CN117343919B

CN117343919B - Flavonoid dihydroxyl site glycosyltransferase and application thereof

Info

Publication number: CN117343919B
Application number: CN202311284570.3A
Authority: CN
Inventors: 易善勇; 陆海波; 李明智
Original assignee: Guangzhou Baishu Biotechnology Co ltd
Current assignee: Guangzhou Baishu Biotechnology Co ltd
Priority date: 2023-10-07
Filing date: 2023-10-07
Publication date: 2024-04-19
Anticipated expiration: 2043-10-07
Also published as: CN117343919A

Abstract

The invention discloses an application of glycosyltransferase coded by dendrobium huoshanense DhUGT genes in biosynthesis of flavonoid 7,4' -di-O-glucoside, and belongs to the technical field of biology. The DhUGT gene nucleotide sequence is shown as SEQ ID NO.1, or is complementary and paired with the sequence shown as SEQ ID NO.1, or is the nucleotide sequence of the amino acid sequence shown as SEQ ID NO. 2. The flavonoid 7,4 '-di-O-glucoside is obtained by utilizing DhUGT protein to perform enzyme-catalyzed biosynthesis in vitro, thus providing a novel method for synthesizing the flavonoid 7,4' -di-O-glucoside.

Description

Flavonoid dihydroxyl site glycosyltransferase and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a glycosylation method of flavonoids.

Background

Flavonoids are a class of active natural compounds widely existing in nature, and are classified into various types of flavones, flavonols, flavanones, isoflavones, etc. according to the difference in the C3 ring structure in the basic skeleton thereof. Modern pharmacological results show that flavonoids have a plurality of pharmacological activities such as antioxidation, anti-inflammatory, antivirus and the like, and are a source treasury for searching new drug development lead compounds. However, naturally occurring flavonoids often have the disadvantages of poor water solubility, low stability, poor selectivity and the like, and greatly limit the search and development of new adaptive drugs. Glycosylation is an important form of structural modification of secondary metabolites, and is one of the main factors leading to structural diversity of flavonoids, and most flavonoid glycosides have smaller toxic and side effects and higher stability and bioavailability compared with aglycones in clinic. Therefore, glycosylation modification of some flavones can help to develop better application direction clinically.

In recent years, research on chemical and biological synthesis methods of glycosylation modification has been greatly advanced; however, the chemical glycosylation reaction has the defects of more byproducts and intermediates, poor regioselectivity and stereoselectivity, low yield, complicated protection deprotection steps, large pollution and the like, and in addition, the chemical synthesis method has a plurality of short plates in mass production of glycoside due to low atomic efficiency and high price of partial catalyst. In contrast, the biosynthesis method using enzyme catalysis has not only stereo and regioselectivity, but also relatively simple operation steps, less pollution to the environment, and accords with the green chemistry concept, so the biosynthesis of the synthesized product using tool enzyme catalysis gradually becomes the research focus in the field. Flavonoid-7, 4 '-di-O-glucosyltransferase, a glycosyltransferase (UDP-glycosyltransferase, UGT) with uridine diphosphate (Uridine diphosphate, UDP) -activated sugar molecules as glycosyl donors, specifically catalyzes the glycosylation of the hydroxyl groups at the 7-position and the 4' -position of the B-ring of flavonoids. Among the flavonoid glycosyltransferases found, those catalyzing glycosylation of hydroxyl groups at the 3 and 7 positions are most abundant, while 7 and 4' flavonoid glycosyltransferases are rarely reported.

Disclosure of Invention

The invention mainly aims at the technical problems and provides a novel flavonoid glycosylation method to solve the defects of the existing chemical synthesis of flavonoid 7,4' -di-O-glucoside series.

Specifically, the invention provides the following technical scheme:

In one aspect, the present invention provides a glycosyltransferase gene having a sequence encoding a nucleotide sequence comprising an amino acid sequence as set forth in SEQ ID NO.4, or a sequence that is complementary to and pairs with a nucleotide sequence encoding an amino acid sequence as set forth in SEQ ID NO. 4.

