A glucosyltransferase obtained by directed evolution, derivatives thereof, uses and methods of preparation

Abstract

The application provides a glucosyltransferase and a derivative thereof obtained by a directed evolution method, application and preparation method, and belongs to the technical field of enzyme engineering. The glucosyltransferase provided by the application is obtained by a directed evolution method, the amino acid sequence of the glucosyltransferase is a sequence shown in SEQ ID NO. 4, and the DNA sequence of the glucosyltransferase comprises a sequence shown in SEQ ID NO. 3. The glucosyltransferase has strong catalytic ability, can significantly and efficiently catalyze flavone to generate flavone-7-O-glycoside, and can significantly reduce the generation of precipitates in plant extracts.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of enzyme engineering, and relates to a glucosyltransferase, in particular to a glucosyltransferase and derivatives thereof obtained by a directed evolution method, application and preparation method. BACKGROUND

[0002] Flavonoids are a class of active natural compounds widely existing in plants. According to the difference of C3 ring structure in the basic skeleton, flavonoids are divided into flavones, flavonols, flavanones, isoflavones and other types. Modern pharmacological results show that flavonoids have many pharmacological activities such as antioxidant, anti-inflammatory, antiviral and the like. However, naturally occurring flavonoids often have the shortcomings of poor water solubility and low stability.

[0003] Flavonoid compounds are also common active ingredients in plant extracts. However, during the preparation of plant extracts, flavonoid compounds often cause the generation of precipitates. The use of organic solvents to prepare plant extracts can increase the solubility and stability of flavonoid compounds, but organic solvents can easily cause environmental pollution, and the residues of organic solvents can harm the human body.

[0004] By glycosylation modification of flavonoids, flavonoid glycosides can be formed to increase the solubility and stability of flavonoid compounds.

[0005] In recent years, great progress has been made in the research of chemical and biosynthetic methods of glycosylation modification. However, chemical glycosylation reactions have many shortcomings such as many by-products and intermediates, poor regioselectivity and stereoselectivity, low yield, complicated protection and deprotection steps, and large pollution. In addition, the low atom economy and the expensive price of some catalysts also make the chemical synthesis method have many shortcomings in large-scale production of glycosides. In contrast, the biosynthetic method using enzyme catalysis not only has stereoselectivity and regioselectivity, but also has relatively simple operation steps and less environmental pollution, which conforms to the concept of green chemistry. Therefore, the use of glucosyltransferase to catalyze flavonoids to synthesize flavonoid glycosides has gradually become the research focus in this field.

[0006] The prior art CN118755685A discloses a mutant AAVT of a glucosyltransferase VcUGT1 screened from blueberries. Compared with the glucosyltransferase VcUGT1, the mutant AAVT has point mutations: S367A, V274A, F82V and I132T. The mutant can significantly improve the catalytic activity of the glycosylation reaction and can efficiently catalyze the substrate to generate the corresponding 7-O-glucoside compound.

[0007] Commonly, most of the glucosyltransferases are catalyzing the formation of glycosides at the 3' position of flavones, and the glucosyltransferases catalyzing the formation of glycosides at the 7' position of flavones are rare. Due to the steric hindrance of the molecular structure, flavone glycosides at the 7' position have better solubility. Therefore, it is urgent to explore more efficient glucosyltransferases which can glycosidize flavones at the 7' position to generate flavone-7-O-glucosides. SUMMARY

[0008] The present application aims at the problem that the prior art lacks efficient glucosyltransferases catalyzing the formation of glycosides at the 7' position of flavones, and provides a glucosyltransferase and its derivative obtained by a directed evolution method, applications and a preparation method. The glucosyltransferase provided by the present application is obtained by a directed evolution method, the amino acid sequence of which is the sequence shown in SEQ ID NO. 4, and the gene sequence of which comprises the sequence shown in SEQ ID NO. 3; the glucosyltransferase has strong catalytic ability and can efficiently catalyze flavones to generate flavone-7-O-glucosides, and can also significantly reduce the generation of precipitates in plant extracts.

[0009] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0010] In one aspect, the present application provides a glucosyltransferase, the amino acid sequence of which is the sequence shown in SEQ ID NO. 4.

[0011] In another aspect, the present application provides a nucleic acid, which encodes the above-mentioned glucosyltransferase.

[0012] Preferably, the nucleic acid has the sequence shown in SEQ ID NO. 3.

[0013] In another aspect, the present application provides a vector, which comprises the above-mentioned nucleic acid.

[0014] In another aspect, the present application provides a cell, which expresses the above-mentioned glucosyltransferase, or the above-mentioned nucleic acid, or the above-mentioned vector.

[0015] In another aspect, the present application provides applications of the above-mentioned glucosyltransferase, the above-mentioned nucleic acid, the above-mentioned vector or the above-mentioned cell.

[0016] Preferably, the applications comprise:

[0017] catalyzing the formation of flavone-7-O-glucosides from flavones;

[0018] preparing plant extracts;

[0019] reducing the generation of precipitates in plant extracts.

