Murine coffeoyl transferase, its coding gene and use thereof

Abstract

The present application relates to the field of bioengineering and medicine, and discloses an enzyme PaBAHD and a gene coding the same. The mouseherb coffee acyltransferase can catalyze the generation of corresponding coffee acylglucoside from glucoside derivatives, especially the generation of acylated flavonoid glycosides such as luteolin-4'-O-beta-D-(6''-E-coffee acyl)-glucoside, quercetin-4'-O-beta-D-(6''-E-coffee acyl)-glucoside, apigenin-4'-O-beta-D-(6''-E-coffee acyl)-glucoside and apigenin-7-O-beta-D-(6''-E-coffee acyl)-glucoside, which are complement inhibitory active ingredients. Therefore, the present application can effectively catalyze the coffee acylation of glucoside derivatives through genetic engineering technology, and provide conditions for the large-scale production of complement inhibitory active ingredients.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering and medicine, and specifically discloses the caffeoyltransferase BAHD protein of *Gnaphalium affine*, its encoding gene, and its applications. Background Technology

[0002] The complement system is one of the important immune defense systems in the human body. Under normal physiological conditions, the main functions of complement are to attack foreign pathogens, clear immune complexes, and maintain homeostasis. However, abnormal activation of the complement system can cause an overreaction of the human immune system, leading to damage and inflammation of normal tissues and is closely related to various diseases such as infections, autoimmune diseases, and tumors. Therefore, the development of complement inhibitors has received considerable attention. In recent years, with the extensive development and in-depth research of natural resources such as traditional Chinese medicine, research on isolating complement inhibitors from natural plants has also increased. Currently, Chinese scholars have isolated compounds with complement-inhibiting activity from plants such as Bupleurum chinense and Eucommia ulmoides. The application of natural plants has a long history, and they have often been tested over a long period of time with relatively few toxic side effects, making them an important source for developing novel, safe, and effective complement inhibitors. However, it should be noted that these natural complement inhibitors belong to the category of plant secondary metabolites, and their content in plants is often low, which seriously limits their in-depth development and application.

[0003] Gnaphalium affine D. Don, commonly known as cudweed, is the dried aerial part of the plant *Gnaphalium affine*, belonging to the genus *Gnaphalium* in the family Asteraceae. It is also called Buddha's Ear Grass, Bone-Chasing Wind, Qingming Vegetable, and Yellow Flower Gnaphalium. It is mainly distributed in provinces south of the Yangtze River in my country, where resources are abundant. It has a sweet and slightly sour taste and is neutral in nature. It possesses the effects of resolving phlegm and relieving cough, dispelling wind and dampness, and detoxifying. It is used for colds and coughs, bronchitis, asthma, hypertension, G6PD deficiency (favism), and rheumatic back and leg pain.

[0004] A systematic chemical composition study of *Gnaphalium affine* revealed that acylated flavonoid glycosides, represented by luteolin-4'-O-β-D-(6”-E-caffeoyl)-glucopyranoside, exhibit significant complement-inhibiting activity. Among these, the complement-inhibiting activity IC50 of luteolin-4'-O-β-D-(6”-E-caffeoyl)-glucopyranoside was [not specified in the original text]. 50 It reaches 0.045 mg / mL, and its activity is comparable to that of the positive control drug heparin sodium (IC50 = 0.048 mg / mL).

[0005] The structural formulas of acylated flavonoid glycosides, the complement-inhibiting active components in Gnaphalium affine, are shown below, including luteolin-4'-O-β-D-(6”-E-caffeoyl)-glucoside, quercetin-4'-O-β-D-(6”-E-caffeoyl)-glucoside, apigenin-4'-O-β-D-(6”-E-caffeoyl)-glucoside and apigenin-7-O-β-D-(6”-E-caffeoyl)-glucoside.

[0006]

[0007] The content of complement-inhibiting active ingredients in *Gnaphalium affine* from different origins was determined, and it was found that the content of such substances was extremely low, with only 315.6 mg obtained from extraction per 25 kg of dried whole herb.

