compound I in Example 7;

[0048] Figure 11 This is the HPLC analysis chart of the high-purity polysaccharide compound II in Example 8. DETAILED DESCRIPTION

[0049] The present invention is described in detail below with reference to the accompanying drawings and embodiments. It is apparent that the embodiments described are only a portion of the embodiments of the present invention, rather than all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0050] In the present invention, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from commercial channels.

[0051] Example 1: Construction and expression of cyclodextrin glucosyltransferase mutants

[0052] The amino acid sequence of cyclodextrin glucosyltransferase is shown in SEQ ID No. 1. A DNA coding sequence (SEQ ID No. 2) was designed based on the codon preference of Escherichia coli. Beijing Qingke Biotechnology Co., Ltd. performed complete gene synthesis and cloned it into the NcoI and XhoI sites of the pET28a plasmid to generate the recombinant plasmid pET-WT. The DNA coding sequence, which lacks a stop codon, is expressed in fusion with the His tag on the plasmid.

[0053] Using the recombinant plasmid pET-WT as a template, reverse amplification of the plasmid was performed using a high-fidelity enzyme and primer pairs 197-F / 197-R, 345-F / 345-R, 407-F / 407-R, and 412-F / 412-R. The primer sequences are shown in Table 1. PCR products with 15-20 bp homology arms were obtained. The PCR product band sizes were determined by agarose gel electrophoresis. After correct bands were recovered from the gel, the residual plasmid template was removed using the restriction endonuclease DpnI. Recombinant plasmids were then constructed by Gibson assembly. The recombinant plasmids were transformed into competent Escherichia coli DH5α cells using the heat shock method, and single clones were selected for sequencing verification. The correct single-mutation recombinant plasmids were designated pET-197H, pET-345N, pET-407M, and pET-412D, respectively. The mutated recombinant plasmid was used to perform multiple rounds of iterative mutation by replacing different primers to obtain the recombinant plasmid pET-HNMD containing the above four mutation sites. The recombinant plasmid was transformed into competent Escherichia coli W3110 (DE3) by the heat shock method, and the positive strains containing the corresponding recombinant plasmids were named EC197H, EC345N, EC407M, EC412D, and ECHNMD, respectively.

[0054] Table 1 Primer sequences

[0055]

[0056] Single colonies of the five recombinant strains were selected and inoculated into LB medium, cultured overnight at 37°C and 200 rpm, and then inoculated into new TB medium at a 3% inoculum size. The culture was cultured at 37°C until the OD reached about 0.6-0.8, cooled to 25°C, and IPTG was added at a final concentration of 0.2 mM to induce recombinant protein expression for 14-18 h.

[0057] The formula of LB medium is: yeast powder 5.0 g / L, peptone 10.0 g / L, and NaCl 10.0 g / L.

[0058] The formula of TB medium is: peptone 12 g / L, yeast powder 24 g / L, glycerol 4 g / L, dipotassium hydrogen phosphate 9.4 g / L, and potassium dihydrogen phosphate 2.2 g / L.

[0059] The cultured cells were collected by centrifugation, resuspended in an equal volume of phosphate-buffered saline (PBS) buffer (pH 7.4), and then disrupted using a high-pressure homogenizer. The supernatant was collected by centrifugation, and the recombinant protein was isolated and purified using a nickel ion chelate column. The protein was then dialyzed at low temperature using a 12 kD semipermeable membrane to remove excess salt. The purified recombinant proteins from strains EC197H, EC345N, EC407M, EC412D, and ECHNMD were the cyclodextrin glucosyltransferase mutants, designated 197H, 345N, 407M, 412D, and HNMD, respectively.

[0060] Example 2: Rutin glycosylation reaction and catalytic verification

[0061] The rutin glycosylation reaction was performed as follows: Cyclodextrin glucosyltransferase and five enzyme mutants produced by recombinant bacterial fermentation were prepared according to the method in Example 1. 50 mL of pH 6.5 phosphate buffer was added to a 250 mL Erlenmeyer flask. Then, 6 g / L rutin, 60 g / L glycosyl donor, and 300 mg / L cyclodextrin glucosyltransferase or its mutant (concentration determined by BCA assay) were added. The reaction was catalyzed at 60°C for 4 h. Samples were diluted with acetonitrile at a specific dilution ratio and HPLC analysis was performed to detect the glycosylation products. The HPLC analysis conditions are shown in the table below.

