Glucosyltransferase obtained through directed evolution method and derivative, application and preparation method thereof

Abstract

The invention provides glucosyltransferase obtained through a directed evolution method and a derivative, application and a preparation method of the glucosyltransferase, and belongs to the technical field of enzyme engineering. The glucosyltransferase provided by the invention is obtained by a directed evolution method, the amino acid sequence of the glucosyltransferase is a sequence as shown in SEQ ID NO.4, and the DNA sequence of the glucosyltransferase comprises a sequence as shown in SEQ ID NO.3; the glucosyltransferase is high in catalytic capacity, flavone can be remarkably and efficiently catalyzed to generate flavone-7-O-glucoside, and meanwhile generation of precipitates in plant extracts can be remarkably reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and relates to a glucosyltransferase, and in particular to a glucosyltransferase obtained by a directed evolution method, and its derivatives, applications and preparation methods. Background Art

[0002] Flavonoids are a class of active natural compounds found widely in plants. Based on differences in the C3 ring structure of their basic skeleton, flavonoids are classified into various types, including flavones, flavonols, flavanones, and isoflavones. Modern pharmacological research indicates that flavonoids possess numerous pharmacological activities, including antioxidant, anti-inflammatory, and antiviral activities. However, naturally occurring flavonoid compounds often suffer from poor water solubility and low stability.

[0003] Flavonoids are also common active ingredients in plant extracts. However, during the preparation of plant extracts, flavonoids often cause precipitation. Using organic solvents to prepare plant extracts can increase the solubility and stability of flavonoids, but organic solvents are prone to environmental pollution, and organic solvent residues can be harmful to the human body.

[0004] By glycosylation of flavonoids to form flavonoid glycosides, the solubility and stability of flavonoid compounds can be increased.

[0005] In recent years, significant progress has been made in the study of chemical and biosynthetic methods for glycosylation modification. However, chemical glycosylation reactions suffer from drawbacks such as numerous byproducts and intermediates, poor regioselectivity and stereoselectivity, low yields, cumbersome protection and deprotection steps, and high pollution levels. Furthermore, low atomic efficiency and the high cost of some catalysts make chemical synthesis methods a major drawback in the large-scale production of glycosides. In comparison, enzyme-catalyzed biosynthesis not only offers stereoselectivity and regioselectivity, but also features relatively simple procedures and minimal environmental pollution, in line with the concept of green chemistry. Therefore, the use of glucosyltransferases to catalyze the synthesis of flavonoid glycosides from flavonoids has gradually become a research focus in this field.

[0006] Prior art CN118755685A discloses a mutant of the glycosyltransferase VcUGT1, AAVT, isolated from blueberries. Compared to VcUGT1, this mutant possesses the following point mutations: S367A, V274A, F82V, and I132T. This mutant significantly enhances the catalytic activity of transglycosylation reactions, efficiently catalyzing the conversion of substrates to the corresponding 7-O-glucoside compounds.

[0007] Most common glucosyltransferases catalyze the formation of flavonoid glycosides at the 3' site, while those that catalyze the formation of flavonoid glycosides at the 7' site are relatively rare. Due to steric hindrance in the molecular structure, flavonoid glycosides at the 7' site have improved solubility. Therefore, the discovery of more efficient glucosyltransferases capable of glycosidating flavonoids at the 7' site to form flavonoid-7-O-glucosides is urgently needed. Summary of the Invention

[0008] The present invention addresses the problem of the prior art lacking an efficient glucosyltransferase that catalyzes the formation of glycosides at the 7' site of flavonoids. The present invention provides a glucosyltransferase obtained by directed evolution, its derivatives, applications, and preparation methods. The glucosyltransferase provided by the present invention is obtained by directed evolution, has an amino acid sequence as shown in SEQ ID NO. 4, and a gene sequence as shown in SEQ ID NO. 3. The glucosyltransferase has strong catalytic ability and can efficiently catalyze flavonoids to form flavonoid-7-O-glycosides, while significantly reducing the formation of precipitates in plant extracts.

[0009] To achieve the above object, the technical solution adopted by the present invention is as follows: In one aspect, the present invention provides a glucosyltransferase, wherein the amino acid sequence of the glucosyltransferase is the sequence shown in SEQ ID NO.4.

[0010] In another aspect, the present invention provides a nucleic acid encoding the above-mentioned glucosyltransferase.

[0011] Preferably, the nucleic acid has the sequence shown in SEQ ID NO.3.

[0012] In another aspect, the present invention provides a vector comprising the above-mentioned nucleic acid.

[0013] In another aspect, the present invention provides a cell, wherein the cell expresses the aforementioned glucosyltransferase, or the aforementioned nucleic acid, or the aforementioned vector.

[0014] On the other hand, the present invention provides uses of the aforementioned glucosyltransferase, the aforementioned nucleic acid, the aforementioned vector or the aforementioned cell.