In a preferred embodiment, the glycosyltransferase gene sequence is a sequence encoding a nucleotide sequence comprising the amino acid sequence shown as SEQ ID NO.3, or a sequence complementary to a nucleotide sequence encoding an amino acid sequence shown as SEQ ID NO. 3.

In a preferred embodiment, the glycosyltransferase gene sequence is a sequence encoding a nucleotide sequence comprising the amino acid sequence shown as SEQ ID NO.2, or a sequence complementary to a nucleotide sequence encoding an amino acid sequence shown as SEQ ID NO. 2.

In a preferred embodiment, the glycosyltransferase gene sequence is a nucleotide sequence encoding the amino acid sequence shown as SEQ ID NO.2, or a sequence that is complementary to and pairs with the nucleotide sequence encoding the amino acid sequence shown as SEQ ID NO. 2.

In a preferred embodiment, the nucleotide sequence of the glycosyltransferase gene is shown as SEQ ID NO.1 or the nucleotide sequence of the glycosyltransferase gene is complementary and matched with the sequence shown as SEQ ID NO. 1.

In another aspect, the present invention provides a glycosyltransferase having an amino acid sequence comprising a sequence as set forth in SEQ ID NO. 4.

In a preferred embodiment, the amino acid sequence of the glycosyltransferase comprises the sequence shown in SEQ ID NO. 3.

In a preferred embodiment, the amino acid sequence of the above glycosyltransferase is a sequence comprising the sequence shown as SEQ ID NO. 2.

In a preferred embodiment, the amino acid sequence of the glycosyltransferase is as shown in SEQ ID NO. 2.

In another aspect, the invention provides the use of a glycosyltransferase gene, which has a nucleic acid sequence encoding a nucleotide sequence comprising an amino acid sequence as shown in SEQ ID NO.4 or a sequence complementary to and paired with a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.4, for the synthesis of flavonoid 7,4' -di-O-glucosides.

In another aspect, the invention provides the use of a glycosyltransferase comprising the amino acid sequence shown in SEQ ID NO.4 for the synthesis of flavonoid 7,4' -di-O-glucoside series glycoside compounds.

In a preferred embodiment, the amino acid sequence of the glycosyltransferase comprises the sequence shown in SEQ ID NO. 2.

In a preferred embodiment, the amino acid sequence of the glycosyltransferase is as shown in SEQ ID NO. 3.

In another aspect, the present invention provides a method for synthesizing flavonoid 7,4' -di-O-glucoside series glycoside compounds, said method comprising the steps of:

1) Obtaining a glycosyltransferase comprising an amino acid sequence as set forth in SEQ ID NO. 4;

2) And (2) catalyzing and synthesizing flavonoid 7,4' -di-O-glucoside series glycoside compounds in an enzyme activity reaction system by utilizing glycosyltransferase in the step (1).

In a preferred embodiment, the above glycosyltransferases are obtained by prokaryotic expression.

In a preferred embodiment, the above glycosyltransferase is obtained by chemical synthesis.

In a preferred embodiment, the enzyme activity reaction system comprises the above glycosyltransferase, UDP-Glc and flavonoid.

In a preferred embodiment, the enzyme activity reaction system further comprises a divalent cation.

In a preferred embodiment, the reaction temperature of the enzyme activity reaction system is 20-42 ℃.

In a preferred embodiment, the pH of the enzyme reaction system is from 8.0 to 10.0.

In a preferred embodiment, the enzymatic reaction system is specifically 20. Mu.g purified glycosyltransferase, 5mM Mg ²⁺, 14mM beta-mercaptoethanol, 5mM UDP-Glc, 0.5mM flavonoid, supplemented to 200. Mu.L with 50mM Tris-HCl buffer pH=9.0, and reacted at 50℃for at least 2h.

In a preferred embodiment, the enzyme activity reaction system is an equal proportion expansion or contraction of the content of each component of the reaction system, and the pH=9.0 is reacted for at least 2 hours at 50 ℃.

In a preferred embodiment, the flavonoid compound is any one of apigenin, luteolin, kaempferol and quercetin.

