A glycosyltransferase mutant and its use in the in vitro enzymatic production of a variety of glycoside compounds

Abstract

The application provides a glycosyltransferase mutant and application of the glycosyltransferase mutant in in-vitro enzymatic production of various glycoside compounds, and belongs to the technical field of novel catalytic enzymes.The application provides a glycosyltransferase VcUGT1 mutant, and the mutant can be used as a biological catalyst, can efficiently utilize various different types of glycosyl donors, and generates corresponding glycoside products, such as apigenin 7-O-glucoside, apigenin 7-O-galactoside, apigenin 7-O-N-acetylglucosamine and apigenin 7-O-xyloside.Therefore, the mutant can increase the diversity of flavonoid glycoside compounds, and has important significance for establishment of a natural medicine library and drug screening.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of novel catalytic enzymes, and particularly relates to a glycosyltransferase mutant and application thereof in in vitro enzyme catalytic production of various glycoside compounds. BACKGROUND

[0002] Flavonoid glycosides are a class of plant secondary metabolites widely existing in nature. Flavonoid glycosides are derivatives of chroman, have a C6-C3-C6 basic skeleton, and form various types of flavones according to the differences in oxidation levels and substituents at different positions. Sugar substituents are divided into O-glycosides and C-glycosides according to glycoside chain types. The most common O-glycoside form is connected by a hydroxyl group on the aglycone and a sugar donor, while C-glycoside is formed by connecting the sugar moiety and the aglycone through a carbon-carbon bond. Most sugar group active donors are UDP-activated sugar-containing compounds, including UDP-glucose, UDP-galactose, UDP-glucuronide, UDP-N-acetylglucosamine, UDP-rhamnose, UDP-xylose and UDP-arabinose. Among them, UDP-glucose is a typical donor, and its glycosyltransferase is called UDP-glycosyltransferase (UGTs). When different sugar donors are combined with flavone aglycone, the water solubility and chemical stability are significantly improved. On the other hand, sugar donors not only play an important role in the structural diversity of flavonoids, but also have an important influence on their biological activity.

[0003] Apigenin, also known as apiin, is a flavonoid compound with various biological activities widely distributed in various vegetables and fruits. Studies have shown that apigenin exists in the form of stable glycosylation, and different sugar donors endow it with higher and broader biological activity. For example, apigenin-7-O-glucoside inhibits the activity of starch synthase in a dose-dependent manner; apigenin-7-O-galactoside exhibits good antioxidant and antitumor activity, and apigenin-7-O-xyloside has certain antibacterial activity.

[0004] Currently, the preparation of flavonoid glycosides containing different glycosyl donors mainly includes: 1) plant tissue extraction method is the most commonly used preparation method at present due to its relatively simple process flow and high separation effect, however, the method is greatly limited by geographical, environmental factors and plant growth cycle, and a large amount of organic waste is produced in the process of separation and purification, which cannot meet the requirements of green, environmental protection and sustainability. In addition, the content of most target flavonoid glycosides in plant tissues is very low, and the production cost is high. 2) Chemical synthesis method is used for flavonoid glycosides which are difficult to separate or have low content in plant tissues, but the traditional chemical synthesis method needs complex protection and deprotection steps, and harmful chemicals are also produced in the process, so it cannot meet the requirements of large-scale preparation. 3) UGTs are used to biosynthesize flavonoid glycoside compounds with different glycosyl donor types, which are concerned due to simple operation, low cost and green environmental protection, and have become the main means for preparing flavonoid glycoside compounds.

[0005] UGTs can directly synthesize flavonoid glycoside compounds by using active glycosyl donors with different flavonoid aglycones as substrates. However, most wild-type UGTs in nature can only use a single type of UDP-sugar as a glycosyl donor, and the catalytic efficiency is low, which greatly restricts the broadness of flavonoid glycosyl donors, thereby limiting the diversity of flavonoid glycoside compounds and their application in the field of biological medicine. Therefore, increasing the glycosyl diversity of flavonoid glycosides through protein engineering technology is an effective way to promote the development of natural medicines. SUMMARY

[0006] The application provides a glycosyltransferase mutant and application thereof in in-vitro enzyme catalysis production of various glycoside compounds, wherein the glycosyltransferase mutant can utilize various glycosyl donors to exert in-vitro enzyme catalysis, and synthesize flavonoid glycoside compounds of multiple donor types.

