Glucosyltransferase obtained by directed evolution method and use thereof

Abstract

The present application relates to a kind of glucose transferase obtained by directed evolution method and its application, belong to the field of bioengineering technology.The present application obtains a kind of glucose transferase UGT197CG by directed evolution method, its amino acid sequence is as shown in SEQ ID NO.4.The glucose transferase UGT197CG of the present application can be converted into flavone-C-glucoside by poor solubility flavonoids, and increase its solubility, thereby reduce the precipitation amount of plant extract and related product.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, and particularly relates to a glucose transferase obtained by a directed evolution method and application thereof. BACKGROUND

[0002] Flavonoids are a class of polyphenolic secondary metabolites widely existing in plant bodies. They are named after the flavone nucleus contained in their molecules. Flavonoids not only give plants colorful colors and physiological functions, but also have important effects on human health. They can reduce oxidative stress damage to cells through free radical scavenging and lipid peroxidation inhibition, thereby delaying the aging process and reducing the incidence of various chronic diseases. They can also regulate lipid metabolism, reduce blood cholesterol and triglyceride levels, inhibit platelet aggregation, improve vascular endothelial function, and help prevent atherosclerosis, hypertension and other cardiovascular diseases. In addition, flavonoids also have anti-inflammatory, antiviral and immunomodulatory effects, can inhibit the release of inflammatory factors, reduce inflammation, and have a relieving effect on inflammatory diseases such as arthritis, and can enhance the activity of immune cells and improve resistance.

[0003] However, due to the molecular structure of flavonoids containing many hydrophobic groups, their solubility in aqueous environments is extremely low, which not only makes it difficult for the human body to absorb and utilize flavonoids, but also makes products containing flavonoids prone to precipitation. Therefore, glycosylation modification of flavonoids, which combines flavone nucleus with glucose, rhamnose and other sugar molecules through glycosidic bonds, can reduce the precipitation of related products and improve their bioavailability. In particular, glycosylation modification of flavonoids in plant extracts is a process problem that needs to be solved urgently.

[0004] Currently, most of the glycosylation processes for flavonoids form O-glycosidic bonds between sugar molecules and the hydroxyl groups of flavonoids. However, the hydroxyl group itself is a hydrophilic group, and further hydrophilic glycosylation modification of the hydrophilic hydroxyl group can only limitedly improve the water solubility of flavonoids. Therefore, modifying the hydrophobic groups of flavonoids to be hydrophilic can better improve their water solubility, for example, C-glycosylation modification of flavonoids, which directly inserts sugar molecules into the flavone skeleton, retains the hydroxyl groups of flavonoids, and can better improve the water solubility of flavonoids.

[0005] Glycosylation modification can be divided into chemical reaction method and biosynthesis method. Chemical glycosylation reaction has the disadvantages of many by-products and intermediates, poor regioselectivity and stereoselectivity, low yield, complicated steps, and large pollution. In comparison, enzyme-catalyzed biosynthesis method has relatively simple operation steps and less environmental pollution, which conforms to the concept of green chemistry. Therefore, the use of glucose transferase to catalyze the synthesis of flavonoid glycosides from flavonoids has gradually become the focus of research in this field.

[0006] Plant extracts often contain a variety of different flavonoids. However, currently available C-glucosyltransferases are all glycosylated for a specific flavonoid substrate. This type of glycosyltransferase cannot effectively solve the problem of precipitation in plant extracts. Therefore, a glycosyltransferase with broad substrate adaptability is needed to C-glycoside modify various flavonoids in plant extracts.

[0007] In summary, there is an urgent need for a glycosyltransferase with broad substrate adaptability that can C-glycoside modify various flavonoids in plant extracts. Summary of the Invention

[0008] The purpose of this invention is to provide a glucosyltransferase obtained by directed evolution and its application, in order to solve the problem that products containing flavonoids prepared in the prior art are prone to precipitation.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] The present invention provides a glucosyltransferase UGT197CG, the amino acid sequence of which is shown in SEQ ID NO.4.

[0011] Preferably, the glucosyltransferase UGT197CG is a mutation occurring at positions 13, 49, 148, 155, 333, and 422 of the amino acid sequence of the wild-type glucosyltransferase UGT708B8.