Compared with the prior art, the invention has the following advantages:

1) Compared with the prior art, the flavonoid 7,4' -di-O-glucoside is obtained by the enzyme-catalyzed biosynthesis method for the first time, and has the advantages of simpler steps, less pollution, more single product and the like compared with the chemical synthesis method.

2) The glycosyltransferase of the invention can convert substrate flavonoid in a short time (within 2 h) to 95 percent

The above.

3) The glycosyltransferase in the invention has a wide temperature range (20-42 ℃) which is suitable for reflection.

4) The glycosyltransferase in the invention has no metal ion dependence, but Mg ²⁺ can improve the catalytic activity.

Drawings

The method of the present invention and its advantageous effects will be described in detail below with reference to the accompanying drawings and detailed description.

FIG. 1 is a catalytic flow diagram of flavonoid-7, 4' -di-O-glucosyltransferase wherein compounds 1-4 are apigenin, luteolin, kaempferol, quercetin, respectively;

FIG. 2 is a diagram showing the result of electrophoresis of a target gene fragment amplified by using dendrobium huoshanense stem cDNA as a template, wherein M is DNA MARKER (2 kbp), and 1 is the target gene fragment;

FIG. 3 is a diagram showing the electrophoretically-verified Nde I and BamHI double-digested recombinant plasmid, wherein M is DNA MARKER (5 kbp), and 1 is pET-28a (+) -DhUGT35 double-digested fragment;

FIG. 4 shows an SDS-PAGE gel electrophoresis of pET-28a (+) -DhUGT fusion protein (Marker size 10-150 kDa), wherein 1 is crude enzyme and 2 is purified protein;

FIG. 5 is a graph showing the catalytic result of pET-28a (+) -DhUGT fusion protein on substrate kaempferol, wherein A is a mass spectrum detection graph of the reaction end product; b is a liquid phase detection diagram of a reaction result;

FIG. 6 is a graph showing the results of enzymatic kinetic parameters of pET-28a (+) -DhUGT fusion protein, wherein A is the reaction temperature, B is the reaction pH, C is the divalent metal ion, D is the reaction time, E is the Michaelis constant of the enzyme pair using kaempferol as the catalytic substrate.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

Example 1

Cloning of the target glycosyltransferase Gene

1. Extracting dendrobium huoshanense stem RNA, reversely transcribing the dendrobium huoshanense stem RNA into cDNA according to the operation instruction of a reverse transcription kit (R333, nanjinouzan biotechnology Co., ltd.), and cloning a target gene by utilizing a PCR technology, wherein specific primer information is as follows:

an upstream primer: 5'ATGGAAAAGCCGCAGCAGC 3'

A downstream primer: 5'TCAGCTTGCTATCTCCTCCACA 3'

The PCR reaction system is as follows: 2X Phanta Max Master Mix (Dye Plus) (P515, nanjinouzan Biotech Co., ltd.) 25. Mu.L, 2. Mu.L each of the upstream and downstream primers, 50-100ng of template, and dd H2O to 50. Mu.L. The PCR amplification procedure was: ① Reacting for 3min at 95 ℃; ② Reacting for 15s at 95 ℃, 45s at 60 ℃ and 60s at 72 ℃ for 35 times in a total cycle; ③ Extending at 72℃for 5min. After the reaction, the size of the product was detected by 1% agarose gel electrophoresis, the electrophoresis pattern is shown in FIG. 1 (M: DL 2000marker;Lane 1:DhUGT35 gene)

2. The amplified product was connected to T carrier pClone Blunt (TSV-007 BS, beijing qing biotechnology Co., ltd.) and transformed into E.coli DH 5. Alpha. And single colony was selected and cultured in LB medium and subjected to bacterial liquid PCR verification, the bacterial liquid PCR reaction system was: 12.5 mu L GREEN TAQ Mix (P131, nanjinouzan Biotechnology Co., ltd.) and 1. Mu.L of each of the upstream and downstream primers, 2. Mu.L of the template (bacterial liquid), were supplemented to 25. Mu.L with dd H ₂ O. Samples with correct bacterial liquid PCR results are sent to sequencing.