[0020] In another aspect, the present application provides a method for producing flavone-7-O-glycoside, comprising catalyzing flavone by the above-mentioned glucosyltransferase.

[0021] In another aspect, the present application provides a method for preparing plant extract, comprising using the above-mentioned glucosyltransferase for extraction.

[0022] In another aspect, the present application provides a method for preparing the above-mentioned glucosyltransferase, comprising obtaining by directed evolution of wild-type glucosyltransferase.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] 1、The glucosyltransferase provided by the present application has strong catalytic ability and can efficiently catalyze flavone to produce flavone-7-O-glycoside.

[0025] 2、The glucosyltransferase provided by the present application can significantly reduce the generation of precipitates in plant extract. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 It is a schematic diagram of pUC19-PcGlcT plasmid.

[0027] Figure 2 It is a schematic diagram of the reaction principle of the present application. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in combination with examples. The specific conditions not mentioned in the examples are carried out according to conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments not mentioned by the manufacturer are conventional products that can be purchased on the market. In order to better illustrate the present application, numerous specific details are given in the following detailed description of the embodiments. The specific embodiments described herein are only used to explain the present application and do not constitute any limitation on the present application. In addition, in the following description, the description of well-known structures and techniques is omitted to avoid unnecessary confusion of the concept of the present application. Such structures and techniques are described in many publications, such as "Molecular Cloning: A Laboratory Manual (Fourth Edition)" (Cold Spring Harbor Laboratory Press), Ausubel, F.M. et al., Current Protocols in Molecular Biology , Greene Publishing Assoc. and Wiley-lnterscience.

[0029] Preparation example: obtaining of glucosyltransferase

[0030] (1) Gene cloning of wild-type glucosyltransferase

[0031] The document "Biochemical Characterization of Parsley Glycosyltransferases Involved in the Biosynthesis of a Flavonoid Glycoside, Apiin" describes a glucose transferase PcGlcT extracted from parsley cells, which is used as a wild-type glucose transferase for the subsequent development work. According to the above document, the DNA sequence of the glucose transferase PcGlcT is obtained as shown in SEQ ID NO. 1, and the amino acid sequence thereof is SEQ ID NO. 2. The DNA fragment of the glucose transferase PcGlcT is synthesized according to SEQ ID NO. 1 by a commercial gene synthesis company.

[0032]

[0033] SEQ ID NO. 2:

[0034] MELNSSSCIVLYPAPGIGHLVSMVELGKLILCHHPDSFSKIIILITNAPHLDTRATAPYMSSVSATIPSITFHQLPTPPLPPNYAPSVKGLDFELIGLNNPNVHQALQTISSTTFKIKAFIIDFICDAAFKVSSALNLPTYYFFTTAASNLSALLYFPTLHQKITVNLKDYNDFVHYPGVPPIFSSENAITVLDRNTMEYKFFMGTAAQMAKSDGIIINTFHSLEPRAITAISDGLCVPDAPTPPIYCIGPLITGKEVDSEEHECLVWLNSQPSKSVVFLCFGSLGAFGEEQLKEIAVGLENSKHRFLWVIKNPPSRQEGDPNTVLPQGFLERTKGRGLVVKSWVPQVAVLNHPSVGGFVTHCGWNSILEGVCAGVPMIGWPLYAEQRVNRVLMVEELRVGLGLVESDGERFVSSDELEKGVRELMNSDNGKRLRNRVVELRDAAKAAMSDEDGSSRIELAKLITKWKE.

[0035] DNA according to the glucosyltransferase PcGlcT. Primers containing enzyme cleavage sites were designed. A Sal I enzyme cleavage site was introduced by the forward primer PcGlcT-WF (SEQ ID NO. 5: 5'-GCGGTCGACTTTTTTCTTAAAATTTCA-3') and a Nde I enzyme cleavage site was introduced by the reverse primer PcGlcT-WR (SEQ ID NO. 6: 5'-TTACATATGCCGACTTTGTTAGTA-3'). The plasmid pUC19 was chosen according to the requirements of the parameters of the subsequent tests and was purchased from a commercial company. The DNA of the glucosyltransferase PcGlcT was used as a template for the PCR amplification using the primers described above (PCR with enzymes and the matching reagents from Takara, item number R010B) and was introduced into the pUC19 plasmid by enzyme cleavage. The reaction system (see Table 1) and the program (see Table 2) for the PCR amplification.

[0036] Table 1 Reaction system for the PCR amplification

[0037]

[0038] Table 2 Program for the PCR amplification

[0039]

[0040] Enzyme digestion was performed using a commercially available rapid digestion kit containing Sal I and Nde I enzymes. The PCR amplification products and pUC19 plasmid obtained in the above steps were digested separately. The digestion temperature was 37℃, and the digestion time was 1 hour. The digestion system is shown in Table 3.