[0008] Plant secondary metabolism engineering and synthetic biology have opened up new possibilities for the enrichment and production of target bioactive compounds. Their fundamental principle is to elucidate the biosynthetic pathways of target bioactive compounds and then achieve high accumulation and stable production by regulating the expression of key enzymes involved in the biosynthesis of target compounds within plants or by autonomously constructing biosynthetic pathways outside plants. Elucidating the biosynthetic pathways is crucial for the efficient biosynthesis and sustainable production of target bioactive ingredients through metabolic engineering or synthetic biology methods.

[0009] If the key enzymes that catalyze the generation of complement-inhibiting active components can be obtained from organisms and expressed, the yield of acylated flavonoid glycosides, the complement-inhibiting active components, can be increased through genetic engineering. Summary of the Invention

[0010] The present invention aims to provide a caffeoyltransferase from *Gnaphalium affine* and its encoding gene.

[0011] This invention also provides applications of the above-mentioned enzymes and genes.

[0012] The technical solution of this invention is as follows:

[0013] Gnaphalium affine caffeoyltransferase (PaBAHD) contains the amino acid sequence of SEQ ID No. 1. Preferably, its amino acid sequence is as shown in SEQ ID No. 1.

[0014] The caffeoyl transferase gene of *Gnaphalium affine* encodes the aforementioned caffeoyl transferase.

[0015] Preferably, the caffeoyl transferase gene from *Gnaphalium affine* contains the nucleotide sequence shown in SEQ ID No. 2 or its homologous sequence. More preferably, its nucleotide sequence is as shown in SEQ ID No. 2 or is a homologous nucleotide encoding the same protein.

[0016] A recombinant vector containing a nucleotide sequence encoding the aforementioned caffeoyl transferase.

[0017] Preferably, the recombinant vector contains the above-mentioned caffeoyl transferase gene from *Gnaphalium affine*.

[0018] A host cell containing the above-mentioned caffeoyl transferase gene or the above-mentioned recombinant vector.

[0019] Preferably, the host cell is Escherichia coli.

[0020] The aforementioned caffeoyl transferase from *Gnaphalium affine*, the caffeoyl transferase gene from *Gnaphalium affine*, recombinant vectors, or host cells can catalyze the generation of corresponding caffeoyl flavonoid glycosides from caffeoyl-CoA and glucoside derivatives, and can be used to prepare caffeoyl flavonoid glycosides.

[0021] Preferably, the caffeoyl glucoside is luteolin-4'-O-β-D-(6"-E-caffeoyl)-glucoside, quercetin-4'-O-β-D-(6"-E-caffeoyl)-glucoside, apigenin-4'-O-β-D-(6"-E-caffeoyl)-glucoside, and apigenin-7-O-β-D-(6"-E-caffeoyl)-glucoside. These are generated by reacting luteolin-4'-O-β-D-glucoside, quercetin-4'-O-β-D-glucoside, apigenin-4'-O-β-D-glucoside, and apigenin-7-O-β-D-glucoside with caffeoyl-CoA, respectively.

[0022] A method for preparing caffeoyl glucoside, using caffeoyl coenzyme A and glucoside derivatives as raw materials, and using caffeoyl transferase of gnaphalium affine, a recombinant vector capable of expressing caffeoyl transferase of gnaphalium affine, or host cells as catalysts.

[0023] This invention obtains the caffeoyltransferase gene from *Gnaphalium affine* and uses it to express a protein, thereby obtaining the encoded caffeoyltransferase. The obtained enzyme exhibits excellent catalytic activity and can effectively catalyze the caffeylation reaction of glucosinolate derivatives, making it one of the important key enzymes in biosynthesis.