The nucleotide sequence obtained by sequencing is shown as SEQ ID NO. 1. As shown in SEQ ID NO.1 and SEQ ID NO.2, the gene sequence contains 1404 nucleotides and codes 467 amino acids.

SEQ ID NO.1

ATGGAAAAGCCGCAGCAGCACATCCTGGTGGCCTGCTATCCTGCCCAAGGCCACATAAACCCCACCCTCCGCCTCTCCAAACGCCTAGCCCATGAAGCAGGAGTTCGAATCACCTTCTCCACCGCCGTCAACGCCCACCGCCGCATGTTCCCAAACCAAGAAGCAGGAGTACTCCATGACGATCTCTTTGCTTACATCCCCTATTCCAATGGTTATGACGACGGCTTCGACCCTAAGATACACGACATTAGAACAACCATGTACGACAGCAAGCAAAATGGCTCCAGAAGCCTCGCCGCCATCGTTGATGATCAAGCCGCACGCGGCCAACCCGTCACCTGCATAATCTACTCTTTCTTCATGTCGTGGGCAGCCGAAATAGCTCGCGAACGCGGCATCCCATCCTTCCTTTTCTGGATCCAGCCCGCCACAGCTTTCGCGGTCTATTGGCACTACTTTCACGACTACAAGAGCATCCTGGCCGCCAACGTTAGTGACCCGTTAAGCGTCGTGAACCTTCCCGGGCTGCCTCCGATGAGGATCCGTGATCTTCCCTCCTTCGTAACCGAAAACTCACCTGACGGACCCTATGCTGTATTTACTCCTTTTATTCGGGAGATGTTTGAGATGCTGGACACGGAGATTTCGATACAGAGCTCGAAGCCAATGGTGATCTCGAACACGGCAGATGAGATGGAAGCGTCGGCGATTATGTCTGTAGCCGATGAGGTAAATATCGTCGGAATTGGTCCGTTGCTGGAGGATACAGTGGAAGGGAGTTTGTTTAAGTCGGATGATAAGAATGAGTACATGGATTGGCTCGATAGGCAGGAGGAGAGGTCGGTGGTGTATGTTTCTTTTGGGAGCATCTCAGTGCTGCAGAAGGATCAGGTAGAGGAAATATGGAAAGGGTTGAGGCGGAGCGGGAGGCCTTATTTATGGGTGGTGAGGAAGGACAATAGGTGGGAAGGGCAGGAACTTGAAGAAGATGAGGATGAGGAAGTGAAGAAGGGGATGGTGGTGGAGTGGTGTTCGCAGGGGAGGGTGCTTGGGCACAAGGCGGTGGGATGTTTCGTGACGCACTGCGGGTGGAATTCAACGGTGGAGAGTCTTTTGTGTGGCGTGCCGACGGTAGCGGTGCCACAATGGACGGATCAGTTAACGAATGCGTGGCTGATGGAGAAGGCGTGGGGGACGGGTGTGAGGGCGGAGGTGAGCAAGGATGGGTTAATGGAAGCAGACGAGTTGCGACGGTGCCTTGAGATTGTGATGGGGCAGGGGGAAAGACCGAAGGAAATTAGGAGGAAAGTGGATATGTGGAAGGAGAAAACTCGTCAGGCGACTGTAGCAGGAGGTTCATCGCATAGAAACCTAATGGCATTTGTGGAGGAGATAGCAAGCTGA

SEQ ID NO.2

MEKPQQHILVACYPAQGHINPTLRLSKRLAHEAGVRITFSTAVNAHRRMFPNQEAGVLHDDLFAYIPYSNGYDDGFDPKIHDIRTTMYDSKQNGSRSLAAIVDDQAARGQPVTCIIYSFFMSWAAEIARERGIPSFLFWIQPATAFAVYWHYFHDYKSILAANVSDPLSVVNLPGLPPMRIRDLPSFVTENSPDGPYAVFTPFIREMFEMLDTEISIQSSKPMVISNTADEMEASAIMSVADEVNIVGIGPLLEDTVEGSLFKSDDKNEYMDWLDRQEERSVVYVSFGSISVLQKDQVEEIWKGLRRSGRPYLWVVRKDNRWEGQELEEDEDEEVKKGMVVEWCSQGRVLGHKAVGCFVTHCGWNSTVESLLCGVPTVAVPQWTDQLTNAWLMEKAWGTGVRAEVSKDGLMEADELRRCLEIVMGQGERPKEIRRKVDMWKEKTRQATVAGGSSHRNLMAFVEEIAS