[0041] Table 3 Enzyme digestion system

[0042]

[0043] The enzyme-digested DNA and pUC19 plasmid were ligated together to obtain the plasmid pUC19-PcGlcT (see schematic diagram of plasmid map). Figure 1 This plasmid served as a template for the wild-type glucosyltransferase. A commercially available T4 ligase kit was used, and ligation was performed at 16°C for 12 hours. The ligation system is shown in Table 4.

[0044] Table 4 Enzyme ligation system

[0045]

[0046] (2) Construction of recombinant expression strains and expression of enzymes

[0047] Using E. coli E. coli BL21(DE3) competent cells were prepared, and the pUC19-PcGlcT plasmid was transformed into the cells using electroporation. The transformed cells were then plated onto solid agar plates containing ampicillin and incubated at 37°C for 24 hours. Single clones were selected from the plates and inoculated into test tubes containing ampicillin-containing LB broth, and incubated at 37°C for 12 hours. The bacterial suspension was then diluted 1:100 and transferred to new test tubes, incubated until the OD600 reached approximately 0.6, and IPTG was added to a final concentration of 0.5 mM. The cells were then incubated at 25°C with shaking at 180 rpm for 24 hours to induce target protein expression. The fermentation broth in the test tubes was then sonicated, centrifuged, and the supernatant was collected to obtain the target enzyme solution.

[0048] (3) Screening method

[0049] The glucosyltransferase studied in this invention uses flavonoids and UDPG as substrates to catalyze the synthesis of flavonoid-7-O-glycosides via glycosylation. In this synthetic pathway, UDPG is hydrolyzed to UDP, thus requiring the addition of UDPG to maintain the reaction. However, since UDPG is relatively expensive, the addition of sucrase synthase can catalyze the synthesis of UDPG from UDP and sucrose, continuously replenishing the reaction system with UDPG and reducing costs.

[0050] In the reaction pathway of sucrose synthase, UDP and sucrose are used as substrates to synthesize UDPG and fructose. Fructose is a reducing sugar, and under heating conditions, it can undergo an oxidation-reduction reaction with light yellow 3,5-dinitrosalicylic acid (DNS) to become brown 3-amino-5-nitrosalicylic acid. 3-amino-5-nitrosalicylic acid has an absorption peak at 540 nm, and can be quickly detected by a UV spectrophotometer. The reaction principle is shown in Figure 2 .

[0051] Therefore, by measuring the OD540 value of the reaction system, the amount of fructose generated in the entire reaction can be quantitatively analyzed, which can indirectly reflect the amount of synthesized flavone-7-O-glycoside, thereby reflecting the activity of the glycosyltransferase. Therefore, the above method can be used for high-throughput and rapid screening of glycosyltransferases.

[0052] In the specific screening operation process, the present application uses luteolin, a common flavone in plants, as the starting material for screening. Luteolin can be synthesized into luteolin-7-O-glucoside by the glucose transferase studied in the present application.

[0053] (4) Verification of enzyme

[0054] In the test tube, 0.01 g / ml of luteolin, 0.1 g / ml of sucrose, 2 mM of UDPG, 50 U of sucrose synthase, 0.5 ml of the above enzyme solution, and buffer solution to make up the reaction system to 5 ml were added. The system was fully reacted at 37°C for 6 hours. The reaction was terminated by boiling in a water bath for 5 minutes, and the supernatant was detected after centrifugation.

[0055] According to the above screening method, the OD540 of the supernatant was detected. Compared with the control group, the OD540 was significantly improved.

[0056] At the same time, the luteolin and luteolin-7-O-glucoside in the supernatant were detected by HPLC method. The content of luteolin was significantly reduced, and the content of luteolin-7-O-glucoside was significantly increased.

[0057] Therefore, it is proved that the target enzyme glucose transferase PcGlcT is successfully expressed, and the OD540 can be used to characterize the activity of the target enzyme in the reaction system.

[0058] (5) Construction of mutation library

[0059] A random mutant library of 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 reagents were purchased from Xiamen Aisbio Technology Co., Ltd.). The PCR amplification reaction system (see Table 5) and procedure (see Table 6) are shown below.

[0060] Table 5. Reaction system for PCR amplification

[0061]

[0062] Table 6. PCR amplification procedure

[0063]

[0064] After obtaining the error-prone mutant DNA fragment of pUC19-PcGlcT, it was used as a large primer, and pUC19-PcGlcT was used as a template for MEGAWHOP PCR (PCR was performed using enzymes and reagents purchased from Takara, catalog number R010B). The PCR amplification reaction system (see Table 7) and procedure (see Table 8) are shown below.

[0065] Table 7. PCR amplification reaction system

[0066]

[0067] Table 8. PCR amplification procedure

[0068]

[0069] The amplification product obtained by MEGAWHOP PCR was incubated with Dpn I restriction enzyme and reacted at 37°C for 12 hours for template digestion. E. coli BL21(DE3) competent cells were then used to introduce the amplification products into the cell line via electroporation. E. coli BL21(DE3) was spread onto a solid plate containing ampicillin and incubated at 37°C for 24 hours.