[0012] The amino acid sequence of the wild-type glucosyltransferase UGT708B8 is shown in SEQ ID NO.2.

[0013] Preferably, the mutation includes changing the 13th amino acid from proline to threonine, the 49th amino acid from serine to arginine, the 148th amino acid from phenylalanine to serine, the 155th amino acid from serine to arginine, the 333rd amino acid from lysine to asparagine, and the 422nd amino acid from serine to cysteine.

[0014] This invention also provides the application of the aforementioned glucosyltransferase UGT197CG in the preparation of plant extracts and related products.

[0015] The present invention also provides a nucleic acid molecule encoding the glucosyltransferase UGT197CG, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0016] The present invention also provides a recombinant plasmid comprising the aforementioned nucleic acid molecule.

[0017] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium includes the recombinant plasmid described above.

[0018] The present invention also provides the application of the aforementioned nucleic acid molecule, recombinant plasmid, and recombinant bacteria in the preparation of plant extracts and related products.

[0019] The present invention has the following technical effects and advantages:

[0020] In the preparation of plant extracts, the glucosyltransferase UGT197CG of the present invention can convert various poorly soluble flavonoids in the extract into flavonoid-C-glucosides, thereby increasing their solubility and reducing the amount of precipitation of plant extracts and related products. Attached Figure Description

[0021] Figure 1 A schematic diagram of the pET-11a expression vector for the recombinant glucosyltransferase gene;

[0022] Figure 2 The results are for the detection of fine extracts treated with glucosyltransferase of mutant ε0197. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to preparation examples and embodiments. Unless otherwise specified, the preparation examples and embodiments are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without a specified manufacturer are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. Such structures and techniques are also described in many authoritative publications, such as *Molecular Cloning: A Laboratory Manual (4th Edition)* (Science Press), edited by [US] MR. Green and J. Sambrook; *A Concise Laboratory Manual of Molecular Biology (5th Edition)* (Science Press), edited by [US] FM. Osber et al.; and *Analytical Chemistry (6th Edition)* (Higher Education Press), edited by Wuhan University; and others.

[0024] In this invention, the pET-11a plasmid was purchased from Shanghai Yuanmu Biotechnology Co., Ltd., the PCR amplification kit and MEGAWHOP PCR kit were purchased from TAKARA Corporation of Japan, the NdeI enzyme, HindIII enzyme, DpnI enzyme digestion kit and T4 ligase kit were purchased from New England Biolabs, and the error-prone PCR kit was purchased from Beijing Biolabs Technology Co., Ltd.

[0025] Preparation example: Obtaining glucosyltransferase

[0026] (1) Gene cloning of wild-type glucosyltransferase UGT708B8

[0027] In the test materials of this invention, the wild-type glucosyltransferase UGT708B8 (NCBI ID: A0A1J7HE57) was derived from the annual herbaceous plant *Lupinus angustifolius*, belonging to the genus *Lupinus* of the Fabaceae family. Its amino acid sequence is disclosed in the literature (Putkaradze N, GalaVD, Vaitkus D, et al. Sequence mining yields 18phloretin C-glycosyltransferases from plants for the efficient biocatalytic synthesis of nothofagin and phloretin-di-C-glycoside[J]. Biotechnol J, 2023, 18(6):e2200609).

[0028] The amino acid sequence of wild-type glucosyltransferase UGT708B8 (NCBI ID: A0A1J7HE57) was codon optimized to obtain the nucleotide sequence encoding wild-type glucosyltransferase UGT708B8, as shown in SEQ ID NO.1;

[0029] SEQ ID NO.1:

[0030]

[0031] The amino acid sequence of wild-type glucosyltransferase UGT708B8 is shown in SEQ ID NO.2;

[0032] SEQ ID NO.2:

[0033] MSEVPPIHVALLPSAGMGHLTPFLRLAAMLVHHNCHVTLITTHPTVSKSESDQISKFHSSFPQVNQLHFHLLPPSDASATKADPFFLRFEAIRSSSHLLPSLLSSVSPPLSSFV YDMTLISPLLPVADSLGVPHYILFTSSATMLSFFSYFPTVSASLPTLNDVEIPGVSSIPRSSIPPMLLVPNSLFGNIFNEDGPKLTKLHGVLINTFEELEAQSLEALNGGKMVKE LPPVYAVGPFVPGEFEKEDQRGAPLKWLDDKAKGSVVYVTFGSRTAMGRDQMREIGEGLVRSGSMFLWVVKGKKVDREEEEEGLEGLLGLELVEKIKERGLVVKEWVDQREILDH EAVGGFVSHCGWNSVVEAAWYGVPIMGWPLGGDQKINAEVVSNKGWGVWNKDWGWEGENVVKGEEIGEAIREMMNDESLRIKAAEVKEAARKAISVGGRGEVTLQKLIEKWNKF.