[0024] Therefore, this invention utilizes genetic engineering technology to catalyze the caffeylation of glucosinolate derivatives, thereby improving the synthesis efficiency of acylated flavonoid glycosides, which are active complement inhibitors. Specifically, it generates acylated flavonoid glycosides such as luteolin-4'-O-β-D-(6”-E-caffeoyl)-glucoside, quercetin-4'-O-β-D-(6”-E-caffeoyl)-glucoside, apigenin-4'-O-β-D-(6”-E-caffeoyl)-glucoside, and apigenin-7-O-β-D-(6”-E-caffeoyl)-glucoside. Thus, this invention provides important enzymes and catalysts for the large-scale production of complement inhibitory active ingredients. Attached Figure Description

[0025] Figure 1 This is a 0.8% agarose gel electrophoresis image of the PCR product of the caffeoyltransferase PaBAHD gene amplified from the cDNA library of *Gnaphalium affine* in Example 1.

[0026] Figure 2 This is a 0.8% agarose gel electrophoresis image of the PCR product of the PaBAHD gene of the monoclonal positive bacterium *Gnaphalium affine* caffeoyltransferase, as shown in Example 1.

[0027] Figure 3 The image shows the PCR product of the PaBAHD gene, a caffeoyltransferase from *Gnaphalium affine*, in the prokaryotic expression vector constructed in Example 2, as shown by 0.8% agarose gel electrophoresis.

[0028] Figure 4 This is an SDS-PAGE electrophoresis image of caffeoyltransferase protein induced by *Gnaphalium affine* in Example 3. M: Protein Marker; 1: Bacterial culture without IPTG induction; 2: Bacterial culture after IPTG induction; 3: Supernatant of lysed cells after induction; 4: Precipitate of lysed cells after induction.

[0029] Figure 5 The TIC chromatogram is shown for the reaction solution of luteolin-4'-O-glucoside catalyzed by PaBAHD21. The chromatograms of the experimental group include the TIC chromatogram and the results after extracting the mass-to-charge ratio of the target compound.

[0030] Figure 6 The TIC chromatogram is shown for the reaction solution of PaBAHD21 catalyzed quercetin-4'-O-glucoside. The chromatograms of the experimental group include the TIC chromatogram and the results after extracting the mass-to-charge ratio of the target compound.

[0031] Figure 7 The TIC chromatogram is shown for the reaction solution of PaBAHD21 catalyzed apigenin-7-O-glucoside. The chromatograms of the experimental group include the TIC chromatogram and the results after extracting the mass-to-charge ratio of the target compound. Detailed Implementation

[0032] Example 1

[0033] 1. RNA extraction from Gnaphalium affine leaves

[0034] (1) Sample collection: Take fresh leaves of cudweed, wrap them in aluminum foil and put them in liquid nitrogen; after quick freezing in liquid nitrogen, take them out and crush them, transfer the sample into a 1.5ml RNase-free centrifuge tube containing two steel balls, and grind them with a ball mill.

[0035] (2) RNA extraction: Sample lysis, extraction and layering, column recovery, washing and centrifugation, and RNA elution (the experimental steps are based on the TransZol Up Plus RNA Kit ER501).

[0036] (3) The concentration and purity of RNA were tested and are shown in Table 1.

[0037] Table 1

[0038] tender leaves of cudweed 353 1.89 1.06

[0039] 2. Construction of cDNA library from Gnaphalium affine leaves

[0040] Total RNA from *Gnaphalium affine* leaves was reverse transcribed into cDNA: Total RNA was placed on ice. RNase-free reagents and consumables were used, and the experimental procedures were performed according to the kit (TaKaRa PrimeScript). TM 1st strand cDNA Synthesis Kit).

[0041] 3. Primer design: The primer sequences used for gene amplification are shown in Table 2 (SEQ ID No. 3-4).

[0042] Table 2

[0043] PaBAHD21-F atggcatcttcatcacccat PaBAHD21-R ctaaatgttcacttgcaatccat

[0044] 4. Gene amplification

[0045] The experimental procedures were performed according to the kit (TOYOBO KOD FX, catalog number: 9503002), with reaction solutions prepared as shown in Table 3. The PCR reaction program is shown in Table 4. PCR products were subjected to 0.8% agarose gel electrophoresis (150V, 15min), and the results are as follows. Figure 1 .