[0070] The single clone was selected from the plate and inoculated into a test tube of liquid LB medium added with ampicillin and cultured at 37°C for 12 hours. The bacterial suspension was taken out at a ratio of 1:100 into a new test tube and cultured to about OD600=0.6, then IPTG was added at a final concentration of 0.5 mM, and the target protein expression was induced by culturing at 25°C and 180 rpm for 24 hours. The fermentation liquid was subjected to ultrasonic crushing, and the supernatant was collected after centrifugation, thereby obtaining the solution of the target enzyme.

[0071] (6) Directional evolution through multiple rounds of high-throughput screening

[0072] According to the above steps, the random mutation library was constructed with pUC19-PcGlcT as the starting point, and a total of 2600 mutant strains were obtained. The target enzyme solution collected from each mutant strain was subjected to high-throughput screening according to the screening method described above. The activities of the mutant enzymes were compared by detecting OD540. Among them, the OD540 value of mutant enzyme A0857 was the highest after the reaction. The content of luteolin-7-O-glucoside was detected by HPLC method, which was increased by 37% compared with the control group.

[0073] Therefore, A0857 was selected as the optimal mutant obtained in the first round of directional evolution screening, and A0857 was used as the template for the next iteration of random mutation library. According to the method for constructing the mutant library described above, the pUC19-PcGlcT plasmid was replaced with the A0857 plasmid, and random mutation library was constructed by error-prone PCR and MEGAWHOP PCR. Subsequently, directional screening was performed by high-throughput screening. A total of 1800 mutant strains were obtained, and B0159 was found to have the highest OD540 value by detecting OD540. The content of luteolin-7-O-glucoside was detected by HPLC method, which was increased by 21% compared with the control group.

[0074] Therefore, B0159 was selected as the optimal mutant obtained in the second round of directional evolution screening, and B0159 was used as the template for the next iteration of random mutation library. According to the method for constructing the mutant library described above, the pUC19-PcGlcT plasmid was replaced with the B0159 plasmid, and random mutation library was constructed by error-prone PCR and MEGAWHOP PCR. Subsequently, directional screening was performed by high-throughput screening. A total of 1100 mutant strains were obtained, and C0562 was found to have the highest OD540 value by detecting OD540. The content of luteolin-7-O-glucoside was detected by HPLC method, which was increased by 13% compared with the control group.

[0075] Therefore, C0562 was selected as the optimal mutant in the third round of directed evolution screening process, and C0562 was used as a template for the next round of random mutation library iteration. According to the method of constructing the mutation library as described above, the pUC19-PcGlcT plasmid was replaced with the C0562 plasmid, and the random mutation library was constructed by error-prone PCR and MEGAWHOP PCR. Subsequently, high-throughput screening was performed. A total of 1200 mutant strains were obtained, and through detection of OD540, it was found that D0374 had the highest OD540 value. Through HPLC method detection of luteolin-7-O-glucoside, its content was increased by 7% compared with the control group.

[0076] Through four rounds of iterative directed evolution, mutant D0374 was obtained. The wild type pUC19-PcGlcT and D0374 mutant corresponding E. coli were respectively cultured according to the foregoing culture method, and 300 ml conical flasks were used for shaking culture to obtain more enzyme extract. Using the foregoing screening method, 100 ml test tubes were used for expanded detection. Through HPLC method detection of luteolin-7-O-glucoside content, D0374 mutant increased by 104% compared with wild type pUC19-PcGlcT corresponding E. coli.

[0077] Finally, mutant D0374 was selected as the end point of directed evolution and was named, and the corresponding E. coli was named BL21(DE3)-UGT374MS, the corresponding plasmid was named pUC-UGT374MS, and the corresponding enzyme was named glucosyltransferase UGT374MS.

[0078] The plasmid pUC-UGT374MS was extracted and sequenced by a commercial sequencing company, and the DNA sequence of glucosyltransferase UGT374MS was SEQ ID NO. 3, and its amino acid sequence was SEQ ID NO. 4.

[0079] SEQ ID NO. 3:

[0080]

[0081] SEQ ID NO. 4:

[0082] MELNSSSCIVLYPAPGIGHQVSMVELGKLILCHHPDSFSKIIILITNAPHLDTRATAPYMISVSATIPSITFHQLPTPPLPPNYAPSVKGLDFELIGLNNPNVHQALQTISSTTFKIKAFIIDFICDAAFKVSSALNLPTHYFFTTAASNLSVLLYFPTLHQKITVNLKDYNDFVHYPGVPPIFSSENAITVLDRNTMEYKFFMGTAAQMAKSDGIIINTFHSLEPTAITAISDGLCVPDAPTPPIYCIGPLITGKEVDSEEHECLVWLNSQPSKSVVFLCFGSLGAFGEEQLKEIAVGLENSKHRFLWVIKNPPSRQEGDPNTVLPQGFLERTKGRGLVVKSWVPQVAVLNHPSVGVFVTHCGWNSILEGVCAGVPMIGWPLYAEQRVNRVLMVEELRVGLGLVESDGERFVSSDELEKGVRELMNSDNGKRLRNRVVELRDAAKAAMSDEDGSSRIELAKLITKWKE.

