Glycosyltransferase combined mutant and application thereof

Abstract

The invention discloses a glycosyl transferase combined mutant and application thereof, and aims to solve the problems that the existing wild PlUGT43 and F196Y single-point mutants thereof have narrow substrate spectrums and cannot efficiently catalyze a plurality of isoflavone derivatives with large structural differences to perform C-glycosylation reaction. The glycosyl transferase combination mutant provided by the invention is a protein which simultaneously contains five mutated sites, namely T81S, N186S, L187A, F196Y and S310P, on the basis of a wild PlUGT43 amino acid sequence as shown in SEQ ID NO.3. The glycosyl transferase combination mutant provided by the invention is a protein which is obtained by mutating five sites, namely T81S, N186S, L187A, F196Y and S310P. Compared with a wild type and an F196Y single-site mutant, the combined mutant has the advantages that the catalytic activity on classical substrates such as daidzein is improved, and efficient C-glycosylation on various substrates such as soybean dihydroaglycone, equol and resveratrol which are difficult to catalyze or extremely low in catalytic efficiency is also realized; the application potential of the glycosyl transferase in the aspect of synthesis of diversified natural products is improved.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering. Specifically, it relates to a glycosyltransferase combination mutant and its application, particularly the application of the glycosyltransferase combination mutant to glycosylate isoflavone derivatives using uridine diphosphate glucose (UDPG) as a glycosyl donor. Background Technology

[0002] In the fields of biomedicine and natural product synthesis, C Glycosides are important compounds for developing novel drugs and functional products due to their unique stability and biological activity. Among them, isoflavones... C Glycosides such as puerarin possess a variety of biological activities, including cardioprotection, vasodilation, protection against ischemia-reperfusion injury, anti-inflammatory activity, antioxidant activity, estrogen-like effects, and anti-tumor activity, and have great development potential.

[0003] Currently, isoflavones C The synthesis of glycosides mainly relies on traditional chemical methods. These methods have several drawbacks: firstly, the routes are lengthy, requiring multiple protection and deprotection steps, demanding stringent reaction conditions, and prone to generating byproducts; secondly, they suffer from poor regioselectivity, making it difficult to precisely control glycosylation sites, resulting in low yields of the target product, high purification costs, and the potential for the violent reaction to damage the product's active backbone.

[0004] To overcome the drawbacks of chemical synthesis methods, enzymatic catalysis has become an ideal alternative due to its high selectivity and mild conditions. Among these, C γ-glycosyltransferases can catalyze the formation of stable C-C bonds, achieving... C The one-step synthesis of glycosides has great application potential. Currently, plant-derived glycosides have been characterized in nature. C There are over 60 types of glycosyltransferases, but very few can effectively catalyze isoflavone substrates; only a handful, such as GgCGT from licorice and PlUGT43 and PlCGT from kudzu, exist. Studies have shown that PlUGT43 has relatively superior catalytic efficiency and specificity and is considered a key biocatalyst.

[0005] A search revealed that C Enzyme modification techniques for glycosyltransferase PlUGT43 have been reported.

[0006] The prior art "A glycosyltransferase mutation and its application" (CN114657160A) uses site-directed saturation mutagenesis technology to modify PlUGT43 glycosyltransferase based on homology modeling and sequence alignment, and selects a glycosyltransferase mutant F196Y with high enzyme activity, which increases the catalytic activity of soybean glycoside and uridine diphosphate glucose (UDPG) condensation reaction by more than 2 times. In addition, it is combined with the UDPG regeneration system to significantly improve the application potential of the glycosyltransferase in the biosynthesis of puerarin.