[0062] Table 2 Chromatographic conditions

[0063]

[0064] First, the glycosyl donor required for verification was selected. The type of glycosyl donor has a great influence on the glycosylation reaction. α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin were used as glycosyl donors. The wild-type enzyme WT was used to catalyze the glycosylation reaction under the above conditions. The results are as follows: Figure 1 As shown, the glycosylation product of rutin is a mixture, with G1-G5 representing rutin with 1-5 glycosyl groups, respectively. During the catalytic reaction, one or more glucose groups are attached to rutin. Glycosylated rutin with one glycosyl group (G1) is the most abundant, decreasing with increasing glycosyl groups. Glycosylated rutin with more than 5 glycosyl groups (G5) is less abundant. When α-cyclodextrin is used as the glycosyl donor, the most glycosylated product is produced, followed by γ-cyclodextrin, and the least is β-cyclodextrin. However, α-cyclodextrin and γ-cyclodextrin are relatively expensive, making them unsuitable as glycosylation donors.

[0065] Using cheap β-cyclodextrin as the glycosyl donor and rutin conversion rate as the indicator, the catalytic effects of the wild-type enzyme WT and different mutants EC197H, EC345N, EC407M, EC412D, and ECHNMD were investigated. Figure 2As shown, the conversion rates of rutin catalyzed by different mutants were different, among which the mutant HNMD catalyzed the least residual rutin with a conversion rate of 81.3%, which had a good catalytic effect.

[0066] Example 3: Optimization of HNMD catalytic conditions of cyclodextrin glucosyltransferase mutants

[0067] Using high concentration of β-cyclodextrin as glycosyl donor (300 g / L), 50 g / L rutin and 1 g / L enzyme HNMD were added. The residual amount of rutin was used as an indicator, and the conditions in Example 2 were used for catalysis. The effects of temperature and pH on the activity of the enzyme mutant HNMD were investigated. The results are shown in Figure 2. Figure 3 、 Figure 4 As shown, the optimum temperature of the mutant is 65°C, and it has good activity in the range of 40-75°C. The optimum pH is 7.5, and it has good activity in the range of pH 5.5-8.5.

[0068] At pH 7.5 and 65°C, 50 g / L rutin, 300 g / L of different inexpensive glycosyl donors (β-cyclodextrin, maltodextrin, dextrin, soluble starch), and 10 g / L of the enzyme mutant HNMD were added. Samples were taken every 2 hours for HPLC analysis until the rutin content stopped decreasing. The catalytic completion time for different glycosyl donors was 6 hours, 8 hours, 16 hours, and 22 hours, respectively. The HPLC analysis results after the completion of the catalytic process are shown in Figure 2. Figure 5 As shown, G1-G6 represent rutin with 1-6 glycosyl groups, respectively. Glycosylation products are primarily polyglycosylated (with greater than 1 glycosyl group), with a maximum of 14 glycosyl groups. Rutin conversion efficiency is related to the molecular weight of the glycosyl donor. The smaller the molecular weight of the glycosyl donor, the higher the rutin conversion efficiency and the more polyglycosylated products.

[0069] Example 4: Screening of different resins for purification of polysaccharidated rutin

[0070] Prepare 100 mL of each of Resin 1 (styrene-based non-polar resin), Resin 2 (styrene-based moderately polar resin), and Resin 3 (styrene-based weakly polar resin). Add 50 g / L rutin and 300 g / L β-cyclodextrin at pH 7.5 and 65°C. React for 6 h to prepare polysaccharidized rutin. Prepare 100 mL of catalyst solution. After the reaction, filter the catalyst solution to remove impurities. Transfer the filtrate to the column (1 BV = 100 mL). After complete adsorption, top up with 3 BV of water to remove impurities. Desorb with 4 BV of 95% ethanol and spin dry.

[0071] Resin purification results are as follows Figure 6 As shown in the figure, the results show that the amount of polysaccharide-containing rutin in the desorbed product of resin 2 is greater than that of resin 1 and resin 3, so resin 2 is selected as the subsequent purification resin.