[0007] The application provides a glycosyltransferase VcUGT1 mutant, and the amino acid sequence is as shown in any one of the following (a) and (b):

[0008] (b) has more than 50% homology with (a), and the 14th amino acid is glycine, the 366th amino acid is histidine, and the 367th amino acid is glycine.

[0009] The application provides a coding gene of the glycosyltransferase VcUGT1 mutant.

[0010] Preferably, the coding gene comprises the nucleotide sequence shown in SEQ ID No. 2.

[0011] The application also provides a biological material comprising the coding gene.

[0012] Preferably, the type of the biological material comprises a recombinant expression vector and a recombinant expression host.

[0013] Preferably, the basic skeleton vector of the recombinant expression vector comprises a eukaryotic expression vector or a prokaryotic expression vector.

[0014] The host cell of the recombinant expression host comprises a bacterial cell, a fungal cell or a plant cell.

[0015] The application also provides the use of the above biological material in the preparation of the above-mentioned glycosyltransferase VcUGT1 mutant.

[0016] The application also provides the use of the glycosyltransferase VcUGT1 mutant prepared by the above-mentioned biological material in the in vitro enzyme catalytic production of flavonoid glycosides.

[0017] The application also provides a method for in vitro enzyme catalytic production of flavonoid glycosides, comprising using apigenin as a substrate acceptor, using the above-mentioned glycosyltransferase VcUGT1 mutant or the glycosyltransferase VcUGT1 mutant prepared by the above-mentioned biological material as an enzyme catalyst to catalyze the glycosylation reaction of a glycosyl donor, thereby preparing a flavonoid glycoside.

[0018] Preferably, the glycosyl donor comprises at least one of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine and UDP-xylose.

[0019] Beneficial effects: The application provides a glycosyltransferase VcUGT1 mutant, which can be obtained in large quantities by an expression system, and the obtained mutant can be used as a biological catalyst. Compared with the wild-type enzyme VcUGT1, using apigenin as a substrate and UDP-glucose as a donor, the mutant can increase the yield of the product apigenin 7-O-glucoside. Compared with the wild-type enzyme VcUGT1, the mutant of the application can efficiently utilize various types of glycosyl donors to generate corresponding glycoside products. For example, using the flavonoid apigenin as a substrate acceptor, UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine and UDP-xylose can be efficiently utilized to generate apigenin 7-O-glucoside, apigenin 7-O-galactoside, apigenin 7-O-N-acetylglucosamine and apigenin 7-O-xyloside, respectively. Therefore, the mutant of the application can increase the diversity of flavonoid glycosides, which is of great significance for the establishment of a natural drug library and drug screening. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is a pET32a-S14G-F366H-S367G plasmid map;

[0021] Figure 2Figure for SDS-PAGE electrophoresis result of pET32a-S14G-F366H-S367G recombinant E. coli expression;

[0022] Figure 3 Figure for high performance liquid chromatography (HPLC) result of wild type and mutant S14G-F366H-S367G with apigenin as substrate and UDP-glucose as donor;

[0023] Figure 4 Figure for mass spectrometry analysis (LC / MS) result of wild type and mutant S14G-F366H-S367G with apigenin as substrate and UDP-glucose as donor;

[0024] Figure 5 Figure for high performance liquid chromatography (HPLC) result of wild type and mutant S14G-F366H-S367G with apigenin as substrate and UDP-galactose as donor;

[0025] Figure 6 Figure for mass spectrometry analysis (LC / MS) result of wild type and mutant S14G-F366H-S367G with apigenin as substrate and UDP-galactose as donor;

[0026] Figure 7 Figure for high performance liquid chromatography (HPLC) result of wild type and mutant S14G-F366H-S367G with apigenin as substrate and UDP-N-acetylglucosamine as donor;