[0007] Although the prior art (CN114657160A) has disclosed the F196Y single-point mutant of PlUGT43, and increased its catalytic efficiency for soybean glycoside by more than 2 times, its substrate spectrum is still extremely narrow, limited to only a few structurally similar isoflavones such as soybean glycoside and genistein. For isoflavone derivatives with large structural differences such as equol, soybean dihydrogen glycoside, and resveratrol, the single-point mutant still has no activity or negligible activity, which severely limits its application in the synthesis of diversified natural products. Therefore, developing a PlUGT43 mutant with broad substrate adaptability is still a technical problem to be solved in the field. SUMMARY

[0008] Although the F196Y single-point mutant in the prior art has improved the activity for soybean glycoside substrate, its substrate spectrum is still narrow, limited to only a few structurally similar isoflavone substrates, and the catalytic efficiency still has room for further improvement. The purpose of the present application is to provide a glycosyltransferase combination mutant, which is obtained by introducing specific mutations at the following five key sites: T81S, N186S, L187A, F196Y and S310P, based on the wild-type PlUGT43 amino acid sequence SEQ ID NO. 3 optimized according to the codon bias of Escherichia coli, through rational design and site-directed mutagenesis. The mutations at the five sites work together to endow the combination mutant with a broadened substrate spectrum and higher catalytic activity.

[0009] As a preferred, the amino acid sequence of the glycosyltransferase combination mutant is shown in SEQ ID NO. 5, and the nucleotide sequence is shown in SEQ ID NO. 6.

[0010] Among them, the wild-type PlUGT43 glycosyltransferase nucleotide sequence is shown in SEQ ID NO. 2, and the wild-type PlUGT43 glycosyltransferase nucleotide sequence optimized according to the codon bias of Escherichia coli is shown in SEQ ID NO. 4.

[0011] The present application provides a recombinant vector comprising a gene encoding the glycosyltransferase combination mutant.

[0012] It is another object of the present application to provide the use of the combination mutant in catalyzing C-glycosylation of isoflavone, dihydroisoflavone, dihydrochalcone or stilbene substrates with co-factor UDPG. C Not only does the combination mutant significantly improve the catalytic activity of the original substrate, but also effectively expands the substrate spectrum.

[0013] The conversion rate of the combination mutant to soybean dihydrogenol, resveratrol is more than 10 times higher than that of the wild type PlUGT43, and more than 5 times higher than that of the control enzyme containing only the F196Y single-point mutation.

[0014] The present application provides a catalyst for preparing C Glycosides. The preparation method comprises: using the above-mentioned glycosyltransferase mutant as a biological catalyst to catalyze C-glycosylation of isoflavone, dihydroisoflavone, dihydrochalcone or stilbene substrates with UDPG, preferably to generate the corresponding 8-C-glucoside.

[0015] As a preferred, the substrate is selected from at least one of daidzin, 7-hydroxyisoflavone, biochanin A, equol, soybean dihydrogenol, phloretin, and resveratrol.

[0016] As a preferred, the reaction conditions are: the addition amount of the glycosyltransferase combination mutant is 0.1-10 mg / mL, the concentration of the substrate is 0.1-50 mM, the concentration of UDPG is 0.15-75 mM, the reaction temperature is 25-35℃, the pH of the reaction is 7.0-9.0, and the reaction time is 0.5-24 h.

[0017] Compared with the prior art, the present application has the following beneficial effects: 1. Effective expansion of the substrate spectrum: the glycosyltransferase combination mutant provided by the present application realizes a breakthrough in expanding the catalytic range of the substrate. It not only has excellent catalytic activity for the known high-efficiency substrate daidzin of the wild type and the F196Y single-point mutant, but more importantly, it exhibits considerable catalytic capacity for a variety of compounds that are originally difficult to be catalyzed or have extremely low catalytic efficiency, including but not limited to soybean dihydrogenol, equol, and resveratrol.

[0018] 2. Significant improvement in catalytic efficiency: for the common substrate (such as daidzin), the substrate conversion rate of the mutant is 3 times that of the wild type PlUGT43, and more than 1 times that of the F196Y single-point mutant; for the newly expanded substrate (such as soybean dihydrogenol), its catalytic activity realizes a qualitative leap from "almost no activity" to "high-efficiency conversion".