[0015] Preferably, the application includes: Catalyzes flavonoids to generate flavonoid-7-O-glycosides; preparing plant extracts; Reduces precipitation in plant extracts.

[0016] In another aspect, the present invention provides a method for producing flavonoid-7-O-glycoside, which comprises catalyzing the production of flavonoids by the above-mentioned glucosyltransferase.

[0017] In another aspect, the present invention provides a method for preparing a plant extract, comprising extracting using the above-mentioned glucosyltransferase.

[0018] On the other hand, the present invention provides a method for preparing the above-mentioned glucosyltransferase, which comprises obtaining the glucosyltransferase through directed evolution of a wild-type glucosyltransferase.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. The glucosyltransferase provided by the present invention has strong catalytic ability and can efficiently catalyze flavonoids to produce flavonoid-7-O-glycosides.

[0020] 2. The glucosyltransferase provided by the present invention can significantly reduce the generation of precipitation in plant extracts. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of pUC19-PcGlcT plasmid.

[0022] Figure 2 Schematic diagram of the reaction principle of the present invention. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below with reference to the examples. If the specific conditions are not specified in the examples, the experiments shall be carried out under conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments without the manufacturer specified are conventional products that can be purchased commercially. In order to better illustrate the present invention, many specific details are given in the specific embodiments below. The specific embodiments described here are only used to explain the present invention and are not intended to constitute any limitation to the present invention. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present invention. Such structures and technologies are described in many publications, such as "Molecular Cloning Laboratory Manual (Fourth Edition)" (Cold Spring Harbor Laboratory Science Press), Ausubel, FM et al., Current Protocols in Molecular Biology , Greene Publishing Assoc., and Wiley-Interscience.

[0024] Preparation Example: Obtaining Glucosyltransferase (1) Gene cloning of wild-type glucosyltransferase The document "Biochemical Characterization of Parsley Glycosyltransferases Involved in the Biosynthesis of a Flavonoid Glycoside, Apiin" describes a glucosyltransferase, PcGlcT, extracted from celery cells. This wild-type glucosyltransferase was used in the present invention for subsequent development. Based on this document, the DNA sequence of the glucosyltransferase, PcGlcT, was obtained as SEQ ID NO. 1, and its amino acid sequence is SEQ ID NO. 2. A commercial gene synthesis company synthesized the PcGlcT DNA fragment according to SEQ ID NO. 1.

[0025]

[0026] SEQ ID NO.2: MELNSSSCIVLYPAPGIGHLVSMVELGKLILCHHPDSFSKIIILITNAPHLDTRATAPYMSSVSATIPSITFHQLPTPPLPPNYAPSVKGLDFELIGLNNPNVHQALQTISSTTFKI KAFIIDFICDAAFKVSSALNLPTYYFFTTAASNLSALLYFPTLHQKITVNLKDYNDFVHYPGVPPIFSSENAITVLDRNTMEYKFFMGTAAQMAKSDGIIINTFHSLEPRAITAISDG LCVPDAPTPPIYCIGPLITGKEVDSEEHECLVWLNSQPSKSVVFLCFGSLGAFGEEQLKEIAVGLENSKHRFLWVIKNPPSRQEGDPNTVLPQGFLERTKGRGLVVKSWVPQVAVLN HPSVGGFVTHCGWNSILEGVCAGVPMIGWPLYAEQRVNRVLMVEELRVGLGLVESDGERFVSSDELEKGVRELMNSDNGKRLRNRVVELRDAAKAAMSDEDGSSRIELAKLITKWKE.

[0027] Primers containing restriction enzyme cleavage sites were designed based on the DNA of the glucosyltransferase PcGlcT. The Sal I restriction site was introduced using the forward primer PcGlcT-WF (SEQ ID NO. 5: 5'-GCGGTCGACTTTTTTCTTAAAATTTCA-3'), and the Nde I restriction site was introduced using the reverse primer PcGlcT-WR (SEQ ID NO. 6: 5'-TTACATATGCCGACTTTGTTAGTA-3'). The pUC19 plasmid, purchased from a commercial company, was selected based on the parameters required for subsequent experiments. PCR amplification was performed using the synthetic DNA of the glucosyltransferase PcGlcT as a template using the aforementioned primers (PCR enzymes and reagents purchased from Takara, Cat. No. R010B). The enzymes were then digested and introduced into the pUC19 plasmid. The PCR amplification reaction system (see Table 1) and procedure (see Table 2) were used.

[0028] Table 1 PCR amplification reaction system

[0029] Table 2 PCR amplification procedures

[0030] A commercially available enzyme digestion kit containing Sal I and Nde I enzymes was used for enzyme digestion. The PCR amplification product and pUC19 plasmid obtained in the above steps were digested separately. The digestion temperature was 37°C for 1 hour, and the digestion system was as shown in Table 3.