Example 2

Conserved domain prediction

The sequence of SEQ ID NO.2 of example 1 was submitted to InterProScan prediction of its conserved domain (CDD and pFAM databases selected) and the result showed that the sequence contained the GT1_ Gtf-like domain of the CDD database and the UDPGT domain of the Pfam database.

The GT1_ Gtf-like domain is the sequence shown as SEQ ID NO.3, and the UDPGT domain is the sequence shown as SEQ ID NO. 4. The above 2 sequences are all glycosyltransferase conserved domains, and may be related to the specific catalysis of SEQ ID NO. 2.

SEQ ID NO.3：

HILVACYPAQGHINPTLRLSKRLAHEAGVRITFSTAVNAHRRMFPNQEAGVLHDDLFAYIPYSNGYDDGFDPKIHDIRTTMYDSKQNGSRSLAAIVDDQAARGQPVTCIIYSFFMSWAAEIARERGIPSFLFWIQPATAFAVYWHYFHDYKSILAANVSDPLSVVNLPGLPPMRIRDLPSFVTENSPDGPYAVFTPFIREMFEMLDTEISIQSSKPMVISNTADEMEASAIMSVADEVNIVGIGPLLEDTVEGSLFKSDDKNEYMDWLDRQEERSVVYVSFGSISVLQKDQVEEIWKGLRRSGRPYLWVVRKDNRWEGQELEEDEDEEVKKGMVVEWCSQGRVLGHKAVGCFVTHCGWNSTVESLLCGVPTVAVPQWTDQLTNAWLMEKAWGTGVRAEVSKDGLMEADELRRCLEIVMGQGERPKEIRRKVDMWKE

SEQ ID NO.4：

EYMDWLDRQEERSVVYVSFGSISVLQKDQVEEIWKGLRRSGRPYLWVVRKDNRWEGQELEEDEDEEVKKGMVVEWCSQGRVLGHKAVGCFVTHCGWNSTVESLLCGVPTVAVPQWTDQLTNAWLMEKAWGTGVRAEVSKDGLMEADELRRCLEIVMG

Example 3

Construction of pET-28a (+) -DhUGT recombinant plasmid and induction expression of fusion protein

1. The glycerol-preserved pET-28a (+) strain solution was taken out from the-80℃refrigerator, 500mL of LB medium, 500. Mu. L KaN ⁺ (50 mg/mL) and 1mL of glycerol strain were added to a 1000mL Erlenmeyer flask, and the mixture was placed in a shaking box at 37℃and cultured at 200rpm for 16 hours. The pET-28a (+) plasmid extracted from the bacterial cells was digested simultaneously with BamH I (1010S, bosun doctor technology (Beijing) Co., ltd.) and Nde I (1161A, bosun doctor technology (Beijing) Co., ltd.) restriction enzymes. The reaction system was 50. Mu.L of the reaction solution containing not more than 1. Mu.g of the plasmid, 1. Mu.L of each of the two restriction enzymes, 5. Mu.L of 10 XK buffer, and the reaction was carried out at 37℃for 3 hours or more with dd H ₂ O, and the linearized vector was recovered by 1% agarose gel electrophoresis and gel recovery kit (DC 301, nannunozan Biotechnology Co., ltd.).

2. The pClone Blunt-DhUGT plasmid with correct sequence is used as a template to amplify a target gene fragment containing a homology arm, and the sequence of an amplification primer is as follows: an upstream primer: 5'gtgccgcgcggcagccatatgATGGAAAAGCCGCAGCAGC 3', downstream primer: 5'acggagctcgaattcggatccTCAGCTTGCTATCTCCTCCACA 3'.