[0019] 3. Wide application prospect: the glycosyltransferase combination mutant provided by the application effectively widens the substrate spectrum, and provides a core enzyme element for green and efficient synthesis of high-value natural products with various structures, which has good application potential in the fields of medicine, health care products and the like. C - glycoside natural products (especially isoflavones and dihydroisoflavones) provide core enzyme elements, which have good application potential in the fields of medicine, health care products and the like. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 . High performance liquid chromatogram and product mass spectrum of glycosylation reaction of PlUGT43 glycosyltransferase catalysis daidzin.

[0021] Figure 2 . High performance liquid chromatogram and product mass spectrum of glycosylation reaction of PlUGT43 glycosyltransferase catalysis 7-hydroxyisoflavone.

[0022] Figure 3 . High performance liquid chromatogram and product mass spectrum of glycosylation reaction of PlUGT43 glycosyltransferase catalysis biochanin A.

[0023] Figure 4 . High performance liquid chromatogram and product mass spectrum of glycosylation reaction of PlUGT43 glycosyltransferase catalysis equol.

[0024] Figure 5 . High performance liquid chromatogram and product mass spectrum of glycosylation reaction of PlUGT43 glycosyltransferase catalysis daidzein.

[0025] Figure 6 . High performance liquid chromatogram and product mass spectrum of glycosylation reaction of PlUGT43 glycosyltransferase catalysis resveratrol.

[0026] Figure 7 . High performance liquid chromatogram and product mass spectrum of glycosylation reaction of PlUGT43 glycosyltransferase catalysis glycitein.

[0027] Figure 8 . High performance liquid chromatogram and product mass spectrum of glycosylation reaction of PlUGT43 glycosyltransferase catalysis phloretin.

[0028] Figure 9 . High performance liquid chromatogram and product mass spectrum of glycosylation reaction of PlUGT43 glycosyltransferase catalysis irisolidon.

[0029] Figure 10 . H nuclear magnetic resonance result of glycitein-8-C-glucoside. 1

[0030] Figure 11 . C nuclear magnetic resonance result of glycitein-8-C-glucoside. 13 ​​

[0031] Figure 12 . H NMR results of 7-hydroxyisoflavone-8-C-glucoside 1 H NMR results of 7-hydroxyisoflavone-8-C-glucoside

[0032] Figure 13 . H NMR results of 7-hydroxyisoflavone-8-C-glucoside 1 H NMR results of 7-hydroxyisoflavone-8-C-glucoside

[0033] Figure 14 . H NMR results of 7-hydroxyisoflavone-8-C-glucoside 13 H NMR results of 7-hydroxyisoflavone-8-C-glucoside

[0034] Figure 15 . H NMR results of 7-hydroxyisoflavone-8-C-glucoside 1 H NMR results of 7-hydroxyisoflavone-8-C-glucoside

[0035] Figure 16 . H NMR results of 7-hydroxyisoflavone-8-C-glucoside 13 H NMR results of 7-hydroxyisoflavone-8-C-glucoside DETAILED DESCRIPTION

[0036] The application will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the application and not intended to limit the scope of the application.

[0037] Example 1. Preparation of glycosyltransferase combination mutant 1. Preparation of wild-type PlUGT43 glycosyltransferase expression engineering bacteria The amino acid sequence SEQ ID NO. 1 (UniProtKB / Swiss-Prot: A0A172J2G3.2) and the nucleotide sequence SEQ ID NO. 2 (GenBank: KU317801.2) of wild-type PlUGT43 glycosyltransferase are both from the NCBI database. Based on the codon usage bias of the E. coli expression system, the above-mentioned nucleotide sequence is codon-optimized to obtain the optimized nucleotide sequence SEQ ID NO. 4. The optimized sequence is synthesized by Shengong Bioengineering (Shanghai) Co., Ltd. and cloned into the pET-28a(+) vector. After sequencing verification, the recombinant plasmid is transformed into BL21(DE3) competent cells by heat shock transformation method, and finally the engineering strain expressing wild-type PlUGT43 glycosyltransferase is constructed. E. coli