[0031] Table 3 Enzyme digestion system

[0032] The digested DNA and pUC19 plasmid were ligated by enzyme to obtain the plasmid pUC19-PcGlcT (see the schematic diagram of the plasmid map for details). Figure 1 This plasmid served as the template for wild-type glucosyltransferase. A commercial T4 ligase kit was used for ligation at 16°C for 12 hours. The enzyme ligation system is shown in Table 4.

[0033] Table 4 Enzyme-linked system

[0034] (2) Construction of recombinant expression strain and enzyme expression Using E. coli E. coli BL21 (DE3), prepare competent cells, use the electroporation method to transform the pUC19-PcGlcT plasmid into the cells, spread onto a solid plate containing ampicillin, and place in an incubator at 37 ° C for 24 hours. Select a single clone from the plate, inoculate it into a test tube of liquid LB medium supplemented with ampicillin, and culture it at 37 ° C for 12 hours. Extract the bacterial suspension into a new test tube at a ratio of 1:100, culture it to about OD600 = 0.6, add IPTG to a final concentration of 0.5mM, culture it at 25 ° C, shake it at 180 rpm for 24 hours to induce the expression of the target protein. Then, ultrasonically disrupt the fermentation broth in the test tube, collect the supernatant after centrifugation, and obtain the target enzyme solution.

[0035] (3) Screening method The glucosyltransferase studied in this study uses flavonoids and UDPG as substrates to catalyze the synthesis of flavonoid-7-O-glycosides through a glycosylation reaction. In this synthetic pathway, UDPG is hydrolyzed to UDP, necessitating the addition of UDPG to maintain the reaction. However, since UDPG is relatively expensive, the addition of sucrose synthase catalyzes the synthesis of UDPG from UDP and sucrose, allowing for a continuous supply of UDPG to the reaction system and reducing costs.

[0036] In the reaction pathway of sucrose synthase, UDP and sucrose serve as substrates to synthesize UDPG and fructose. Fructose is a reducing sugar that, under heating conditions, can undergo a redox reaction with pale yellow 3,5-dinitrosalicylic acid (DNS) to convert it into brown 3-amino-5-nitrosalicylic acid. 3-amino-5-nitrosalicylic acid has an absorption peak at 540nm and can be quickly detected using a UV spectrophotometer. The reaction principle is shown in Figure 2 .

[0037] Therefore, by measuring the OD540 value of the reaction system and quantitatively analyzing the fructose produced throughout the reaction, the amount of synthesized flavonoid-7-O-glycoside can be indirectly reflected, thereby reflecting the activity of the glycosyltransferase. Therefore, this method can be used for high-throughput rapid screening of glycosyltransferases.

[0038] In the specific screening process, the present invention uses luteolin, a common flavonoid in plants, as a screening reaction starting material. Luteolin can be synthesized into luteolin-7-O-glucoside by the glucosyltransferase studied in the present invention.

[0039] (4) Enzyme verification To a test tube, add 0.01 g / ml luteolin, 0.1 g / ml sucrose, 2 mM UDPG, 50 U sucrose synthase, 0.5 ml of the above enzyme solution, and buffer to a 5 ml reaction volume. Incubate in a 37°C water bath for 6 hours to allow the reaction to fully react. Terminate the reaction by boiling in water for 5 minutes. Centrifuge and collect the supernatant for analysis.

[0040] According to the above screening method, the OD540 of the supernatant was detected. Compared with the control group, the OD540 was significantly increased.

[0041] At the same time, HPLC was used to detect luteolin and luteolin-7-O-glucoside in the supernatant. The content of luteolin was significantly reduced, while that of luteolin-7-O-glucoside was significantly increased.

[0042] Therefore, it was shown that the target enzyme glucosyltransferase PcGlcT was successfully expressed, and OD540 could be used to characterize the activity of the target enzyme in the reaction system.

[0043] (5) Construction of mutation library A random mutation library for the glucosyltransferase PcGlcT was constructed using error-prone PCR. The forward primer sequence was PcGlcT-MF (SEQ ID NO. 7: 5'-GAGGTACATCGATGCCTCTGC-3'), and the reverse primer sequence was PcGlcT-MR (SEQ ID NO. 8: 5'-GCAGAGGCATCGATGTACCTC-3'). The template was the pUC19-PcGlcT plasmid. Error-prone PCR amplification was performed using rTaq DNA polymerase (rTaq DNA polymerase and its associated reagents were purchased from Xiamen Esper Technology Co., Ltd.). The PCR amplification reaction system (see Table 5) and protocol (see Table 6) are shown below.