[0046] Table 3 PCR Reaction Solution Formulation Table

[0047] Template (cDNA) 1μL F(10μM) 1.5μL R(10μM) 1.5μL 2×KOD FX buffer 25μL 2mM dNTPs 10μL KOD FX 1μL Nuclase-free Water Fill to 50μL

[0048] Table 4 PCR reaction procedure

[0049]

[0050] 5. Ligate the amplified product to the pMD19-T vector

[0051] The amplification products were produced using Premix Taq enzyme (TaKaRa Premix LA). Continue amplification by adding a T to the end of the product sequence amplified by KOD FX enzyme, and add samples according to Table 5.

[0052] Table 5

[0053] PaBAHD21 amplification products 40μL Premix Taq 1μL

[0054] The mixture was then reacted at 72°C for 20 minutes.

[0055] Ligate the amplified product to the pMD19-T vector: Add samples according to Table 6, mix gently, and incubate at room temperature for 1 hour. After the reaction is complete, place on ice (refer to the TaKaRa pMD kit for experimental procedures). TM 19-T Vector Cloning Kit).

[0056] Table 6

[0057] PCR products 2.5μL pMD19-T 0.5μL Solution I 2μL

[0058] 6. Vector transformation of Escherichia coli

[0059] The experimental procedure was performed according to the kit (Video Biotech TOP10 Chemically Competent Cell).

[0060] (1) Take out Escherichia coli TOP10 competent cells from the -80℃ freezer, thaw at room temperature, and then quickly place them on ice;

[0061] (2) Take 5 μL of the ligation product and add it to 50 μL of competent cells (performed in a clean bench);

[0062] (3) Place on ice for 30 minutes, heat shock at 42℃ for 90 seconds, and immediately place on ice for 5 minutes;

[0063] (4) Add 500 μL of antibiotic-free LB culture medium and incubate at 37°C and 200 rpm for about 1 hour on a shaker.

[0064] (5) Centrifuge at 5000 rpm for 1 min at room temperature, retain 100 μL of supernatant, and resuspend by pipetting;

[0065] (6) Spread the suspension onto LB solid medium containing 100 mg / L Amp+ and invert it in a 37°C constant temperature incubator for 12-16 h;

[0066] (7) Select 8 single colonies and inoculate them into 300 μL of LB liquid medium containing 100 mg / L Amp+, and incubate at 37℃ and 200 rpm for more than 3 hours.

[0067] 7. Detection of monoclonal positive bacteria

[0068] a. The reaction solution preparation is shown in Table 7, and the PCR reaction procedure is shown in Table 8. The PCR products were subjected to 0.8% agarose gel electrophoresis (150V, 15min). The results are as follows: Figure 2 .

[0069] Table 7 PCR reaction solution formulation

[0070] Template (bacterial solution) 1μL M13-F (10μM) 1μL M13-R (10μM) 1μL 2×Flash PCR MasterMix(Dye) 10μL ddH2O Add to 20 μL

[0071] Table 8 PCR Reaction Procedure

[0072]

[0073] 8. Sequencing

[0074] Select positive bacteria with matching bands, and send 100 μL for sequencing (Sangon Biotech). Sequence alignment was performed using SnapGene software. The nucleotide sequence of the sequencing results is shown in SEQ ID No. 2. The protein sequence it encodes is shown in SEQ ID No. 1.