[0038] 2. Preparation of PlUGT43 glycosyltransferase mutant and its expression engineering bacteria 2.1. Mutation of PlUGT43 glycosyltransferase ​The three-dimensional structure of wild-type PlUGT43 glycosyltransferase was predicted using the AlphaFold3 online website, and then through molecular docking and molecular dynamics simulation, it was speculated that the 81 th threonine, 186 th asparagine, 187 th leucine, 196 th phenylalanine and 310 th serine located around the channel were the key potential mutation sites for regulating the substrate channel. The above optimized nucleotide sequence SEQ ID NO. 4 was used as a template, and site-directed mutagenesis was designed for the five sites, and double-point, three-point, four-point and five-point mutants were constructed by site combination. Table 1 is the primer used for mutation. After each mutant was verified by sequencing, it was transformed into E. coli BL21(DE3) competent cells for expression, and glycosyltransferase mutant expression engineering bacteria were obtained.

[0039] 3. Protein expression of PlUGT43 glycosyltransferase The wild-type and mutant PlUGT43 glycosyltransferase expression engineering bacteria constructed above were inoculated into 5 mL LB liquid medium containing 50 μg / mL kanamycin, and incubated at 37℃, 200 rpm for 12 h to obtain seed liquid. 5 mL of seed liquid was transferred to 500 mL LB liquid medium containing the same concentration of kanamycin, and incubated under the same conditions until the OD 600 was about 0.6. Then the culture temperature was reduced to 16℃, IPTG was added to a final concentration of 0.1 mM, and expression was induced for 16 h. The bacterial liquid at the end of induction was centrifuged at 8000 rpm for 10 min, and the supernatant culture medium was discarded. The bacterial cells were collected and stored at -20℃ for use.

[0040] 4. Protein purification of PlUGT43 glycosyltransferase The collected bacteria were added to 8 mL lysis buffer (10 mM imidazole, 50 mM Tris, 500 mM NaCl, pH 8.0) at 1 g wet weight, resuspended by shaking, and then ice-bathed for 30 min. Ultrasonic disruption was performed for 20 min under ice-bathing conditions. Then, centrifugation was performed at 4°C and 12000 rpm for 10 min, and the supernatant was collected, which was the crude enzyme solution. Ni-NTA affinity chromatography material was used to purify the target protein. 2 mL of Ni-NTA filler was packed into a column, and the chromatography column was equilibrated with 15 mL of lysis buffer. The crude enzyme solution was loaded at a flow rate of 1 mL / min, and then 15 mL of washing buffer (20 mM imidazole, 50 mM Tris, 500 mM NaCl, pH 8.0) was used to wash away the non-specifically adsorbed impurities. Finally, the target protein was eluted with elution buffer (250 mM imidazole, 50 mM Tris, 500 mM NaCl, pH 8.0). The collected eluate was treated with a desalting column, and replaced with reaction buffer (50 mM Tris, 100 mM NaCl, pH 8.0) to remove high concentrations of salt ions and imidazole. The obtained pure enzyme solution was stored at 4°C for standby use.

[0041] Example 2. Activity detection of PlUGT43 glycosyltransferase wild type and mutants The purified PlUGT43 glycosyltransferase (wild type or mutant) was added to the 200 L reaction system at a final concentration of 1 mg / mL, wherein the final concentration of the substrate was 0.2 mM, and the final concentration of UDPG was 0.3 mM. No UDPG was added to the blank control group, and three parallel experiments were set up in the experimental group. The reaction system was incubated at 30°C and 666 rpm under constant temperature oscillation conditions for 15 h. After the reaction was completed, an equal volume of methanol was added to terminate the reaction, and after vortex mixing, centrifugation was performed at 12000 rpm for 20 min. The supernatant was analyzed by high performance liquid chromatography.