[0044] Table 5 PCR amplification reaction system

[0045] Table 6 PCR amplification program

[0046] After obtaining the error-prone mutant DNA fragment of pUC19-PcGlcT, we used it as a megaprimer and pUC19-PcGlcT as a template for MEGAWHOP PCR (PCR using enzymes and reagents purchased from Takara, Catalog No. R010B). The PCR amplification reaction system (see Table 7) and protocol (see Table 8) are shown below.

[0047] Table 7 PCR amplification reaction system

[0048] Table 8 PCR amplification program

[0049] The amplified product obtained by MEGAWHOP PCR was digested by adding Dpn I endonuclease and reacting at 37°C for 12 hours. E. coli BL21 (DE3) competent cells, and then the amplified product was introduced into E. coli BL21 (DE3) was plated onto a solid plate containing ampicillin and cultured in an incubator at 37°C for 24 hours.

[0050] Single colonies were selected from the plates and inoculated into test tubes containing liquid LB medium supplemented with ampicillin. Culture was performed at 37°C for 12 hours. The bacterial suspension was aspirated at a 1:100 ratio into a new tube and cultured to approximately OD600 = 0.6. IPTG was added to a final concentration of 0.5 mM and cultured at 25°C with a shaker at 180 rpm for 24 hours to induce target protein expression. The fermentation broth was ultrasonically disrupted and centrifuged, and the supernatant was collected to obtain the target enzyme solution.

[0051] (6) Directed evolution through multiple rounds of high-throughput screening Following the above steps, a random mutation library was constructed using pUC19-PcGlcT as a starting point, yielding a total of 2,600 mutant strains. The target enzyme solution collected from each mutant strain was subjected to high-throughput screening according to the aforementioned screening method. The activities of the mutant enzymes were compared by measuring OD540. Among them, mutant enzyme A0857 had the highest OD540 value after the reaction. HPLC analysis of luteolin-7-O-glucoside revealed a 37% increase in its content compared to the control group.

[0052] Therefore, A0857 was selected as the optimal mutant obtained during the first round of directed evolution screening, and A0857 was used as a template for the next round of random mutation library iteration. According to the aforementioned method for constructing a mutation library, the pUC19-PcGlcT plasmid was replaced with the A0857 plasmid, and the random mutation library was constructed by error-prone PCR and MEGAWHOP PCR. Subsequently, high-throughput screening was performed for directed screening. A total of 1,800 mutant strains were obtained this time. By testing OD540, it was found that B0159 had the highest OD540 value. The HPLC method was used to detect luteolin-7-O-glucoside, and its content was increased by 21% compared with the control group.

[0053] Therefore, B0159 was selected as the optimal mutant obtained during the second round of directed evolution screening, and B0159 was used as a template for the next round of iteration of the random mutation library. According to the aforementioned method for constructing a mutation library, the pUC19-PcGlcT plasmid was replaced with the B0159 plasmid, and the random mutation library was constructed by error-prone PCR and MEGAWHOP PCR. High-throughput screening was then performed. A total of 1,100 mutant strains were obtained this time, and by testing OD540, it was found that C0562 had the highest OD540 value. The HPLC method was used to detect luteolin-7-O-glucoside, and its content was increased by 13% compared with the control group.

[0054] Therefore, C0562 was selected as the optimal mutant obtained during the third round of directed evolution screening, and C0562 was used as a template for the next round of random mutation library iteration. According to the aforementioned method for constructing a mutation library, the pUC19-PcGlcT plasmid was replaced with plasmid C0562, and the random mutation library was constructed using error-prone PCR and MEGAWHOP PCR. High-throughput screening was then performed. A total of 1,200 mutant strains were obtained. By testing OD540, it was found that D0374 had the highest OD540 value. The HPLC method was used to detect luteolin-7-O-glucoside, and its content was 7% higher than that of the control group.

[0055] Through four rounds of iterative directed evolution, mutant D0374 was obtained. E. coli corresponding to wild-type pUC19-PcGlcT and mutant D0374 were cultured in 300ml conical flasks using the aforementioned shaking method to obtain larger amounts of enzyme extract. Using the aforementioned screening method, scale-up testing was performed in 100ml test tubes. HPLC analysis of luteolin-7-O-glucoside content revealed a 104% increase in mutant D0374 compared to the wild-type pUC19-PcGlcT-derived E. coli.

[0056] Finally, mutant D0374 was selected as the end point of directed evolution and named. The corresponding Escherichia coli was named BL21(DE3)-UGT374MS, the corresponding plasmid was named pUC-UGT374MS, and the corresponding enzyme was named glucosyltransferase UGT374MS.

[0057] Plasmid pUC-UGT374MS was extracted and sequenced by a commercial sequencing company. The DNA sequence of glucosyltransferase UGT374MS was obtained as SEQ ID NO.3, and the amino acid sequence thereof was obtained as SEQ ID NO.4.