[0072] Example 5: Desorption and removal of glycosyl donors using different gradient ethanol

[0073] Glycosyl donors in polysaccharidized rutin were further removed by optimizing different ethanol gradients. 50 g / L rutin and 300 g / L β-cyclodextrin were added at pH 7.5 and 65°C for 6 h to prepare 100 mL of catalytic solution for polysaccharidized rutin. The filtrate was passed through resin 2 (1 BV = 100 mL). After complete adsorption, impurities were removed with water. Desorption was then performed using different ethanol gradients. The desorbed solutions were then dried and analyzed.

[0074] The polysaccharide-modified rutin was detected according to the method in Example 2. The residual glycosyl donor was detected using a phenyl-bonded silica gel column and a differential refractive index detector. The analytical conditions were as follows: Column: Phenyl-bonded silica gel column (Venusil XBP Polar-Phenyl 4.6 × 250 mm, 5 μm); Column temperature: 40°C; Flow rate: 1.0 mL / min; Injection volume: 10 μL; Mobile phase: methanol-water (90:10); Differential refractive index detector.

[0075] The results of polysaccharide rutin detection are as follows Figure 7 As shown in the figure, the results showed that after desorption with 30% ethanol, the main product was polysaccharidized rutin. When the ethanol elution concentration was too high, rutin was easily eluted. At the same time, the residual amount of glycosyl donor detected by 40% ethanol desorption solution and 50% ethanol desorption solution was less than 0.1%. Therefore, 30% ethanol was selected as the desorbent in the subsequent step.

[0076] Take 100 mL of upstream liquid and filter it. Pass the filtrate through resin 2 (1 BV = 100 mL). After complete adsorption, remove impurities with water. After completion, continue desorption with 30% ethanol. Collect the desorbed liquid at intervals of 0.5 BV. Stop desorption when polysaccharide-sylated rutin is no longer detected. Then, spin-dry the desorbed liquid and measure the results.

[0077] The results are as follows Figure 8 As shown, the composition of polyglycosylated rutin in the desorption solution is affected by the desorption volume. The lower the desorption volume, the higher the polyglycosylated rutin content, while the polyglycosylated rutin content is no longer detectable after 2.5 BV. The residual amount of glycosyl donor in the desorption solution was also tested. At 1.0 BV, 0.5% residual glycosyl donor could still be detected, while after 1.5 BV, the residual glycosyl donor was less than 0.1%. Therefore, considering all factors, the desorption volume of 30% ethanol was selected to be 1.5-2.5 BV.

[0078] Example 6: Preparation of high-purity polysaccharide rutin

[0079] At pH 7.5 and 65°C, 50 g / L rutin, 300 g / L β-cyclodextrin, and 10 g / L enzyme mutant HNMD were added and reacted at 65°C and pH 7.5 for 6 hours. After the reaction, impurities were removed by filtration, and the filtrate was passed through resin 2 (1 BV = 1 L). After complete adsorption, impurities were removed with 4 BV of water, and then desorbed with 4 BV of 30% ethanol. 1.5-2.5 BV of desorbed liquid was collected and concentrated at 50°C. After concentration, the sample was vacuum-dried at 50°C to obtain the sample. The sample was tested for the content of polysaccharide-based rutin and dextrin by liquid chromatography. The test results of polysaccharide-based rutin are as follows: Figure 9 As shown, G1-G6 represent rutin with 1-6 glycosyl groups connected, respectively. It can be seen that the content in the product sample contains products connected with different glycosyl groups, and the proportion of products connected with multiple glycosyl groups is high. The yield of polyglycosylated rutin in this embodiment is 0.71 g / g rutin, the residual amount of rutin is 3.0%, and the residual amount of glycosyl donor is 0.08%, and the purity is high.

[0080] Example 7: Preparation of High-Purity Polysaccharide Compound I

[0081] At pH 7.5 and 65°C, 50 g / L of compound I (chemical structure shown in formula III), 300 g / L of β-cyclodextrin, and 10 g / L of enzyme mutant HNMD were added and reacted at 65°C and pH 7.5 for 6 h. After the reaction, impurities were removed by filtration, and the filtrate was passed through resin 2 (1 BV = 1 L). After complete adsorption, impurities were removed with 4 BV of water, and then desorbed with 4 BV of 30% ethanol. 1.5-2.5 BV of desorbed liquid was collected and concentrated at 50°C. After concentration, the sample was vacuum-dried at 50°C to obtain the sample. The sample was tested for the content of polysaccharide compound I and dextrin by liquid chromatography at a detection wavelength of 290 nm. Other conditions were the same as in Examples 2 and 5. The detection results of polysaccharide compound I are shown in Figure 2. Figure 10 As shown, the product sample contains products with different glycosyl groups, and the proportion of products with multiple glycosyl groups is relatively high. The yield of polyglycosylated Compound I in this example is 0.75 g / g Compound I, the residual amount of Compound I is 0.8%, and the residual amount of glycosyl donor is 0.07%, indicating high purity.