[0042] The high performance liquid chromatography analysis conditions were as follows: a Waters 2695 high performance liquid chromatography system was used, the chromatographic column was Agilent 5 HC-C18 (250 mm x 4.6 mm, 5 m) with a column temperature of 35°C, a flow rate of 0.8 mL / min, and a sample size of 20 L. The mobile phase A was phosphoric acid aqueous solution (pH 3.0), and the mobile phase B was methanol. The elution program was set according to Table 2; the detection wavelengths of different substrates are shown in Table 3.

[0043] Table 2 Liquid phase elution program Table 3 Detection wavelengths of different substrates The substrate conversion rate was calculated according to the liquid phase results, and the relative activity of the wild type protein was taken as 100%, and the relative activity of the mutant was calculated, and the relative activity of each mutant was shown in Table 4. The substrate conversion rates of the wild type, F196Y single mutant and optimal combination mutant were shown in Table 5. 突变体 / A 野生型 ×100%, and the relative activity of each mutant was shown in Table 4. The substrate conversion rates of the wild type, F196Y single mutant and optimal combination mutant were shown in Table 5.

[0044] Table 4 Relative activity of PlUGT43 glycosyltransferase wild type and mutants on different substrates Table 5 Substrate conversion rates of PlUGT43 glycosyltransferase wild type, F196Y single mutant and optimal combination mutant on different substrates The experimental results show that the catalytic performance of the five-site combination mutant is comprehensively and significantly improved compared with the wild type and the F196Y single mutant.

[0045] Compared with the wild type: (1) the conversion rates of daidzin and 7-hydroxyisoflavone are increased by more than 2 times; (2) the conversion rate of daidzein is increased from less than 3% to 41.9%, and the relative activity is increased by more than 19 times, realizing the breakthrough from "almost no catalytic activity" to "high-efficiency catalysis"; (3) the conversion rate of resveratrol is increased from 1.9% to 13.1%, and the relative activity is increased by more than 6 times.

[0046] Compared with the prior art F196Y single mutant: the conversion rates of equol, daidzein and resveratrol are increased by 2 times, 5 times and 7 times, respectively. It is particularly worth pointing out that the five-site combination mutant realizes the significant growth of catalytic activity for daidzein and resveratrol which are also difficult to be catalyzed by the F196Y single mutant, which can be explained as not a simple additive effect.

[0047] The above data fully prove that the five-site combination mutant not only significantly widens the substrate spectrum, but also realizes significant improvement in catalytic efficiency.

[0048] Example 3. Preparation of carbon glycoside product by large-scale reaction using PlUGT43 glycosyltransferase combination mutant 1. Preparation of product crude product by large-scale reaction using PlUGT43 glycosyltransferase combination mutant According to the protein expression method of PlUGT43 glycosyltransferase described in Example 1, about 100 g of wet bacterial cells were collected, resuspended in 1000 mL of reaction buffer, and ice-bathed for 30 min. Then, the bacterial cells were ultrasonically broken in batches under ice-bath condition for 40 min. Subsequently, the bacterial cells were centrifuged at 4℃ and 12000 rpm for 10 min, and the supernatant was collected to obtain the crude enzyme solution.

[0049] Large-scale reactions were carried out using the crude enzyme solution described above. The total reaction volume was 1000 mL, with a final substrate concentration of 0.5 mM and a final UDPG concentration of 0.75 mM. The reaction was carried out at 30 °C and 300 rpm for 16 h. After the reaction, twice the volume of methanol was added to precipitate the protein, and the mixture was sonicated for 3 min to fully release the product encapsulated in the precipitate. The mixture was then centrifuged at 8000 rpm for 10 min, and the supernatant was collected to obtain the crude product.

[0050] 2. Purification of crude C-glycoside products The crude product was purified by first concentrating it and then extracting it with ethyl acetate. The organic phase was washed twice each with ultrapure water and saturated sodium chloride solution. The washed organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Finally, the product was purified by silica gel column chromatography using dichloromethane / methanol (80:20 v / v) as the eluent to obtain the final product.