[0027] Figure 8 Figure for mass spectrometry analysis (LC / MS) result of wild type and mutant S14G-F366H-S367G with apigenin as substrate and UDP-N-acetylglucosamine as donor;

[0028] Figure 9 Figure for high performance liquid chromatography (HPLC) result of wild type and mutant S14G-F366H-S367G with apigenin as substrate and UDP-xylose as donor;

[0029] Figure 10 Figure for mass spectrometry analysis (LC / MS) result of wild type and mutant S14G-F366H-S367G with apigenin as substrate and UDP-xylose as donor. DETAILED DESCRIPTION

[0030] The present application provides a glycosyltransferase VcUGT1 mutant, the amino acid sequence is as shown in any one of the following: (a) SEQ ID No. 1: MSSHHHFLLLSCPGQGHLNPTLELAKKLTRADAHVTLATTVHGLRNITTLPAIENLSYATFSNGYDDGPDPTASYTVVMSEFKRVGSSTLTQLLTSLSTEGRPVTFLIYSILLPWAATVAREFHLPSAFLSIQSATSFAIYHSYFNTNNGLYPDKTTPPISIKLPGLPLFSSNEIPSFLLPDSPFVSVVPTFQEHIQTLEEDPNPVILANTFDELEQESIGAVDDMKVIPIGPLILSDRDPLQLNLFEKSTDYIQWLDSKPEKSVIYVSFGSMVVLQKKQIEEIFHGLLKTHRPFLWVIRKTEYQGEIKDLIETELNEEDGLIVPWCSQLEVLNHVSTGCFLTHCGWNSVLESLVAGVPVVGLPQHGDQMTNVKMVEEVWGNGVRAAVNGEGVVEREEVGRCLEVVMGGGEKGEEIRRKCEKWKGLGKGAMVEGAQGGSSHKNLEQFLERLG;

[0031] (b) has more than 50% homology with (a), and the 14th amino acid is glycine (Gly), the 366th amino acid is histidine (His), and the 367th amino acid is glycine (Gly).

[0032]

[0033] The present application provides a gene encoding the above-mentioned glycosyltransferase VcUGTl mutant.

[0034] The present application preferably uses the plasmid containing the gene encoding the glycosyltransferase VcUGTl shown in SEQ ID No. 4 as a template, and uses the primers shown in Table 1 to introduce point mutations.

[0035] Table 1 Primers used for cloning mutants

[0036] Primer name Primer sequence (5'-3') SEQ ID No. F366H-F cgcagCATgcggatcagatgactaa 5 F366H-R gatccgcATGctgcggcaaacccac 6 S367G-F cgcagcatGGCgatcagatgactaa 7 S367G-R ttagtcatctgatcGCCatgctgcg 8 S14G-F gccctGGCcaaggccacctcaacc 9 S14G-R ggccttgGCCagggcaggagaggag 10

[0037]

[0038] The application also provides a biological material containing the above-mentioned coding gene.

[0039] The type of the biological material of the application preferably includes a recombinant expression vector and a recombinant expression host, and the basic backbone vector of the recombinant expression vector preferably includes a eukaryotic expression vector or a prokaryotic expression vector, and more preferably includes a pET series vector, and in the embodiments, pET-32a is taken as an example for illustration, but it cannot be merely identified as the entire protection scope of the application.

[0040] The host cell of the recombinant expression host of the application preferably includes a bacterial cell, a fungal cell or a plant cell, and in the embodiments, a bacterium such as E. coli is taken as an example for illustration, but it cannot be merely identified as the entire protection scope of the application.

[0041] The application also provides an application of the above-mentioned biological material in preparing the above-mentioned glycosyltransferase VcUGT1 mutant.

[0042] In one specific embodiment of the application, pET-32a is preferably used as an expression vector to connect the mutant nucleotide sequence shown in SEQ ID No. 2 and the vector, and the mutant plasmid constructed is amplified in E. coli Top10. Then the mutant plasmid is extracted and transformed into E. coli BL21 to express the mutant protein shown in SEQ ID No. 1, and then extraction and purification are performed.