[0082] Example 8: Preparation of High-Purity Polysaccharide Compound II

[0083] At pH 7.5 and 65°C, 50 g / L of compound II (chemical structure shown in formula IV), 300 g / L of β-cyclodextrin, and 10 g / L of enzyme mutant HNMD were added and reacted at 65°C and pH 7.5 for 6 h. After the reaction, impurities were removed by filtration, and the filtrate was passed through resin 2 (1 BV = 1 L). After complete adsorption, impurities were removed with 4 BV of water, and then desorbed with 4 BV of 30% ethanol. 1.5-2.5 BV of desorbed liquid was collected and concentrated at 50°C. After concentration, the sample was vacuum-dried at 50°C to obtain the sample. The sample was tested for the content of polysaccharide compound II and dextrin by liquid chromatography, and the detection conditions were the same as those in Examples 2 and 5. The detection results of polysaccharide compound II are shown in Figure 2. Figure 11 As shown, it can be seen that the product sample contains products with different sugar groups attached, and the proportion of products with multiple sugar groups attached is relatively high. The yield of polyglycosylated compound II in this example is 0.81 g / g compound II, the residual amount of compound II is 1.2%, and the residual amount of glycosyl donor is 0.06%, indicating high purity.

[0084] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, for those of ordinary skill in the art, various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A cyclodextrin glucosyltransferase mutant, characterized in that: The mutant is obtained by simultaneously mutating the amino acids at positions 197, 345, 407, and 412 of the amino acid sequence shown in SEQ ID No. 1; The mutations are to change the amino acid sequence shown in SEQ ID No. 1 at position 197 to H, at position 345 to N, at position 407 to M, and at position 412 to D.

2. A genetically engineered bacterium expressing the cyclodextrin glucosyltransferase mutant according to claim 1, characterized in that: The genetically engineered bacteria are obtained by cloning the gene encoding the mutant into the NcoI and XhoI sites of the pET-28a vector and then transforming the gene into Escherichia coli.

3. A method for preparing polysaccharidized flavonoids, characterized in that: The method comprises the following steps: S1. preparing the cyclodextrin glucosyltransferase mutant according to claim 1, or the cyclodextrin glucosyltransferase mutant obtained by fermentation using the genetically engineered bacteria according to claim 2; S2, adding glycosyl donor, flavonoid and cyclodextrin glucosyltransferase mutant to carry out catalytic reaction; S3, removing the glycosyl donor by separation and purification to obtain high-purity polyglycosylated flavonoids; The flavonoids are rutin or dihydroflavonol having the structure shown in the following formula I, or flavonol having the structure shown in the following formula II: (Formula I); wherein R1-R6 groups are -H or -OH; (Formula II) Wherein R1-R6 groups are -H or -OH.

4. The preparation method according to claim 3, characterized in that The dihydroflavonol has the structure described in Formula III below: (Formula III) The flavonol has the structure described in Formula IV below: (Formula IV).

5. The preparation method according to claim 3, characterized in that The glycosyl donor in step S2 is one or more of β-cyclodextrin, maltodextrin, and soluble starch.

6. The preparation method according to claim 3, characterized in that In step S2, the amount of the glycosyl donor added is 1-300 g / L, the amount of flavonoids added is 1-100 g / L, and the amount of enzyme added is 0.1-10 g / L.

7. The preparation method according to claim 3, characterized in that The temperature of the catalytic reaction in step S2 is 40-75° C., the reaction pH is 5.5-8.5, and the reaction time is 1-24 h.

8. The preparation method according to claim 3, characterized in that The purification conditions described in step S3 are: using a styrene-type medium-polarity resin for purification, with a sample loading of 1 BV; using water as an impurity remover, with a dosage of 3-5 BV; using 30% ethanol as a desorbent, collecting 1.5-2.5 BV of desorbate, and then performing rotary evaporation to dry the desorbate to obtain high-purity polysaccharidized flavonoids.

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