[0051] 3. Structural verification of the C-glycoside product 3.1. Mass spectrometry structure verification LC / MS analysis was performed using a Shimadzu LC-40D liquid chromatograph in tandem with an AB Sciex X 500B mass spectrometer. Chromatographic separation was performed on an Agilent ZORBAX SB-C18 column (4.6 mm × 250 mm, 5). The mass spectrometry was performed on a mobile phase consisting of an aqueous solution containing 0.1% formic acid (solvent A) and methanol (solvent B), at a flow rate of 0.8 mL / min. The gradient elution program is shown in Table 2. Mass spectrometry detection was performed using IDA mode.

[0052] In the reactions of the PlUGT43 glycosyltransferase combinatorial mutant with various substrates (daidzein, 7-hydroxyisoflavone, chickpea sprout A, equol, daidzein dihydrogenase, resveratrol, genistein, phloretin, and iris flavonoids), the corresponding substrates were successfully detected. C - Glycosylation products, mass spectrometry results are attached to the instruction manual. Figures 1-9 exhibit.

[0053] 3.2. Verification by Nuclear Magnetic Resonance Structure 1 H and 13 C NMR spectra were obtained from Bruker AVANCE III-500 type ( 1 H, 500 MHz) or BrukerAVANCE NEO 600 ( 1 H, 600 MHz; 13C, 150 MHz) were collected on a nuclear magnetic resonance spectrometer. The samples were dissolved in deuterated methanol with tetramethylsilane as an internal standard.

[0054] 3.3. 1H NMR data of the products 1 H and 13 C NMR data of the products Using the PlUGT43 glycosyltransferase combinatorial mutants, glycosylation products of daidzein, tectorigenin, 7-hydroxyisoflavone, and biochanin A were successfully prepared and analyzed by nuclear magnetic resonance. The 1H NMR data of each product is shown in the following table. 1 H and 13 C NMR data of the products Figures 10-16 The 1H NMR spectra are shown in the following figures.