[0075] SEQ ID No.1: MASSSPIITVVQVSKVSPPPEALQNRSLELSFFDMVWLLRPPVHHVFFYEIPNLTKTEFVTTILPDLNRSLSTTLQHFFPYAGNLIVFHTPTKKPEIRYVQGDHVNVTY SECNLDFSDLTGNHPRSCEKFYHLIPPLGQCVKLPDHTKIPIYSVQVTLFPNHGISIGMTKHHSVSDASAQLGFLKAWTSISKCGSDESFLARKTFPCYDRVICYPTQEDEILG RANLESLKNQEYQPPHLYGPSAKVRATFIMTASMISQLKKLVLTQLPTVSYVSSFTVSCAYIWTCLARSCKTELEVFLFFIDCRARMDPPVPATYFGNCIKPCITVATGSSLAE NEGFVGVAKLMGENLQKVLNTEGGIVMAKPPSAEVLSKPIIGVAGSPKVNFYDMDFGWGDPKKQEIISLDYGVGSISMGASKESKEDVEIGICLSPTEMQAFVHFFNDGLQVNI

[0076]

[0077] Example 2 Construction of prokaryotic expression vector

[0078] 1. Plasmid extraction

[0079] Take 20 μL of correctly sequenced bacterial culture PaBAHD21-pMD19-T and add it to 20 mL of LB (Amp + 100 mg / L) medium. Incubate overnight at 37°C and 200 rpm in a shaker. Simultaneously, take 20 μL of bacterial culture pET32a+ and add it to 20 mL of LB (Amp + 100 mg / L) medium. Incubate overnight at 37°C and 200 rpm in a shaker. Lyse the bacterial cells, centrifuge, collect the supernatant, collect the supernatant on a column, wash, centrifuge, and elute to obtain the plasmid solution (plasmid extraction procedure follows the EasyPure PlasmidMiniPrep Kit EM101).

[0080] 2. Double digestion of pET32a+ plasmid

[0081] The pET32a+ vector was double-digested with Hind III-HF (NEB) and SacI-HF (NEB) for seamless cloning of PaBAHD21. The reaction system is shown in Table 9.

[0082] Table 9

[0083]

[0084]

[0085] The reaction solution was mixed thoroughly and digested at 37°C for 40 min. The enzyme digestion results were detected by agarose gel electrophoresis. The corresponding fragments were excised, and the linear plasmid solution was recovered from the gel. The concentration and purity of the linear plasmid solution were determined using a micro-ultraviolet spectrophotometer for further calculations. The linear plasmid solution can be stored at -20°C for one week.

[0086] 3. Seamless cloning primer design

[0087] Primers used for seamless cloning (with underlined gagctc and aagctt restriction endonuclease sites) are shown in Table 10 (SEQ ID No. 5-6).

[0088] Table 10

[0089] PET32a-PaBAHD21-F <![CDATA[tccgaattc gagctc ATGGCATCTTCATCACCCATCATCA]]> PET32a-PaBAHD21-R <![CDATA[tgcggccgc aagctt AATGTTCACTTGCAATCCATCATTG]]>

[0090] 4. Amplification of gene fragments

[0091] The experimental procedures were performed according to the instructions of the kit (TOYOBO KOD FX, catalog number: 9503002). The PCR reaction solution formulation and PCR procedure were as shown in Tables 11 and 12.

[0092] Table 11 PCR Reaction Solution Formulation Table

[0093] Template (PaBAHD21-pMD19-T plasmid) 1μL F(10μM) 1.5μL R(10μM) 1.5μL 2×KOD FX buffer 25μL 2mM dNTPs 10μL KOD FX 1μL Nuclase-free Water Fill to 50μL

[0094] Table 12 PCR Reaction Procedure

[0095]

[0096]

[0097] The PCR products were electrophoresed on a 0.8% agarose gel (150V, 15min). The results are as follows: Figure 3 .

[0098] 5. Seamless cloning

[0099] a. Prepare the reaction solution according to Table 13, mix gently, and incubate in a metal bath at 50°C for 30 minutes. After the reaction, immediately place on ice or cool to 4°C (refer to the kit for steps). plus One-step PCR Cloning Kit (nearshore).

[0100] Table 13

[0101]

[0102] b. Transform the recombinant product into E. coli competent cells Trans1-T1 (the experimental procedure is the same as the Trans1-T1 Phage Resistant Chemically Competent Cell CD501 kit).