[0058] SEQ ID NO.3:

[0059] SEQ ID NO.4: MELNSSSCIVLYPAPGIGHQVSMVELGKLILCHHPDSFSKIIILITNAPHLDTRATAPYMISVSATIPSITFHQLPTPPLPPNYAPSVKGLDFELIGLNNPNVHQALQTISSTTFKI KAFIIDFICDAAFKVSSALNLPTHYFFTTAASNLSVLLYFPTLHQKITVNLKDYNDFVHYPGVPPIFSSENAITVLDRNTMEYKFFMGTAAQMAKSDGIIINTFHSLEPTAITAISDG LCVPDAPTPPIYCIGPLITGKEVDSEEHECLVWLNSQPSKSVVFLCFGSLGAFGEEQLKEIAVGLENSKHRFLWVIKNPPSRQEGDPNTVLPQGFLERTKGRGLVVKSWVPQVAVLN HPSVGVFVTHCGWNSILEGVCAGVPMIGWPLYAEQRVNRVLMVEELRVGLGLVESDGERFVSSDELEKGVRELMNSDNGKRLRNRVVELRDAAKAAMSDEDGSSRIELAKLITKWKE.

[0060] By comparing the amino acid sequence of the wild-type glucosyltransferase PcGlcT (SEQ ID NO. 2), it can be found that the glucosyltransferase UGT374MS of the present invention, through qualitative evolution, obtains mutations in 6 amino acid sites, namely L20Q, S61I, Y141H, A153V, R227T, and G358V.

[0061] Preparation Example: Preparation of glucosyltransferase UGT374MS enzyme solution A BL21(DE3)-UGT374MS bacterial suspension was added to a 5L fermenter at a ratio of 1:100. The cells were cultured in TB medium at 37°C to an OD600 of approximately 0.6. Lactose was then added to a final concentration of 5 mM at 25°C for 24 hours to induce target protein expression. The fermentation broth was ultrasonically disrupted, centrifuged, and the supernatant collected to obtain the UGT374MS glucosyltransferase enzyme solution, which can be used to prepare plant extracts. During the preparation of plant extracts, plant material can be physically crushed, ultrasonically disrupted, and then enzymatically extracted to release the plant cell contents. Plant cell contents naturally contain sucrose and UDP. The addition of sucrose synthase continuously generates UDPG. The UGT374MS glucosyltransferase can then convert poorly soluble flavonoids and UDPG into flavonoid-7-O-glycosides, increasing their solubility and reducing precipitation in the plant extract.

[0062] Example 1: Application of Glucosyltransferase in the Preparation of African Cardamom Seed Extract African cardamom seeds (AFRAMOMUM MELEGUETA) were washed with water and oven-dried at 40°C until the surface moisture was removed. The seeds were then pulverized using a low-temperature mill and passed through a 10-mesh sieve. 100 g of the sieved seeds were mixed with 10 g of a complex enzyme consisting of cellulase, xylosidase, pectinase, galactosidase, and protease in a mass ratio of 1:1:1:1:1. 5 g of UGT374MS glucosyltransferase and 5 g of sucrose synthase were added, and the volume was made up to 300 g of water. The mixture was stirred at 25°C for 5 hours to obtain a crude extract. The crude extract was heated to 60°C for 60 minutes to inactivate the enzymes. The inactivated crude extract was then transferred to a filtration unit and filtered through a 0.5 μm membrane filter, followed by a second filtration through a 0.22 μm membrane filter. The resulting filtrate was the refined extract. Glycerol was added to the refined extract to make up to 1000 g, and the extract was divided and sealed in a sterile room to obtain the African cardamom seed extract.

[0063] The African cardamom seed extract obtained using this method produced no precipitation after standing for 2 hours and was found to contain 92 μg / g of 6-gingerol.

[0064] Comparative Example 1: Preparation of African cardamom seed extract African cardamom seeds (AFRAMOMUM MELEGUETA) were washed with water and oven-dried at 40°C until the surface moisture was removed. The seeds were then pulverized using a low-temperature grinder and passed through a 10-mesh sieve. 100 g of the sieved seeds were mixed with 10 g of a complex enzyme consisting of cellulase, xylosidase, pectinase, galactosidase, and protease in a mass ratio of 1:1:1:1:1. 300 g of water was added and the mixture was stirred continuously at 25°C for 5 hours to obtain a crude extract. The crude extract was heated to 60°C for 60 minutes to inactivate the enzymes. The inactivated crude extract was transferred to a filtration apparatus and filtered through a 0.5 μm membrane and then a 0.22 μm membrane. The resulting filtrate was the refined extract. Glycerol was added to the refined extract to a total of 1000 g. The resulting aliquots were sealed in a sterile container to obtain the African cardamom seed extract.