[0083] By comparing the amino acid sequence of wild-type glucosyltransferase PcGlcT (SEQ ID NO. 2), it can be found that the glucosyltransferase UGT374MS of the present application is obtained by qualitative evolution, and 6 amino acid sites are mutated, which are L20Q, S61I, Y141H, A153V, R227T, and G358V.

[0084] Preparation Example: Preparation of glucosyltransferase UGT374MS enzyme solution

[0085] Using 5L fermentor, add BL21(DE3)-UGT374MS bacteria suspension into the fermentor with 1:100 ratio, use TB medium, cultivate at 37℃ until OD600=0.6, add lactose with final concentration of 5mM, cultivate at 25℃ for 24 hours to induce the expression of target protein. Ultrasonically break the fermentation broth, collect the supernatant after centrifugation, and obtain the glucose transferase UGT374MS enzyme solution which can be used for preparing plant extract. In the process of preparing plant extract, the plant raw material can be released by physical crushing, ultrasonic breaking, enzymatic extraction and other methods. The plant cell content itself contains sucrose and UDP, and by adding sucrose synthase, UDPG is continuously generated. Thus, the glucose transferase UGT374MS can synthesize flavone-7-O-glycoside from flavone with poor solubility and UDPG, increase the solubility, and reduce the precipitation of plant extract.

[0086] Example 1: Application of glucose transferase in preparing AFRICAN MELAGUETA seed extract

[0087] Take AFRICAN MELAGUETA seed (AFRAMOMUM MELEGUETA), wash with clean water, and dry in an oven at 40℃ until the surface is free of moisture. Use a low-temperature pulverizer to crush, and sieve the crushed AFRICAN MELAGUETA seed particles through a 10-mesh sieve. Take 100g of sieved AFRICAN MELAGUETA seed particles, and add 10g of complex enzyme. The complex enzyme consists of cellulase, xylosidase, pectinase, galactosidase, and protease with a mass ratio of 1:1:1:1:1. Add 5g of enzyme solution of glucose transferase UGT374MS and 5g of enzyme solution of sucrose synthase, make up to 300g of water, and continuously stir at 25℃ for 5h to obtain a crude extract. Heat the crude extract to 60℃ and keep for 60 minutes to inactivate the enzymes in the system. Transfer the inactivated crude extract to a filtration device, first filter with a 0.5μm filter membrane, and then filter with a 0.22μm filter membrane. The obtained filtrate is a refined extract. Add glycerol to the refined extract to make up to 1000g, and seal and store in a sterile room to obtain AFRICAN MELAGUETA seed extract.

[0088] The AFRICAN MELAGUETA seed extract obtained by this method is placed for 2 hours without precipitation. After detection, it contains 92μg / g of 6-gingerol.

[0089] Comparative Example 1: Preparation of AFRICAN MELAGUETA seed extract

[0090] Take AFRICAN MELEGUETA, wash with clean water, and place in an oven at 40°C to dry until the surface is free of moisture. Use a low-temperature pulverizer to pulverize, and sieve the pulverized AFRICAN MELEGUETA particles through a 10-mesh sieve. Take 100 g of the sieved AFRICAN MELEGUETA particles and add 10 g of a composite enzyme. The composite enzyme consists of cellulase, xylosidase, pectinase, galactosidase, and protease in a mass ratio of 1:1:1:1:1. Add water to make up 300 g, and continuously stir at 25°C for 5 h to obtain a crude extract. Heat the crude extract to 60°C and maintain for 60 min to inactivate the enzymes in the system. Transfer the inactivated crude extract to a filtration device, first filter once using a 0.5-μm filter membrane, and then filter twice using a 0.22-μm filter membrane. The obtained filtrate is a refined extract. Add glycerol to the refined extract to make up 1000 g, and seal and store in a sterile room to obtain an AFRICAN MELEGUETA extract.

[0091] The above method differs from the method of Example 1 only in that 5 g of enzyme solution of glucose transferase UGT374MS and 5 g of enzyme solution of sucrose synthase are not added, and 300 g of water is directly made up. The AFRICAN MELEGUETA extract obtained using this method has precipitates generated after 20 min of storage. Detection shows that it contains 0.8 μg / g of 6-gingerol.