[0043] The application also provides an application of the glycosyltransferase VcUGT1 mutant prepared by using the above-mentioned biological material in in-vitro enzyme catalysis to produce flavone glycoside compounds.

[0044] The mutant of the application can be used as a catalytic enzyme to catalyze a variety of glycosyl donors to occur glycosylation reaction with apigenin as a substrate acceptor, thereby preparing a variety of flavone glycoside compounds, greatly improving the broadness of flavone glycoside donors, and providing a basis for the variety of flavone glycoside compounds and the application in the field of biological medicine.

[0045] The application also provides a method for in-vitro enzyme catalysis to produce flavone glycoside compounds, which comprises using apigenin as a substrate acceptor, using the above-mentioned glycosyltransferase VcUGT1 mutant or the glycosyltransferase VcUGT1 mutant prepared by using the above-mentioned biological material as an enzyme catalyst to catalyze a glycosyl donor to occur glycosylation reaction, thereby preparing a flavone glycoside compound.

[0046] The sugar donor of the present application preferably comprises at least one of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine and UDP-xylose. The mutant of the present application can efficiently utilize UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine and UDP-xylose to generate apigenin 7-O-glucoside, apigenin 7-O-galactoside, apigenin 7-O-N-acetylglucosamine and apigenin 7-O-xyloside, respectively.

[0047] The enzyme catalytic reaction of the present application, taking 200 μL as an example, preferably comprises: 50 mM Tris-HCl buffer with pH = 7.5, mutant protein amount of 20-50 μg, final concentration of apigenin substrate of 0.5 mM, final concentration of sugar donor of 1 mM, 0.5 mM CaCl2, and reaction temperature of 25°C, and reaction time of 16 h.

[0048] In order to further illustrate the present application, the application of the glycosyltransferase mutant and the in vitro enzyme catalytic production of various glycoside compounds provided by the present application are described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0049] Example 1: Obtaining of the glycosyltransferase mutant

[0050] 1. The glycosyltransferase mutant S14G-F366H-S367G is designed by using the wild-type glycosyltransferase pET32a-VcUGT1 plasmid derived from highbush blueberry as a template. The S14, F366 and S367 sites are mutated to obtain the mutant pET32a-S14G-F366H-S367G plasmid. Then the amplified mutant plasmid is transformed into E. coli Top10, and positive clones are screened and detected to obtain the recombinant engineering bacteria containing the pET32a-S14G-F366H-S367G plasmid.

[0051] (1) The wild-type glycosyltransferase VcUGT1 shown in SEQ ID No. 4 is inserted into the BamHI and XhoI sites of pET32a to construct the pET32a-VcUGT1 plasmid;

[0052] (2) The pET32a-VcUGT1 plasmid is used as a template, and the full-form gold FastMutagenesis System kit is used to first mutate the amino acid 14th site from serine to glycine, and the primer sequence is shown in Table 1.

[0053] PCR reaction system as follows: 1 ng pET32a-VcUGT1 plasmid, 25 μL 2 x TransStart@FastPfu Fly PCR SuperMix, 1 μL primer F (10 μM), 1 μL primer R (10 μM), and ddH2O to 50 μL. PCR amplification conditions: pre-denaturation 94 °C, 3 min; denaturation 94 °C, 20 s; annealing, 55 °C, 20 s; extension 72 °C, 2 min, a total of 25 cycles; complete extension 72 °C, 10 min; 4 °C storage. After the PCR reaction, 10 μL of the amplified product was subjected to 1% agarose gel electrophoresis detection. If the target plasmid was correctly amplified, the next step of DMT digestion experiment was performed.