[0055] Daidzein-8-C-glucoside: 1 H NMR (600 MHz, MeOD+H2O) δ 8.06 (s, 1H), 7.39(s, 1H), 7.32 (d, J = 8.6 Hz, 2H), 6.83 (d, J = 8.6 Hz, 2H), 5.03 (d, J =10.0 Hz, 1H), 4.31 (t, J = 9.2 Hz, 1H), 3.86 (s, 2H), 3.83 – 3.79 (m, 1H),3.74 – 3.69 (m, 2H), 3.53 (t, J = 9.5 Hz, 2H), 3.46 (s, 1H), 3.29 (s, 3H). 13 CNMR (150 MHz, MeOD+H2O) δ 177.80, 157.79, 153.39, 131.44, 130.89, 125.03,124.56, 116.64, 116.25, 111.91, 103.70, 82.11, 80.02, 75.78, 72.16, 71.44,64.03, 62.43, 61.97, 60.29, 56.42, 49.84. LC-MS (ESI) m / z: calcd. for C 22 H 22 O 10 [M+H] + : 447.13, found: 447.28. Tectorigenin-8-C-glucoside: 1H NMR (600 MHz, MeOD) δ 8.08 (s, 1H), 7.42 –7.37 (m, 1H), 7.12 (d, J = 8.1 Hz, 1H), 6.90 – 6.86 (m, 1H), 6.81 – 6.77 (m,1H), 4.97 (d, J = 9.7 Hz, 1H), 4.32 (t, J = 9.2 Hz, 1H), 3.88 (d, J = 11.8Hz, 2H), 3.80 (d, J = 5.9 Hz, 1H), 3.57 (d, J = 9.2 Hz, 1H), 3.52 (s, 1H), 3.46 (s, 1H), 3.38 (s, 3H), 3.12 (t, J = 7.7 Hz, 1H), 2.88 (t, J = 7.7 Hz,1H), 2.42 (s, 1H). LC-MS (ESI) m / z : calcd. for C 22 H 22 O 11 [M+H] + : 463.12, found: 463.27. 7-Hydroxyisoflavone-8-C-glucoside: 1 H NMR (600 MHz, MeOD) δ 8.25 (s, 1H), 8.05(d, J = 8.9 Hz, 1H), 7.59 – 7.55 (m, 2H), 7.48 – 7.43 (m, 2H), 7.41 (d, J =7.4 Hz, 1H), 6.98 (d, J = 8.9 Hz, 1H), 5.12 (d, J = 9.9 Hz, 1H), 4.26 – 4.21(m, 1H), 3.91 (dd, J = 12.0, 2.4 Hz, 1H), 3.83 – 3.78 (m, 1H), 3.58 (dd, J =9.5, 8.3 Hz, 2H), 3.54 – 3.49 (m, 1H). 13C NMR (150 MHz, MeOD) δ 177.88, 158.63, 154.86, 133.56, 130.20, 129.39, 129.01, 127.82, 125.47, 118.25, 117.14, 112.93, 82.61, 80.15, 75.77, 72.75, 71.64, 62.61. LC-MS (ESI) m / z : calcd. for C 21 H 21 O8 [M+H] + : 401.12, found: 401.43. Scytalone A-8-C-glucoside: 1 H NMR (600 MHz, MeOD) δ 8.18 (s, 1H), 7.50(d, J = 8.3 Hz, 2H), 7.00 (d, J = 8.3 Hz, 2H), 6.31 (d, J = 1.2 Hz, 1H), 4.95(d, J = 9.9 Hz, 1H), 4.12 (s, 1H), 3.89 (d, J = 12.0 Hz, 1H), 3.85 (s, 3H),3.74 (dd, J = 12.3, 5.6 Hz, 1H), 3.50 (d, J = 8.1 Hz, 2H), 3.45 (s, 1H). LC-MS (ESI) m / z : calcd. for C 22 H 22 O 10 [M+H] + : 447.13, found: 447.34. It should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit. Those skilled in the art should understand that, on the basis of obtaining the glycosyltransferase combination mutant and the coding gene thereof described in the present application, the combination mutant can be further constructed into various expression vectors known in the art, and transformed into suitable host cells (such as Escherichia coli, yeast, etc.) to express the combination mutant. The combination mutant can also be prepared into an immobilized enzyme by using conventional immobilization methods in the art (such as carrier adsorption, covalent cross-linking, embedding, etc.). These extended applications based on the combination mutant of the present application all fall within the concept and protection scope of the present application.

Claims

1. A glycosyltransferase combinatorial mutant, characterized in that, The combined mutant contains mutations at five sites: T81S, N186S, L187A, F196Y, and S310P, based on the amino acid sequence SEQ ID NO.3 of the wild-type PlUGT43 glycosyltransferase.

2. The glycosyltransferase combinatorial mutant according to claim 1, characterized in that, The amino acid sequence of the combined mutant is shown in SEQ ID NO.

5.

3. A gene encoding a gene, characterized in that, The encoding gene encodes the glycosyltransferase combination mutant of claim 1.

4. The encoding gene according to claim 3, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

6.

5. A recombinant vector, characterized in that, It includes the encoding gene as described in claim 3 or 4.

6. The combined mutant of claim 1 as a catalyst in C - Application in glycosylation reactions, characterized by The C Glycosylation is a reaction in which isoflavones, dihydroisoflavones, dihydrochalcones, or stilbene substrates are reacted with uridine diphosphate glucose under suitable reaction conditions and buffer.

7. The application according to claim 6, characterized in that, The substrate is selected from at least one of daidzein, 7-hydroxyisoflavone, chickpea sprout A, equol, daidzein dihydrogenase, phloretin, and resveratrol.

8. The application according to claim 6, characterized in that, The suitable reaction conditions are as follows: the amount of the glycosyltransferase combination mutant added is 0.1-10 mg / mL, the concentration of the substrate is 0.1-50 mM, the concentration of uridine diphosphate glucose is 0.15-75 mM, the reaction temperature is 25℃-35℃, the reaction pH is 7.0-9.0, and the reaction time is 0.5-24 h.

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