[0065] The above method differs from the method in Example 1 only in that 5 g of glucosyltransferase UGT374MS enzyme solution and 5 g of sucrose synthase enzyme solution are omitted, and the mixture is directly supplemented with 300 g of water. The African cardamom seed extract obtained using this method formed a precipitate after standing for 20 minutes. Testing revealed a precipitate content of 0.8 μg / g 6-gingerol.

[0066] Example 2: Application of Glucosyltransferase in the Preparation of Saffron Extract Saffron (also known as Crocus sativus) petals are washed with clean water and dried in a low-temperature dryer at 20°C until the surface is free of moisture. 100g of dried saffron petals are mixed with 10g of a complex enzyme consisting of cellulase, xylosidase, pectinase, galactosidase, and protease in a mass ratio of 1:1:1:1:1. 5g of UGT374MS glucosyltransferase enzyme solution and 5g of sucrose synthase enzyme solution are added, followed by 850g of water. The mixture is stirred continuously at 25°C for 5 hours to obtain a crude extract. The crude extract is then heated to 60°C for 60 minutes to inactivate the enzymes. The inactivated crude extract is then transferred to a filtration unit and filtered through a 0.5μm membrane filter, followed by a second filtration through a 0.22μm membrane filter. The resulting filtrate is the refined extract. 1,3-Propanediol was added to the refined extract to make up to 1000 g, and the extracts were divided and sealed in a sterile room to obtain a saffron flower extract.

[0067] The crocus flower extract obtained using this method produced no precipitation after being allowed to stand for 2 hours and was found to contain 0.35 g / L of total flavonoids.

[0068] Comparative Example 2: Preparation of Saffron Extract Crocus flower petals were washed with clean water and dried in a low-temperature dryer at 20°C until the surface moisture was removed. 100 g of dried crocus flower petals were mixed with 10 g of a complex enzyme consisting of cellulase, xylosidase, pectinase, galactosidase, and protease in a mass ratio of 1:1:1:1:1. 850 g of water was added and the mixture was stirred continuously at 25°C for 5 hours to obtain a crude extract. The crude extract was heated to 60°C for 60 minutes to inactivate the enzymes. The inactivated crude extract was transferred to a filtration apparatus and filtered through a 0.5 μm membrane and then a 0.22 μm membrane. The resulting filtrate was the refined extract. 1,3-Propanediol was added to the refined extract to make up to 1000 g. The aliquots were sealed in a sterile container to obtain the crocus flower extract.

[0069] The above method differs from the method in Example 2 only in that 5g of glucosyltransferase UGT374MS enzyme solution and 5g of sucrose synthase enzyme solution are omitted, and 850g of water are directly added. The crocus flower extract obtained using this method formed a precipitate after standing for 20 minutes. Testing revealed a total flavonoid content of 0.02g / L.

[0070] Example 3: Application of Glucosyltransferase in the Preparation of Marshmallow Root Extract The roots of Althaea officinalis (ALTHAEA OFFICINALIS) were washed with water and oven-dried at 40°C until the surface moisture was removed. The roots were then pulverized using a low-temperature mill and the pulverized granules were passed through a 10-mesh sieve. 50 g of the sieved granules were mixed with 10 g of a complex enzyme consisting of cellulase, xylosidase, pectinase, galactosidase, and protease in a mass ratio of 1:1:1:1:1. 5 g of UGT374MS glucosyltransferase and 5 g of sucrose synthase were added, and the volume was made up to 500 g of water. The mixture was stirred at 25°C for 5 hours to obtain a crude extract. The crude extract was heated to 60°C for 60 minutes to inactivate the enzymes. The inactivated crude extract was then transferred to a filtration unit and filtered through a 0.5 μm membrane and then a 0.22 μm membrane. The resulting filtrate was the purified extract. Glycerol was added to the refined extract to make up to 1000 g, and the extract was divided and sealed in a sterile room to obtain the marshmallow root extract.

[0071] The Marshmallow root extract obtained by the method does not produce precipitation after being placed for a long time for 2 hours.

[0072] Comparative Example 3: Preparation of Marshmallow Root Extract Wash the roots of marshmallow (Althaea officinalis) with clean water and oven-dry at 40°C until the surface is free of moisture. Pulverize the roots using a low-temperature grinder, and pass the pulverized marshmallow root particles through a 10-mesh sieve. Add 50 g of the sieved marshmallow root particles and 10 g of a complex enzyme consisting of cellulase, xylosidase, pectinase, galactosidase, and protease in a mass ratio of 1:1:1:1:1. Add 500 g of water and stir continuously at 25°C for 5 hours to obtain a crude extract. The crude extract is heated to 60°C for 60 minutes to inactivate the enzymes. The inactivated crude extract is then transferred to a filtration unit and filtered through a 0.5 μm membrane followed by a second filtration through a 0.22 μm membrane. The resulting filtrate is the refined extract. Glycerol was added to the refined extract to make up to 1000 g, and the extract was divided and sealed in a sterile room to obtain the marshmallow root extract.