[0103] c. Spread the plating, pick spots, add 500 μL LB (Amp + 100 mg / L), incubate at 37℃ and 200 rpm overnight, perform bacterial testing, send to Sangon Biotech for analysis, and perform sequence alignment.

[0104] 6. Extract plasmids and transform them into expression strain BL21.

[0105] The correctly sequenced PaBAHD21-pET32a-Trans1-T1 and pET32a+ bacterial cultures were transferred (20 μL) to 20 mL of LB broth (Amp + 100 mg / L) and incubated overnight at 37°C and 200 rpm. The plasmid was extracted the next day and re-transformed into BL21 cells (using the same method as Trans1-T1 competent cells). The plating was plated and incubated overnight at 37°C. Spots were picked, and positive clones were retained.

[0106] Example 3 Protein Induction and Purification

[0107] 1. Protein-induced expression

[0108] a. Take 10 μL of each of the bacterial cultures PaBAHD21-pET32a-BL21 and add them to 0.5 mL of LB medium (containing Amp + 100 mg / L), incubate overnight, and activate twice;

[0109] b. Take 20 μL of bacterial culture and add it to 5 mL of LB medium (containing ampicillin Amp 100 mg / L) and incubate at 37℃ for about 6 h at 200 rpm; take 1 mL of bacterial culture and add it to 500 mL of LB medium (containing Amp + 100 mg / L) and incubate at 37℃ for 200 rpm until the OD600 value reaches 0.5-0.6, and take 1 mL of bacterial culture and store it at -20℃ as a control;

[0110] c. Add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.2 mmol / L, incubate at 80 rpm and 16 ℃ for 48 h, and take 1 mL of bacterial culture and store it at -20 ℃ as a control.

[0111] d. Centrifuge at 5,000 rpm for 15 min to enrich bacterial cells, collect them in 50 mL centrifuge tubes, resuspend in 20 mL PBS buffer (pH=7.4), centrifuge again, discard the supernatant; resuspend the bacterial cells in 20 mL PBS buffer (pH=7.4).

[0112] e.200w (variable stage 3, power 35%), 2s ultrasound, 4s interval, ultrasound disruption on ice for 15min, bacterial culture temperature must not exceed 4℃;

[0113] f. Centrifuge the sonicated buffer at 4°C and 12,000 rpm for 15 min, and collect the supernatant, which is the crude protein solution. (In the preliminary experiment, take 1 mL of the sonicated buffer, centrifuge at 4°C and 12,000 rpm for 15 min, collect the supernatant, which is the crude protein solution, and resuspend the precipitate with an equal volume of 1×PBS buffer.)

[0114] 2. SDS-PAGE electrophoresis analysis

[0115] Take 20 μL of the supernatant and precipitate obtained in the previous experiment and add 4 μL of protein loading buffer (6× protein loading buffer). After mixing, boil in water for 8 min (heat denaturation). Centrifuge at 12,000 rpm for 2 min and take the supernatant for SDS-PAGE electrophoresis analysis.

[0116] a. Prepare a 10% SDS-PAGE gel. The formulas for the separating gel and the stacking gel are shown in Table 14, where the table lists the amounts of both gels. First, add the separating gel to the gel mixing tank and allow it to solidify for approximately 30 minutes. Then, add the stacking gel and quickly insert the comb. Use immediately after solidification, or wrap the gel in moistened absorbent paper and store at 4°C.

[0117] Table 14 Formulations for separating gels and concentrating gels

[0118] Mini-Q H2O 5.0mL Mini-Q H2O 3.7mL 30% Acr-Bis(29:1) 4.3mL 30% Acr-Bis(29:1) 0.67mL 1M Tris·HCl (pH=8.8) 3.5mL 1M Tris·HCl (pH=6.8) 0.5mL 10% SDS 0.1mL 10% SDS 0.04mL 10% AP 0.1mL 10% AP 0.04mL TEMED 0.004mL TEMED 0.004mL

[0119] b. Take 8 μL of the denatured sample supernatant and add it evenly to the lane of the stacking gel. Use the Bio-Rad electrophoresis apparatus and the electrophoresis program is: 80V for 20 min, 120V for 60 min.