3. The product was recovered with a gel recovery kit and the gene of interest was ligated into a linearized vector by homologous recombination enzyme (C112, nanjinouzan Biotechnology Co., ltd.) and the ligation product was transformed into DH 5. Alpha. Competent cells by: sucking 9 mu L of the connection product, slowly pumping into the center of 100 mu L of competent cells, flicking the tube wall, throwing the bacterial liquid to the bottom of the tube through the light swing of the wrist, and then placing the tube on crushed ice for standing for 30min. Centrifuge tubes were placed in a 42℃water bath for 45s, and shaking should be avoided during this process. 500. Mu.L of LB medium was added to the tube, and the mixture was shaken at 37℃and 200rpm for 1 hour, followed by application to the surface of the solid LB medium. After the clone grows out, the bacterial liquid PCR detection is carried out, and the bacterial liquid with correct result is sent to a biological company for sequencing.

4. As shown in FIG. 3, the recombinant plasmid obtained was digested with Nde I and BamH I restriction enzymes to obtain two bands, wherein a pET-28a (+) linearized vector fragment was located around 5k, and a band between 1 and 2k was the target gene fragment, indicating that a gene was successfully inserted into the vector pET-28a (+).

3. The obtained recombinant plasmid is transformed into escherichia coli BL21 (DE 3) cells, positive clones are selected and cultured in LB culture medium, when OD ₆₀₀ = 0.6-0.8 (taking sterile LB culture medium as a control), isopropyl thiogalactoside (IPTG) is added to a final concentration of 0.5mM, and the target protein is induced to be expressed at 16 ℃ overnight.

4. And collecting the bacteria after induced expression, suspending in Tris-HCl buffer solution with pH value of 8.0, and performing ultrasonic crushing to obtain supernatant which is crude enzyme solution. Purifying target protein by using nickel affinity chromatography column according to His-Tag label on fusion protein, eluting target protein by using imidazole solution with different concentrations, and detecting molecular weight and purity of target protein by 10% SDS-PAGE electrophoresis. As shown in FIG. 4, the crude enzyme electrophoresis result has more bands, and the expression of the target gene cannot be judged; the purified protease solution has single band in an electrophoresis chart, the size of the band is consistent with the molecular weight of a theoretical protein plus a recombination label, the protein is successfully expressed in supernatant, and the purified protein can be used for subsequent experiments. To increase the concentration of the purified protein, the target protein was concentrated by centrifugation using a 30kDa ultrafiltration tube, and the protein concentration was detected using BCA protein quantification kit (E112, nanjinouzan Biotechnology Co., ltd.). The concentration was 1.65. Mu.g/mL.

Example 4

In vitro enzyme activity assay

1. Enzymatic activity reaction system: 10. Mu.g of purified protein, 5mM Mg ²⁺, 14mM beta-mercaptoethanol, 5mM UDP-Glc, 0.5mM flavonoids (apigenin, luteolin, kaempferol, quercetin) were added to a 1.5ml centrifuge tube, 50mM Tris-HCl buffer, pH=8.0 was used to supplement 200. Mu.L, reacted for 1h in a 30℃water bath, after the reaction was completed, three times of ethyl acetate was used to extract, the combined extracts were dried by spin-drying at 45℃on a rotary evaporator, then sonicated with methanol, centrifuged at 14000rpm for 5min, and the supernatant was taken for LC-MS detection.

2. HPLC conditions

HPLC model: waters ACQUITY Arc A

Mobile phase: phase A: 0.1% formic acid in water; and B phase: acetonitrile.

Elution gradient ：0-5min：5％-20％B;5-8min：20％-22％B;8-17min：22-25％B;17-23min：25-35％B;23-25min：35-50％B;25-32min：50-95％B;32-37min：95％ B;37-38min：95-5％ B;38-42min：5％ B.

Detection wavelength: 336nm.