[0054] (3) DMT enzyme digestion of PCR product, gel recovery and transformation

[0055] In the remaining 40 μL PCR amplification product, 1 μL DMT enzyme was added to digest the original template plasmid, and the reaction conditions were 37 °C, 1 h. After the reaction, the Gel Extraction Kit agarose gel recovery kit was used to recover and purify the digestion product according to the instructions. The gel recovery product was detected for concentration on a nanodrop and stored in a -20 °C refrigerator. 100 μL of the competent cells Top10 (TSC-C12) was thawed on ice, 5 μL of the purified digestion product was added, mixed gently, and incubated on ice for 30 min. It was quickly transferred to an ice water bath after 42 °C water bath for 60 s, and incubated for 5 min. Antibiotic-free LB medium 800 μL was added, and incubated at 37 °C, 200 rpm for 1 h. About 100 μL of the bacterial solution was spread on LB solid medium containing 50 μg / mL ampicillin, and incubated at 37 °C for 12 h. Three single colonies on the plate were picked into LB medium (50 μg / ml ampicillin), and incubated at 37 °C, 200 rpm for 12 h. Then 1 mL of the bacterial solution was taken for sequencing. The successfully mutated pET32a-VcUGT1-S14G plasmid was used as a template to PCR amplify the F366H mutant using the same method, and finally the pET32a-VcUGT1-S14G-F366H-S367G mutant plasmid shown in the formula was obtained. Figure 1

[0056] Example 2 Expression and purification of glycosyltransferase mutant

[0057] ​The recombinant vector pET32a-VcUGTl-S14G-F366H-S367G constructed in Example 1 was transformed into E. coli BL21 competent cells by chemical transformation. About 100 μL of the bacterial solution was spread on LB solid medium containing 50 μg / mL ampicillin and incubated at 37°C for 12 h. A single colony on the plate was picked into 5 mL of LB medium (50 μg / mL ampicillin) and incubated at 37°C, 200 rpm for 12 h. Then, it was transferred into 100 mL of LB medium (50 μg / mL ampicillin) and incubated at 37°C, 200 rpm for 4 h. When the OD 600 = 0.7, 10 μL of 1 M inducer IPTG was added and the culture was induced at 16°C, 180 rpm for 18 h.

[0058] After the induction, the bacterial solution was centrifuged at 4°C, 6000 rpm for 15 min to collect the bacterial cells. The bacterial cells were resuspended in PBS buffer and then broken by ultrasonic treatment in an ice water bath. The supernatant was collected by centrifugation at 4°C, 12000 rpm for 20 min, and then filtered through a 0.45 μm water filter. The His-cobalt ion column affinity purification was performed.

[0059] The purification steps were as follows: the cobalt ion column stored at 4°C was taken out and the storage solution in the column was drained. The cobalt ion column was washed with 20 mL of ddH2O. The column was first equilibrated with 10 mL of Equilibration Buffer, and then the 20 mL of filtered supernatant was passed through the column. The column was then washed with 8 mL of Equilibration Buffer and 15 mL of Wash buffer containing 15 mM imidazole, in sequence. Then, the target protein was eluted with 8 mL of Elution Buffer (containing 150 mM imidazole), which was collected in 1.5 mL centrifuge tubes in small portions (numbered 1-8). The molecular weight of the purified protein was verified by SDS-PAGE gel electrophoresis (Example 3), and its concentration was determined using a BCA protein concentration detection kit. Finally, the purified protein was concentrated and desalted using a Millipore ultrafiltration tube. An equal amount of 50% glycerol was added to each tube of the purified protein, mixed well, and stored at -80°C. Figure 2

[0060] Example 3 Detection of the catalytic activity of wild-type enzyme VcUGTl and mutant S14G-F366H-S367G on UDP-glucose

[0061] ​The catalytic activity of wild-type enzyme VcUGT1 and its mutant S14G-F366H-S367G was detected in a 1.5 mL EP tube, and the reaction system was: 50 mM Tris-HCl (pH 7.5), 0.5 mM CaCl2, 1 mM UDP-glucose, 0.5 mM apigenin, finally 30 μg of pure enzyme was added, and the total volume was 200 μL. The reaction condition was 25°C, 16h. After the reaction was completed, an equal volume of methanol was added to terminate the reaction, and centrifuged at 12000 rpm for 30 min. The supernatant was collected and filtered with a 0.22 μm organic filter membrane for HPLC, LC / MS analysis.