[0034] Nucleotides were synthesized using a commercial gene synthesis company according to SEQ ID NO. 1. Primers containing restriction enzyme sites were designed based on the nucleotide sequence of glucosyltransferase UGT708B8. The forward primer UGT708B8-WF, which incorporates an NdeⅠ restriction site, is shown in SEQ ID NO. 5. The reverse primer UGT708B8-WR, which incorporates a HindⅢ restriction site, is shown in SEQ ID NO. 6. The pET-11a plasmid was selected according to the relevant parameters required for subsequent experiments. Using the synthesized glucosyltransferase UGT708B8 nucleotides as a template, amplification was performed using the above primers via a standard PCR procedure.

[0035] Next, double digestion was performed using a commercially available NdeI and HindIII enzyme digestion kit. The PCR amplification products and pET-11a plasmid obtained in the above steps were digested separately. The digestion temperature was 37℃, and the digestion time was 1 hour.

[0036] Finally, the digested nucleotide fragments were ligated to the pET-11a plasmid using a commercially available T4 ligase kit at 16°C for 12 hours. The resulting plasmid was pET-11a-UGT708B8. Figure 1 As shown, this plasmid serves as a plasmid template for wild-type glucosyltransferase.

[0037] Table 1. Nucleotide sequences of each primer

[0038] Name Sequence (5'~3') Restriction enzyme SEQ ID NO. UGT708B8-WF ACGCATATGATGAGCGAAGTGCCGCCGA Nde I 5 UGT708B8-WR GGAAGCTTAAATTTGTTCCATTTTTCAAT Hind III 6 UGTC-MF TTCGCAGCAGCAGCCATCTGCT —— 7 UGTC-MR AGCAGATGGCTGCTGCTGCGAA —— 8

[0039] (2) Construction of recombinant expression strains and expression of enzymes

[0040] Competent cells were prepared using *E. coli* BL21(DE3). The pET-11a-UGT708B8 plasmid was transformed into the cells using electroporation. The cells were then plated onto solid agar plates containing ampicillin and incubated at 37°C for 24 hours. Single colonies were selected from the plates and inoculated into test tubes containing ampicillin-containing LB broth, incubated at 37°C for 12 hours. IPTG was added to a final concentration of 0.5 mmol / L to induce expression of the target protein. The cells were then incubated at 25°C and 180 rpm for 24 hours. The fermentation broth in the test tubes was centrifuged, the supernatant was discarded, and the precipitate was collected to obtain *E. coli* cells. An appropriate amount of deionized water was added, and the cells were sonicated. After centrifugation, the supernatant was collected to obtain the solution of the target enzyme.

[0041] (3) Screening methods and functional verification of glucosyltransferases

[0042] The glucosyltransferase studied in this invention uses flavonoids and UDPG as substrates to catalyze the synthesis of flavonoid-C-glucoside 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.

[0043] This invention is specifically applied to the preparation of plant extracts. Therefore, in the specific screening process, this invention uses a plant extract, specifically a crude extract of Lysimachia christinae, as the reaction starting material. Lysimachia christinae contains many types of flavonoids, and the extract is prone to precipitation.

[0044] (3a) Preparation of crude extract of Lysimachia christinae:

[0045] After drying Lysimachia christinae, dried Lysimachia christinae was obtained. 2g of cellulase, 1g of pectinase, and 1g of protease were mixed to obtain a mixed enzyme. 1g of dried Lysimachia christinae, 7.5g of water, and 0.3g of the mixed enzyme were mixed and ultrasonically treated at 300W for 15 minutes. Then, the mixture was stirred in a reaction vessel at 25℃ for 150 minutes, followed by stirring at 60℃ for 10 minutes to inactivate the enzyme. The mixture was then filtered through a 0.5μm filter membrane and a 0.22μm filter membrane to obtain a crude extract of Lysimachia christinae. 8.5g of the crude extract was mixed with 1.5g of 1,3-propanediol to obtain a crude extract of Lysimachia christinae.