[0120] c. After the reaction is complete, remove the gel, carefully cut off the stacking gel, and place the separating gel in a glass petri dish containing Coomassie Brilliant Blue quick staining solution. Shake on a side for approximately 10-30 minutes. Remove and place in water, then microwave on medium heat for 30 seconds to destain. Repeat this process several times until the protein bands are clearly visible. The results are as follows: Figure 4 .

[0121] d. Results Analysis: The predicted molecular weight of PaBAHD21-pET32a protein is 70.9 kDa. Figure 4 The results showed that the target protein PaBAHD21 was present in the bacterial culture, supernatant and precipitate induced by IPTG, and there was no band at the corresponding position in the bacterial culture before induction.

[0122] 3. Desalting crude protein

[0123] The obtained supernatant crude protein solution was filtered through a 0.22 μm microporous membrane; the collected supernatant crude protein was concentrated using a Millipore molecular sieve concentration column (50 kDa) to concentrate the protein sample, which also served to remove salt.

[0124] Then centrifuge at 4°C to a concentration of less than 5000×g, reducing the volume to less than 0.5-1mL. Place the concentrated protease on ice for subsequent experiments.

[0125] Sequencing revealed that the amino acid sequence of the obtained caffeoyl transferase from *Gnaphalium affine* is shown in SEQ ID No. 1.

[0126] Example 4: In vitro enzyme function verification

[0127] Control group: pET32a+-BL21 protein expression induced

[0128] Experimental group: PaBAHD21-pET32a protein was induced to express.

[0129] (Note: In the preliminary experiment, the crude protein solution from Example 3 "Protein Induced Expression" was used to verify enzyme activity, and purification was not necessary; in the formal experiment, the enzyme-catalyzed reaction used a purified enzyme PBS solution, and the control group was replaced with PBS buffer of the same pH value.)

[0130] Substrates: Quercetin-4'-O-glucoside, luteolin-4'-O-glucoside, apigenin-7-O-glucoside.

[0131] The reaction system is shown in Table 15. The reaction conditions are: constant temperature mixer, 37℃, 300rpm, 2h.

[0132] Table 15 In vitro recombinant enzyme activity reaction system

[0133] 6mM Caffeoyl-CoA 2μL 20mM DTT 5μL PaBAHD21 crude enzyme 50μL Substrate 3μL 1×PBS (pH=7.4) Add to 100μL

[0134] After the reaction was complete, the reaction was quenched with 100 μL of anhydrous ethanol. The mixture was centrifuged at 12000 × g and 4 °C for 30 min. 100 μL of the supernatant was transferred to a vial for analysis.

[0135] The samples were qualitatively analyzed using an Agilent 1290 ultra-high performance liquid chromatograph and an Agilent 6530 triple quadrupole mass spectrometer (Agilent, USA).

[0136] Chromatographic conditions: Waters ACQUITY UPLC T3 column (Waters, USA); column temperature: 35℃; injection volume: 1 μL

[0137] Mass spectrometry conditions: ESI ion source; negative ion mode scanning range m / z: 100-1700 Da; negative ion mode low energy: 6 V, high energy: 30-60 V; ion source temperature 350 ℃, capillary voltage 3.5 kV (negative ions), nebulizer gas pressure 45 Psi, drying gas flow rate 11 L / min, sheath gas flow rate 11 L / min, sheath gas temperature 350 ℃, fragment charge 140 V, collision energy 30 V.

[0138] The analytical conditions are shown in Table 16, and the TIC spectra of the standard, control, and experimental groups are shown in Table 16. Figure 5-7 As shown, the spectra of the experimental group include the TIC spectrum and the results after extracting the mass-to-charge ratio of the target compound.