[0062] HPLC detection method: the liquid chromatography column was Welch Ultimate LP-C18 column (250mm×4.6mm, 5.0μm). The mobile phase was methanol (A) containing 0.1% formic acid and 0.1% formic acid water (B), and the gradient elution program was as follows: 0-1.5min, 35% A; 1.5-7min, 35-80% A; 7-12min, 80% A, 12-13min, 80-35% A; 13-17min, 35% A. The detection wavelength was 290nm, the flow rate was 1.0mL / min, the column temperature was 30°C, and the sample injection amount was 20μL.

[0063] LC / MS detection method: In order to further confirm the obtained product, Agilent Q-TOF 6540 liquid chromatography mass spectrometry (LC / MS) was used for detection. The mass spectrometry condition: the ion source was in ion mode, the voltage was 3500V; the fragmentation voltage was 175V; the cone hole voltage was 65V; the radio frequency voltage was 750V, and the range was negative ion mode (m / z 150-1200). The chromatography condition: the column used was Welch Ultimate LP-C18 column (250mm×4.6mm, 5.0μm), and the flow rate was 1.0ml / min. The mobile phase was methanol (A) containing 0.1% formic acid and 0.1% formic acid water (B), and the gradient was 0-1.5min, 35% A; 1.5-7min, 35-80% A; 7-12min, 80% A, 12-13min, 80-35% A; 13-17min, 35% A.

[0064] From Figure 3 and Figure 4 It can be seen that the mutant S14G-F366H-S367G catalyzes apigenin to generate apigenin 7-O-glucoside (product a) with high efficiency of UDP-glucoside, and the conversion rate reaches 65%. The utilization rate of wild-type enzyme to UDP-glucose is low, and the conversion rate is about 6%. The mutant S14G-F366H-S367G has higher utilization rate of UDP-glucose than the wild-type, and has stronger catalytic activity.

[0065] Example 4 Detection of catalytic activity of wild type enzyme VcUGT1 and mutant S14G-F366H-S367G on UDP-galactose

[0066] The detection of catalytic activity of wild type enzyme VcUGT1 and mutant S14G-F366H-S367G was carried out in 1.5 mL EP tubes with the reaction system of 50 mM Tris-HCl (pH 7.5), 0.5 mM CaCl2, 1 mM UDP-galactose, 0.5 mM apigenin, and finally 30 μg of pure enzyme, with a total volume of 200 μL. The reaction condition was 25 °C for 16 h. After the reaction was completed, an equal volume of methanol was added to terminate the reaction, and centrifuged at 12000 rpm for 30 min. The supernatant was collected and filtered with a 0.22 μm organic filter membrane before being used for HPLC and LC / MS analysis.

[0067] The HPLC detection method and LC / MS detection method are as described in Example 3.

[0068] From Figure 5 and Figure 6 It can be seen that mutant S14G-F366H-S367G catalyzes apigenin to generate apigenin 7-O-galactoside (product b) from UDP-galactose with high efficiency, with a conversion rate of 30%. The wild type enzyme has no catalytic activity on UDP-galactose. Compared with the wild type, mutant S14G-F366H-S367G increases the sugar donor versatility.

[0069] Example 5 Detection of catalytic activity of wild type enzyme VcUGT1 and mutant S14G-F366H-S367G on UDP-N-acetylglucosamine

[0070] The detection of catalytic activity of wild type enzyme VcUGT1 and mutant S14G-F366H-S367G was carried out in 1.5 mL EP tubes with the reaction system of 50 mM Tris-HCl (pH 7.5), 0.5 mM CaCl2, 1 mM UDP-N-acetylglucosamine, 0.5 mM apigenin, and finally 30 μg of pure enzyme, with a total volume of 200 μL. The reaction condition was 25 °C for 16 h. After the reaction was completed, an equal volume of methanol was added to terminate the reaction, and centrifuged at 12000 rpm for 30 min. The supernatant was collected and filtered with a 0.22 μm organic filter membrane before being used for HPLC and LC / MS analysis.