[0092] Example 2: Application of glucose transferase to preparation of saffron extract

[0093] Take CROCUS SATIVUS, wash with clean water, and place in a low-temperature dryer at 20°C to dry until the surface is free of moisture. Take 100 g of the dried CROCUS SATIVUS petals and add 10 g of a composite enzyme. The composite enzyme consists of cellulase, xylosidase, pectinase, galactosidase, and protease in a mass ratio of 1:1:1:1:1. Add 5 g of enzyme solution of glucose transferase UGT374MS and 5 g of enzyme solution of sucrose synthase, and add water to make up 850 g. Continuously stir at 25°C for 5 h to obtain a crude extract. Heat the crude extract to 60°C and maintain for 60 min to inactivate the enzymes in the system. Transfer the inactivated crude extract to a filtration device, first filter once using a 0.5-μm filter membrane, and then filter twice using a 0.22-μm filter membrane. The obtained filtrate is a refined extract. Add 1,3-propanediol to the refined extract to make up 1000 g, and seal and store in a sterile room to obtain a CROCUS SATIVUS flower extract.

[0094] The CROCUS SATIVUS flower extract obtained using this method has no precipitates generated after 2 h of long-term storage. Detection shows that it contains 0.35 g / L of total flavonoids.

[0095] Comparative Example 2: Preparation of saffron extract

[0096] Take the petals of crocus, wash with clean water, and place in a low-temperature dryer at 20°C to dry until the surface is free of moisture. Take 100 g of the dried crocus petals and add 10 g of a complex enzyme. The complex enzyme consists of cellulase, xylosidase, pectinase, galactosidase, and protease in a mass ratio of 1:1:1:1:1. Add 850 g of water and continue stirring at 25°C for 5 h to obtain a crude extract. Heat the crude extract to 60°C and maintain for 60 min to inactivate the enzymes in the system. Transfer the inactivated crude extract to a filtration device, first filter once using a 0.5 μm filter membrane, and then filter twice using a 0.22 μm filter membrane. The resulting filtrate is a refined extract. Add 1,3-propanediol to the refined extract to make up 1000 g, and then seal and store in a sterile room to obtain a crocus flower extract.

[0097] The above method differs from the method of Example 2 only in that 5 g of enzyme solution of glucose transferase UGT374MS is not added, and 5 g of enzyme solution of sucrose synthase is not added, and 850 g of water is directly added. The crocus flower extract obtained using this method has a precipitate after being left to stand for 20 min. It was detected that the precipitate contains 0.02 g / L of total flavonoids.

[0098] Example 3: Application of glucose transferase to preparation of a hollyhock root extract

[0099] Take the roots of hollyhock (ALTHAEA OFFICINALIS), wash with clean water, and place in an oven at 40°C to dry until the surface is free of moisture. Use a low-temperature pulverizer to pulverize the hollyhock roots, and sieve the pulverized hollyhock root particles through a 10-mesh sieve. Take 50 g of the sieved hollyhock root particles and add 10 g of a complex enzyme. The complex enzyme consists of cellulase, xylosidase, pectinase, galactosidase, and protease in a mass ratio of 1:1:1:1:1. Add 5 g of enzyme solution of glucose transferase UGT374MS and 5 g of enzyme solution of sucrose synthase, and add 500 g of water. Continue stirring at 25°C for 5 h to obtain a crude extract. Heat the crude extract to 60°C and maintain for 60 min to inactivate the enzymes in the system. Transfer the inactivated crude extract to a filtration device, first filter once using a 0.5 μm filter membrane, and then filter twice using a 0.22 μm filter membrane. The resulting filtrate is a refined extract. Add glycerol to the refined extract to make up 1000 g, and then seal and store in a sterile room to obtain a hollyhock root extract.

[0100] The hollyhock root extract obtained using this method has no precipitate after being left to stand for 2 h.

[0101] Comparative Example 3: Preparation of a hollyhock root extract

[0102] Take the roots of medicinal hollyhock (ALTHAEA OFFICINALIS), wash with clean water, and place in an oven at 40°C to dry until the surface is free of moisture. Use a low-temperature pulverizer to pulverize, and sieve the pulverized medicinal hollyhock root particles through a 10-mesh sieve. Take 50 g of the sieved medicinal hollyhock root particles and add 10 g of a composite enzyme. The composite enzyme consists of cellulase, xylosidase, pectinase, galactosidase, and protease in a mass ratio of 1:1:1:1:1. Add water to make up 500 g, and continuously stir at 25°C for 5 h to obtain a crude extract. Heat the crude extract to 60°C and maintain for 60 min to inactivate the enzymes in the system. Transfer the inactivated crude extract to a filtration device, first filter once using a 0.5-μm filter membrane, and then filter twice using a 0.22-μm filter membrane. The obtained filtrate is a refined extract. Add glycerol to the refined extract to make up 1000 g, and seal and store in a sterile room to obtain a medicinal hollyhock root extract.

[0103] The above method differs from the method of Example 3 only in that no enzyme solution 5 g of glucose transferase UGT374MS is added, and no enzyme solution 5 g of sucrose synthase is added, and water is directly made up to 500 g. The medicinal hollyhock root extract obtained using this method has precipitates generated after 20 min of storage.