3. LC-MS conditions:

Chromatographic column model: waters ACQUITY UPLC BEH C ₁₈ column (2.1X100 mm,1.7 μm)

Mobile phase: phase A: 0.1% formic acid in water; and B phase: acetonitrile.

Elution gradient ：0-0.5min：5％ B;0.5-1.5min：5-10％ B;1.5-4min：10-25％ B;4-7min：25-35％ B;7-9.5min：35-65％ B;9.5-10.5min：65-85％ B;10.5-11.5min：85-95％ B;11.5-12.5min：95％ B;12.5-13.5min：95-5％ B;13.5-15min：5％ B.

Mass spectrometry conditions: an electrospray ion source; the acquisition mode is AutoMS ²; a negative ion mode; capillary voltage 3500V; sheath gas temperature is 350 ℃ and flow rate is 11L/min; the temperature of the drying gas is 325 ℃, and the flow rate is 8L/min; the scanning range of the mass-to-charge ratio is 100-1700m/z; a collision voltage 175V; the collision energy is 15-50eV.

5. Detection result:

By comparing the retention time and relative molecular mass of the product standard, the catalytic end product mass-to-charge ratio of the recombinase pET-28a (+) -DhUGT to the flavonoid substrate is found to be 324 more than that of the substrate (the increased molecular weight of the product is the difference between two glucose and two water molecules removed), and the product is proved to be flavonoid diglucoside. Because apigenin, luteolin, kaempferol and quercetin have the same skeleton structure, only the-H and-OH of 2 side chains are combined differently, and the glycosyltransferase catalyzes one of them, the other 3 compounds can be catalyzed. Taking kaempferol as an example, the product is kaempferol-7, 4' -di-O-glucoside according to the retention time of the product standard substance, and the detection result of the catalytic reaction is shown in figure 5. Example 5

Enzymatic dynamic parameter detection of recombinant protein pET-28a (+) -DhUGT35

To further understand the enzymatic properties of recombinant proteins, we explored the catalytic conditions of the enzyme in terms of pH, temperature, metal ions, reaction time, etc. Wherein, kaempferol is used as a reaction substrate, UDP-Glc is used as a sugar donor, and beta-mercaptoethanol is used as an antioxidant.

1. Temperature: to a Tris-HCl (50 mM, pH=8.0) reaction system having a total volume of 200. Mu.L, 14mM beta-mercaptoethanol, 5mM UDP-Glc, 10. Mu.g of protein, 0.5mM of substrate was added, and the reaction was carried out at 4, 20, 30, 37, 40, 42, 50, 60, 70℃for 1 hour, and the optimum temperature of the reaction was examined.

2. PH: the optimum pH of the reaction was examined by including 14mM beta-mercaptoethanol, 5mM UDP-Glc, 10. Mu.g of protein, 0.5mM of substrate in a total volume of 200. Mu.L of 50mM of different buffers (citric acid-sodium citrate at pH=5.0-6.0, sodium dihydrogen phosphate-disodium hydrogen phosphate at pH=6.0-8.0, tris-HCl at pH=7.0-9.0, sodium carbonate-sodium bicarbonate at pH=9.0-10.0), and reacting for 1 hour in different types of buffers at 37 ℃.

3. Metal ions: to a total volume of 200. Mu.L Tris-HCl (50 mM, pH=9.0) reaction system was added 14mM beta-mercaptoethanol, 5mM UDP-Glc, 10. Mu.g purified protein, 0.5mM substrate, 5mM divalent metal ion, and reacted at 37℃for 1 hour. The effect of different divalent metal ions, EDTA.2Na, on the catalytic efficiency was observed.

4. Reaction time: 14mM beta-mercaptoethanol, 5mM UDP-Glc, 10. Mu.g protein, 0.5mM substrate and 5mM Mg ²⁺ were added to a Tris-HCl (50 mM, pH=9.0) reaction system with a total volume of 200. Mu.L, and the reaction was carried out at 37℃for 5, 10, 20, 30, 60, 120, 240, 480 and 720 minutes to examine the change rule of the enzyme catalytic efficiency in different reaction times.