[0139] Table 16 Analysis Conditions

[0140] 0.400 98％ 2％ 7.00 0.400 80％ 20％ 11.00 0.400 78％ 22％ 20.00 0.400 40％ 60％ 25.00 0.400 35％ 65％ 28.00 0.400 35％ 65％ 30.00 0.400 5％ 95％ 33.00 0.400 5％ 95％ 33.10 0.400 98％ 2％ 38.00 0.400 98％ 2％

[0141] Results analysis:

[0142] like Figure 5As shown, the luteolin-4'-O-β-D-(6”-E-caffeoyl)-glucoside standard eluted at 12.90 min, and the same peak appeared at 12.36 min in the enzyme activity system with PaBAHD21 enzyme added, and the secondary mass spectrometry fragmentation mode was the same. No corresponding peak appeared in the control enzyme activity reaction system with pET32a+ blank carrier. Therefore, PaBAHD21 can catalyze the acylation of luteolin-4'-O-glucoside to luteolin-4'-O-β-D-(6”-E-caffeoyl)-glucoside.

[0143]

[0144] like Figure 6 The quercetin-4'-O-β-D-(6”-E-caffeoyl)-glucoside standard showed a peak at 13.78 min. The same peak appeared at 13.78 min in the enzyme activity system with PaBAHD21 added, and the secondary mass spectrometry fragmentation mode was the same. No corresponding peak appeared in the control enzyme activity reaction system with pET32a+ blank carrier. Therefore, PaBAHD21 can catalyze the acylation of quercetin-4'-O-glucoside to quercetin-4'-O-β-D-(6”-E-caffeoyl)-glucoside.

[0145]

[0146] like Figure 7 The apigenin-7-O-β-D-(6”-E-caffeoyl)-glucoside standard showed a peak at 13.55 min. In the enzyme activity system with PaBAHD21 added, the same peak appeared at 13.52 min, and the secondary mass spectrometry fragmentation mode was the same. No corresponding peak appeared in the control enzyme activity reaction system with pET32a+ blank carrier. Therefore, PaBAHD21 can catalyze the acylation of apigenin-7-O-glucoside to apigenin-7-O-β-D-(6”-E-caffeoyl)-glucoside.

[0147]

[0148] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. Any equivalent substitutions or obvious modifications made within the technical field to which this invention pertains, without departing from the concept of the present invention, and which have the same or similar performance or use, should fall within the protection scope of this invention.

Claims

1. A caffeoyl transferase from gnaphalium affine, characterized in that, Its amino acid sequence is shown in SEQ ID No.

1.

2. The caffeoyl transferase gene of *Gnaphalium affine*, characterized in that, The caffeoyl transferase of Gnaphalium affine as described in claim 1 is encoded.

3. The caffeoyl transferase gene from *Gnaphalium affine* according to claim 2, characterized in that, It contains the nucleotide sequence shown in SEQ ID No.

2.

4. The caffeoyl transferase gene of *Gnaphalium affine* according to claim 2 or 3, characterized in that, Its nucleotide sequence is shown in SEQ ID No. 2 or is a homologous nucleotide encoding the same protein.

5. A recombinant vector, characterized in that, It contains a nucleotide sequence encoding the caffeoyl transferase of claim 1.

6. The recombinant vector according to claim 5, characterized in that, It contains the caffeoyl transferase gene of *Gnaphalium affine* as described in any one of claims 2-4.

7. A host cell, characterized in that, It contains the caffeoyl transferase gene of *Gnaphalium affine* as described in any one of claims 2-4 or the recombinant vector as described in claim 5 or 6.

8. The use of the caffeoyl transferase of Gnaphalium affine according to claim 1, the caffeoyl transferase gene of Gnaphalium affine according to any one of claims 2-4, the recombinant vector according to claim 5 or 6, or the host cell according to claim 7 in the preparation of caffeoyl flavonoid glycosides.

9. A method for preparing caffeoyl flavonoid glycosides, characterized in that, Using caffeoyl-CoA and glucoside derivatives as raw materials, and using the caffeoyl transferase of Gnaphalium affine as described in claim 1, the recombinant vector as described in claim 5 or 6, or the host cell as described in claim 7 as a catalyst.

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