[0104] Example 4: Application of glucose transferase to preparation of a composite plant extract

[0105] Take purple violet, mother chrysanthemum, dandelion, peony root, malva sylvestris, and honeysuckle flower, and compound them in a mass ratio of 2:2:1:1:1:1, wash with clean water, and place in an oven at 40°C to dry until the surface is free of moisture. Use a low-temperature pulverizer to pulverize, and sieve the pulverized composite plant particles through a 10-mesh sieve. Take 50 g of the sieved composite plant particles and add 10 g of a composite enzyme. The composite enzyme consists of cellulase, xylosidase, pectinase, galactosidase, and protease in a mass ratio of 1:1:1:1:1. Add enzyme solution 5 g of glucose transferase UGT374MS and enzyme solution 5 g of sucrose synthase, and add water to make up 500 g, and continuously stir at 25°C for 5 h to obtain a crude extract. Heat the crude extract to 60°C and maintain for 60 min to inactivate the enzymes in the system. Transfer the inactivated crude extract to a filtration device, first filter once using a 0.5-μm filter membrane, and then filter twice using a 0.22-μm filter membrane. The obtained filtrate is a refined extract. Add glycerol to the refined extract to make up 1000 g, and seal and store in a sterile room to obtain a composite plant extract.

[0106] The composite plant extract obtained using this method has no precipitates generated after 2 h of long-term storage.

[0107] Comparative Example 4: Preparation of a composite plant extract

[0108] Take purple flower, mother chrysanthemum, dandelion, peony root, malve flower, honeysuckle flower for compounding, the mass ratio is 2:2:1:1:1:1, wash with clean water, place in the oven at 40℃ to dry until the surface is no water. Use low temperature pulverizer to crush, the crushed compound plant particles are sieved through 10 mesh. Take 50g of sieved compound plant particles, 10g of complex enzyme. The composition of the complex enzyme is cellulase, xylosidase, pectinase, galactosidase, and protease, and the mass ratio is 1:1:1:1:1. Make up 500g of water, continuously stir at 25℃ for 5h to obtain the crude extract. Heat the crude extract to 60℃ and keep for 60 minutes to inactivate the enzymes in the system. Transfer the inactivated crude extract to the filtration device, first use 0.5μm filter membrane for primary filtration, then use 0.22μm filter membrane for secondary filtration, the obtained filtrate is the refined extract. Add glycerol to the refined extract, make up 1000g, seal in a sterile room to obtain the compound plant extract.

[0109] The above method is different from the method of example 4 only in that 5g of enzyme solution of glucose transferase UGT374MS is not added, and 5g of enzyme solution of sucrose synthase is not added, and 500g of water is directly made up. The compound plant extract obtained by using this method has precipitate generated after 20 minutes of placement.

[0110] Example 5: Application of glucose transferase in preparation of buckwheat seed extract

[0111] Take buckwheat seed (POLYGONUM FAGOPYRUM), wash with clean water, place in the oven at 40℃ to dry until the surface is no water. Use low temperature pulverizer to crush, the crushed buckwheat seed particles are sieved through 10 mesh. Take 100g of sieved buckwheat seed particles, 10g of complex enzyme. The composition of the complex enzyme is cellulase, xylosidase, pectinase, galactosidase, and protease, and the mass ratio is 1:1:1:1:1. Add 5g of enzyme solution of glucose transferase UGT374MS, add 5g of enzyme solution of sucrose synthase, make up 990g of water, continuously stir at 25℃ for 5h to obtain the crude extract. Heat the crude extract to 60℃ and keep for 60 minutes to inactivate the enzymes in the system. Transfer the inactivated crude extract to the filtration device, first use 0.5μm filter membrane for primary filtration, then use 0.22μm filter membrane for secondary filtration, the obtained filtrate is the refined extract. Add 1,2-hexanediol to the refined extract, make up 1000g, seal in a sterile room to obtain the buckwheat seed extract.

[0112] The buckwheat seed extract obtained by using this method has no precipitate generated after 2 hours of long time placement. After detection, it contains 11.2g / 100g of total carbohydrates.

[0113] Comparative example 5: preparation of buckwheat seed extract

[0114] Polygonum fagopyrum was taken, washed with clean water, and dried in an oven at 40°C until the surface was free of moisture. A low-temperature pulverizer was used for pulverization, and the pulverized Polygonum fagopyrum particles were sieved through a 10-mesh sieve. 100 g of the sieved Polygonum fagopyrum particles and 10 g of a composite enzyme were taken. The composite enzyme consisted of cellulase, xylosidase, pectinase, galactosidase, and protease in a mass ratio of 1:1:1:1:1. 990 g of water was added, and stirring was continued at 25°C for 5 h to obtain a crude extract. The crude extract was heated to 60°C and maintained for 60 min to inactivate the enzymes in the system. The inactivated crude extract was transferred to a filtration device, and first filtration was performed using a 0.5-μm filter membrane, and second filtration was performed using a 0.22-μm filter membrane. The obtained filtrate was a refined extract. 1,2-hexanediol was added to the refined extract, 1000 g was added, and the mixture was sealed and stored in a sterile room to obtain a Polygonum fagopyrum extract.