[0073] The above method differs from the method in Example 3 only in that 5 g of glucosyltransferase UGT374MS enzyme solution and 5 g of sucrose synthase enzyme solution are not added, and 500 g of water is directly added. The Marshmallow root extract obtained using this method produced a precipitate after standing for 20 minutes.

[0074] Example 4: Application of Glucosyltransferase in the Preparation of Composite Plant Extracts Viola yedoensis, chamomile, dandelion, peony root, mallow, and honeysuckle are blended in a mass ratio of 2:2:1:1:1:1. The mixture is washed with clean water and oven-dried at 40°C until the surface moisture is removed. The blended plant particles are pulverized using a low-temperature grinder and passed through a 10-mesh sieve. 50g of the sieved blended plant particles are added to 10g of a complex enzyme containing cellulase, xylosidase, pectinase, galactosidase, and protease in a mass ratio of 1:1:1:1:1. 5g of UGT374MS glucosyltransferase and 5g of sucrose synthase are added, and the mixture is made up to 500g of water. The mixture is stirred at 25°C for 5 hours to obtain a crude extract. The crude extract is heated to 60°C for 60 minutes to inactivate the enzymes. The inactivated crude extract was transferred to a filtration device and filtered using a 0.5 μm membrane filter, followed by a second filtration using a 0.22 μm membrane filter. The resulting filtrate was the refined extract. Glycerol was added to the refined extract to a total volume of 1000 g, and the resulting aliquots were sealed in a sterile chamber to obtain the compounded plant extract.

[0075] The compound plant extract obtained by this method does not produce any precipitation after being placed for a long time for 2 hours.

[0076] Comparative Example 4: Preparation of composite plant extracts Viola yedoensis, chamomile flower, dandelion, peony root, mallow flower, and honeysuckle flower were blended in a mass ratio of 2:2:1:1:1:1. The mixture was washed with clean water and oven-dried at 40°C until the surface moisture was removed. The blended plant particles were pulverized using a low-temperature grinder and passed through a 10-mesh sieve. 50g of the sieved blended plant particles were mixed with 10g of a complex enzyme containing cellulase, xylosidase, pectinase, galactosidase, and protease in a mass ratio of 1:1:1:1:1. 500g of water was added and the mixture was stirred continuously at 25°C for 5 hours to obtain a crude extract. The crude extract was heated to 60°C for 60 minutes to inactivate the enzymes. The inactivated crude extract was transferred to a filtration device and filtered through a 0.5μm membrane, followed by a second filtration through a 0.22μm membrane. The resulting filtrate was the refined extract. Glycerol was added to the refined extract to make up to 1000 g, and the extracts were divided and sealed in a sterile room to obtain a compound plant extract.

[0077] The above method differs from the method in Example 4 only in that 5 g of glucosyltransferase UGT374MS enzyme solution and 5 g of sucrose synthase enzyme solution are not added, and 500 g of water is directly added. The composite plant extract obtained using this method produced a precipitate after being allowed to stand for 20 minutes.

[0078] Example 5: Application of Glucosyltransferase in the Preparation of Buckwheat Seed Extract Buckwheat seeds (POLYGONUM FAGOPYRUM) were washed with water and oven-dried at 40°C until the surface moisture was removed. The seeds were then pulverized using a low-temperature mill and passed through a 10-mesh sieve. 100 g of the sieved buckwheat seeds were mixed with 10 g of a complex enzyme consisting of cellulase, xylosidase, pectinase, galactosidase, and protease in a mass ratio of 1:1:1:1:1. 5 g of UGT374MS glucosyltransferase and 5 g of sucrose synthase were added, and the volume was made up to 990 g of water. The mixture was stirred at 25°C for 5 hours to obtain a crude extract. The crude extract was heated to 60°C for 60 minutes to inactivate the enzymes. The inactivated crude extract was then transferred to a filtration unit and filtered through a 0.5 μm membrane and then a 0.22 μm membrane. The resulting filtrate was the purified extract. 1,2-Hexanediol was added to the refined extract to make up to 1000 g, and the extracts were divided and sealed in a sterile room to obtain buckwheat seed extract.

[0079] The buckwheat seed extract obtained using this method showed no precipitation after being stored for 2 hours and was found to contain 11.2 g / cm of total carbohydrates.