5. Miss constant K _m: the total volume of the enzyme activity reaction system is 200 mu L, which comprises the following steps: 5. Mu.g of protein was reacted with 5mM UDP-Glc at a substrate concentration of 50-300. Mu.M (50, 100, 150, 200, 250, 300, 350, 400. Mu.M, respectively) with a pH=9.0, 50mM Tris-HCl supplement to 200. Mu.L, at 37℃for 10min, immediately after the completion of the reaction, the reaction was stopped with an equal volume of precooled methanol, centrifuged at 14000rpm for 5min, and 20. Mu.L was subjected to HPLC detection under the liquid phase conditions as above. The Miq constants were calculated by the Miq equation and the double reciprocal plot method (Lineweaver-Burk plot).

6. Detection result:

The optimal reaction temperature of the recombinant protein pET-28a (+) -DhUGT for catalyzing kaempferol is 37 ℃, and the catalytic efficiency is obviously reduced along with the temperature rise when the reaction is carried out between 40 and 70 ℃; the optimal reaction pH of the recombinant protein was 9.0, and the reaction rate in Tris-HCl was much higher than in sodium carbonate-sodium bicarbonate; the recombinant protein has no dependence on metal ions, but Mg ²⁺ can greatly improve the catalytic efficiency; the catalytic efficiency of the recombinant protein is within 2 hours, the catalytic rate and the reaction time are positively correlated, and the conversion rate of the substrate can reach more than 95% within 2 hours, so that 2 hours is selected as the optimal reaction time of the catalytic reaction. Finally, the recombinant protein is calculated to have a value of 26.00 mu M for kaempferol K _m. The results of the enzymatic kinetics are shown in FIGS. 6A-E.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to the embodiments described above will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A glycosyltransferase gene has the nucleotide sequence of the amino acid sequence shown as SEQ ID NO.2 or the sequence of the complete complementary pairing with the nucleotide sequence of the amino acid sequence shown as SEQ ID NO. 2.

2. A glycosyltransferase has an amino acid sequence shown in SEQ ID NO. 2.

3. The application of glycosyltransferase gene in synthesizing flavonoid 7,4' -di-O-glucoside series glycoside compounds is provided, wherein the glycosyltransferase gene has a nucleotide sequence of an amino acid sequence shown as SEQ ID NO.2 or a sequence of full complementary pairing with the nucleotide sequence of the amino acid sequence shown as SEQ ID NO. 2.

4. The application of glycosyltransferase in synthesizing flavonoid 7,4' -di-O-glucoside series glycoside compounds is provided, and the glycosyltransferase sequence is shown as SEQ ID NO. 2.

5. A method for synthesizing flavonoid 7,4' -di-O-glucosides, said method comprising the steps of:

1) Obtaining a glycosyltransferase comprising an amino acid sequence as set forth in SEQ ID NO. 2;

2) And 3) catalyzing and synthesizing flavonoid 7,4' -di-O-glucoside series glycoside compounds in an enzyme activity reaction system by utilizing the glycosyltransferase in the step 1).

6. A method for synthesizing a flavonoid 7,4' -di-O-glucoside series glycoside compound according to claim 5, wherein the glycosyltransferase is obtained by prokaryotic expression or by chemical synthesis in step 1).

7. The method for synthesizing a flavonoid 7,4' -di-O-glucoside series glycoside compound according to claim 5, wherein the enzyme activity reaction system in the step 2) contains the glycosyltransferase, UDP-Glc and flavonoid compound in the step 1).

8. The method for synthesizing a flavonoid 7,4' -di-O-glucoside series glycoside compound according to claim 5, wherein the enzyme activity reaction system in the step 2) further contains a divalent cation.

9. The method for synthesizing a flavonoid 7,4' -di-O-glucoside series glycoside compound according to claim 5, wherein the reaction temperature of the enzyme-activated reaction system in the step 2) is 20 to 42 ℃.

10. The method for synthesizing a flavonoid 7,4' -di-O-glucoside series glycoside compound according to claim 5, wherein the pH of the enzyme-activated reaction system in the step 2) is 8.0 to 10.0.