[0115] The above method was different from the method of Example 5 only in that 5 g of enzyme solution of glucosyltransferase UGT374MS was not added, and 5 g of enzyme solution of sucrose synthase was not added, and 990 g of water was directly added. The Polygonum fagopyrum extract obtained using the method had precipitates generated after being left to stand for 20 min.

[0116] Example 6: Application of glucosyltransferase to preparation of Althaea officinalis leaf extract

[0117] Althaea officinalis leaves were taken, washed with clean water, and dried in an oven at 40°C until the surface was free of moisture. A low-temperature pulverizer was used for pulverization, and the pulverized Althaea officinalis leaf particles were sieved through a 10-mesh sieve. 50 g of the sieved Althaea officinalis leaf particles and 10 g of a composite enzyme were taken. The composite enzyme consisted of cellulase, xylosidase, pectinase, galactosidase, and protease in a mass ratio of 1:1:1:1:1. 5 g of enzyme solution of glucosyltransferase UGT374MS and 5 g of enzyme solution of sucrose synthase were added, 550 g of water was added, and stirring was continued at 25°C for 5 h to obtain a crude extract. The crude extract was heated to 60°C and maintained for 60 min to inactivate the enzymes in the system. The inactivated crude extract was transferred to a filtration device, and first filtration was performed using a 0.5-μm filter membrane, and second filtration was performed using a 0.22-μm filter membrane. The obtained filtrate was a refined extract. 1,3-propanediol was added to the refined extract, 1000 g was added, and the mixture was sealed and stored in a sterile room to obtain an Althaea officinalis leaf extract.

[0118] The Althaea officinalis leaf extract obtained using the method had no precipitates generated after being left to stand for 2 h.

[0119] Comparative Example 6: Preparation of Althaea officinalis leaf extract

[0120] Take the leaves of Alpinia officinarum Hance (ALTHAEA OFFICINALIS), wash them with clean water, and dry them in an oven at 40°C until the surface is free of moisture. Use a low-temperature pulverizer to pulverize them, and sieve the pulverized Alpinia officinarum Hance leaves through a 10-mesh sieve. Take 50 g of the sieved Alpinia officinarum Hance leaf particles, and add 10 g of a composite enzyme. The composite enzyme consists of cellulase, xylosidase, pectinase, galactosidase, and protease, and the mass ratio is 1:1:1:1:1. Add water to make up 550 g, and continuously stir at 25°C for 5 h to obtain a crude extract. Heat the crude extract to 60°C and keep it at 60°C for 60 min to inactivate the enzymes in the system. Transfer the inactivated crude extract to a filtration device, first filter it once using a 0.5-μm filter membrane, and then filter it twice using a 0.22-μm filter membrane. The obtained filtrate is a refined extract. Add 1,3-propanediol to the refined extract to make up 1000 g, and seal and store it in a sterile room to obtain an Alpinia officinarum Hance leaf extract.

[0121] The above method is different from the method of Example 6 only in that 5 g of the enzyme solution of glucose transferase UGT374MS is not added, and 5 g of the enzyme solution of sucrose synthase is not added, and 550 g of water is directly added. The Alpinia officinarum Hance leaf extract obtained using the method has precipitates generated after being placed for 20 min.

[0122] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those of ordinary skill in the art do not deviate from the essence and scope of the technical solutions of the present application.

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 of claim 1.

3. The nucleic acid of claim 2, wherein, The nucleic acid is the sequence shown in SEQ ID NO.

3.

4. A vector, characterized by, The vector comprises the nucleic acid of claim 2 or 3.

5. A cell, comprising: The cell expresses the glucosyltransferase of claim 1, or comprises the nucleic acid of any one of claims 2-3, or comprises the vector of claim 4.

6. Use of the glucosyltransferase of claim 1, the nucleic acid of any one of claims 2-3, the vector of claim 4, or the cell of claim 5, characterized in that, The application comprises: Catalyzing flavones to generate flavone-7-O-glycosides.

7. A method of producing a flavone-7-O-glycoside, characterized by, The method comprises catalyzing flavones to generate flavone-7-O-glycosides by the glucosyltransferase of claim 1.

8. A method of preparing a plant extract, characterized in that, The method is a treatment using the glucosyltransferase of claim 1, and the plant is Aframomum melegueta seed, Crocus sativus, Alcea rosea, Viola yedoensis, Matricaria recutita, Taraxacum officinale, Paeonia lactiflora, Malva sylvestris, Lonicera japonica, Fagopyrum esculentum or Hedychium coccineum; comprising the following steps: S1, drying and crushing the plant to obtain plant particles; S2, treating the plant particles obtained in step S1 with enzymes to release the contents of the plant cells; adding the glucosyltransferase of claim 1 and stirring to obtain a crude extract; S3, filtering the crude extract obtained in step S2 to obtain a plant extract.

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