[0071] The HPLC detection method and LC / MS detection method are as described in Example 3.

[0072] From Figure 7 and Figure 8It can be seen that mutant S14G-F366H-S367G catalyzes apigenin to generate apigenin 7-O-xylose (product d) using UDP-xylose efficiently, and the conversion rate is about 85%. The wild-type enzyme has no catalytic activity on UDP-xylose. Compared with the wild-type, mutant S14G-F366H-S367G increases the sugar donor versatility and the structural richness of flavonoid glycosides.

[0073] Example 6 Detection of catalytic activity of wild-type enzyme VcUGT1 and mutant S14G-F366H-S367G on UDP-xylose

[0074] The detection of catalytic activity of wild-type enzyme VcUGT1 and mutant S14G-F366H-S367G was carried out in a 1.5 mL EP tube, and the reaction system was: 50 mM Tris-HCl (pH 7.5), 0.5 mM CaCl2, 1 mM UDP-xylose, 0.5 mM apigenin, and finally 30 μg of pure enzyme was added, and the total volume was 200 μL. The reaction condition was 25℃, 16h. After the reaction was completed, an equal volume of methanol was added to terminate the reaction, and centrifuged at 12000 rpm for 30 min. The supernatant was collected and filtered with a 0.22 μm organic filter membrane for HPLC and LC / MS analysis.

[0075] The HPLC detection method and LC / MS detection method are as described in Example 3.

[0076] From Figure 9 and Figure 10 It can be seen that mutant S14G-F366H-S367G catalyzes apigenin to generate apigenin 7-O-xylose (product d) using UDP-xylose efficiently, and the conversion rate is about 85%. The wild-type enzyme has no catalytic activity on UDP-xylose. Compared with the wild-type, mutant S14G-F366H-S367G increases the sugar donor versatility and the structural richness of flavonoid glycosides.

[0077] Although the above examples have described the present application in detail, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which are within the protection scope of the present application.

Claims

1. A glycosyltransferase VcUGT1 mutant, characterized in that, The amino acid sequence is shown in SEQ ID No.

1.

2. The encoding gene of the glycosyltransferase VcUGT1 mutant as described in claim 1.

3. The encoding gene according to claim 2, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID No.

2.

4. Biological material comprising the encoding gene as described in claim 2 or 3.

5. The biomaterial according to claim 4, characterized in that, The types of biomaterials include recombinant expression vectors and recombinant expression hosts.

6. The biomaterial according to claim 5, characterized in that, The basic scaffold vector of the recombinant expression vector includes a eukaryotic expression vector or a prokaryotic expression vector; The host cells of the recombinant expression host include bacterial cells or fungal cells.

7. The use of the encoding gene of claim 2 or 3 or the biological material of any one of claims 4 to 6 in the preparation of the glycosyltransferase VcUGT1 mutant of claim 1.

8. The application of the VcUGT1 mutant glycosyltransferase prepared using the biomaterials described in any one of claims 4 to 6 in the in vitro enzymatic production of flavonoid glycosides, characterized in that, The flavonoid glycosides include: apigenin 7-O-glucoside, apigenin 7-O-galactoside, apigenin 7-ON-acetylglucosamine, and apigenin 7-O-xylose.

9. A method for the in vitro enzyme-catalyzed production of flavonoid glycosides, characterized in that, The method includes using apigenin as a substrate acceptor, and using the glycosyltransferase VcUGT1 mutant of claim 1 or the glycosyltransferase VcUGT1 mutant prepared from any one of the biological materials of claims 4 to 6 as an enzyme catalyst to catalyze the glycosylation reaction of the glycosyl donor to prepare flavonoid glycosides. The glycosyl donor includes at least one of the following: UDP-glucose, UDP-galactose, UDP- N - Acetylglucosamine and UDP-xylose; The flavonoid glycosides include: apigenin 7-O-glucoside, apigenin 7-O-galactoside, apigenin 7-ON-acetylglucosamine, and apigenin 7-O-xylose.

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