[0080] Comparative Example 5: Preparation of buckwheat seed extract Buckwheat seeds (POLYGONUM FAGOPYRUM) were washed with water and oven-dried at 40°C until the surface moisture was removed. The seeds were then pulverized using a low-temperature grinder and passed through a 10-mesh sieve. 100 g of the sieved buckwheat seeds were mixed with 10 g of a complex enzyme consisting of cellulase, xylosidase, pectinase, galactosidase, and protease in a mass ratio of 1:1:1:1:1. 990 g of water was added and the mixture was stirred continuously at 25°C for 5 hours to obtain a crude extract. The crude extract was heated to 60°C for 60 minutes to inactivate the enzymes. The inactivated crude extract was then transferred to a filtration unit and filtered through a 0.5 μm membrane followed by a second filtration through a 0.22 μm membrane. The resulting filtrate was the refined extract. 1,2-Hexanediol was added to the refined extract to make up to 1000 g, and the extracts were divided and sealed in a sterile room to obtain buckwheat seed extract.

[0081] The above method differed from the method in Example 5 only in that 5 g of glucosyltransferase UGT374MS enzyme solution and 5 g of sucrose synthase enzyme solution were not added, and 990 g of water was directly added. The buckwheat seed extract obtained using this method formed a precipitate after standing for 20 minutes.

[0082] Example 6: Application of Glucosyltransferase in the Preparation of Alpinia glabra Leaf Extract Take leaves of Althaea officinalis, wash them with water, and oven-dry them at 40°C until the surface moisture is removed. Use a low-temperature grinder to pulverize the leaves, and pass the pulverized leaves through a 10-mesh sieve. Add 50g of the sieved leaves and 10g of a complex enzyme consisting of cellulase, xylosidase, pectinase, galactosidase, and protease in a mass ratio of 1:1:1:1:1. Add 5g of UGT374MS glucosyltransferase and 5g of sucrose synthase, and make up to 550g of water. Stir continuously at 25°C for 5 hours to obtain a crude extract. Heat the crude extract to 60°C for 60 minutes to inactivate the enzymes. Transfer the inactivated crude extract to a filtration unit, filter it once through a 0.5μm filter membrane, and then filter it again through a 0.22μm filter membrane. The resulting filtrate is the refined extract. 1,3-propylene glycol was added to the refined extract to make up to 1000 g, and the extract was divided and sealed in a sterile room to obtain the Alpinia glabra leaf extract.

[0083] The Alpinia galanga leaf extract obtained by this method does not produce any precipitation after being left for a long time of 2 hours.

[0084] Comparative Example 6: Preparation of Alpinia glabra leaf extract Wash the leaves of Althaea officinalis (Althaea officinalis) with clean water and oven-dry at 40°C until the surface moisture is removed. Pulverize the leaves using a low-temperature grinder, and pass the pulverized leaves through a 10-mesh sieve. Add 50g of the sieved leaves and 10g of a complex enzyme containing cellulase, xylosidase, pectinase, galactosidase, and protease in a mass ratio of 1:1:1:1:1. Add 550g of water and stir continuously at 25°C for 5 hours to obtain a crude extract. Heat the crude extract to 60°C for 60 minutes to inactivate the enzymes. The inactivated crude extract is then transferred to a filtration unit and filtered through a 0.5μm membrane, followed by a second filtration through a 0.22μm membrane. The resulting filtrate is the refined extract. 1,3-propylene glycol was added to the refined extract to make up to 1000 g, and the extract was divided and sealed in a sterile room to obtain the Alpinia glabra leaf extract.

[0085] The above method differed from the method in Example 6 only in that 5 g of glucosyltransferase UGT374MS enzyme solution and 5 g of sucrose synthase enzyme solution were not added, and 550 g of water was directly added. The Alpinia glabra leaf extract obtained using this method produced a precipitate after standing for 20 minutes.

[0086] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A glucosyltransferase, characterized in that The amino acid sequence of the glucosyltransferase is the sequence shown in SEQ ID NO.

4.

2. A nucleic acid, characterized in that The nucleic acid encodes the glucosyltransferase according to claim 1.

3. The nucleic acid according to claim 2, characterized in that The nucleic acid has the sequence shown in SEQ ID NO.

3.

4. A carrier, characterized in that The vector comprises the nucleic acid according to claim 2 or 3.

5. A cell, characterized in that The cell expresses the glucosyltransferase according to claim 1, or comprises the nucleic acid according to any one of claims 2-3, or comprises the vector according to claim 4.

6. Use of the glucosyltransferase according to claim 1, the nucleic acid according to any one of claims 2 to 3, the vector according to claim 4 or the cell according to claim 5.

7. The use according to claim 6, characterized in that The applications include: Catalyzes flavonoids to generate flavonoid-7-O-glycosides; preparing plant extracts; Reduces precipitation in plant extracts.

8. A method for producing flavonoid-7-O-glycoside, characterized in that: The method comprises catalyzing the production of flavonoids by the glucosyltransferase of claim 1 .

9. A method for preparing a plant extract, characterized in that: The method comprises extracting using the glucosyltransferase according to claim 1.

10. The method for preparing the glucosyltransferase according to claim 1, wherein The preparation method comprises obtaining the wild-type glucosyltransferase through directed evolution.

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