[0046] When the crude extract of Lysimachia christinae is left to stand at room temperature for 30 minutes, a flocculent precipitate composed of flavonoids is clearly formed, indicating that the crude extract of Lysimachia christinae readily precipitates and can be used as a substrate for determining and screening the enzyme activity of glycosyltransferases.

[0047] (3b) Enzyme function verification:

[0048] Take 0.2g of wild-type glucosyltransferase UGT708B8 solution prepared in step (2), 8g of crude extract of Lysimachia christinae prepared in step (3a), 0.5g of sucrose, 2mmol / L of UDP and 50U of sucrose synthase as the experimental group. Sonicate at 400W for 20min, shake for 120min, and inactivate the enzyme in a water bath at 60℃ for 5min to terminate the reaction. After standing at room temperature for 24h, centrifuge and take the supernatant to obtain the fine extract.

[0049] Flavonoid C-glucosides in the fine extract were determined by HPLC. Due to the large variety of flavonoids, representative compounds, isoharonine and Nothofagin (1-(2,4,6-trihydroxyphenyl)-3-(4-hydroxyphenyl)propane-1,3-dione), were selected. Isoharonine is a C-glucoside of luteolin, and Nothofagin is a C-glucoside of phlorizin. The precipitates after centrifugation were dried at 50°C for 1 hour and then weighed. A solution without wild-type glucosyltransferase UGT708B8 was used as a control group. The results are shown in Table 2.

[0050] Table 2. Functional validation results of wild-type glucosyltransferase UGT708B8

[0051] Ingredient Experimental group content (mg) Control group content (mg) Precipitation 249 387 Isorhapontin 37.2 0.17 Nothofagin 28.6 0.09

[0052] The results showed that the wild-type glucosyltransferase UGT708B8 significantly reduced the precipitation content of the crude extract of Lysimachia christinae and increased the content of flavonoid-C-glucoside in the refined extract. This indicates that our target enzyme, glucosyltransferase UGT708B8, was successfully expressed. Furthermore, the activity of the target enzyme in the reaction system can be characterized by the weight of the precipitate, facilitating high-throughput screening in subsequent directed evolution processes.

[0053] (4) Directed evolution—constructing a mutant library

[0054] A random mutant library of glucosyltransferase UGT708B8 was constructed using error-prone PCR. Primer sequences are shown in Table 1. The forward primer sequence is UGTC-MF, as shown in SEQ ID NO. 6; the reverse primer sequence is UGTC-MR, as shown in SEQ ID NO. 6; the template is the pET-11a-UGT708B8 plasmid, and error-prone PCR amplification was performed using rTaq DNA polymerase. The reaction system and procedure for error-prone PCR amplification are as follows:

[0055]

[0056]

[0057]

[0058] After obtaining the error-prone mutant nucleotide fragment pET-11a-UGT708B8, it was used as a large primer, and pET-11a-UGT708B8 was used as a template for MEGAWHOP PCR. The PCR amplification reaction system and procedure are as follows:

[0059] 2 x PrimeStar Buffer (Mg 2+ plus) 50.0 μL dNTP Mixture (2.5 mM) 10.0 μL pET-11a-UGT708B8 5.0 μL Error-prone PCR product as large primer 5.0 μL PrimeStar HS DNA polymerase 1 μL ddH2O 29 μL

[0060]

[0061]

[0062] The amplification products obtained by MEGAWHOP PCR were incubated with Dpn I restriction enzyme at 37°C for 12 h, followed by template digestion. E. coli BL21(DE3) competent cells were then prepared, and the amplification products were subsequently introduced into E. coli BL21(DE3) cells using electroporation. The cells were then plated onto solid plates containing ampicillin and incubated at 37°C for 24 h.

[0063] Single clones were selected from the plate and inoculated into test tubes containing liquid LB medium supplemented with ampicillin, and cultured at 37°C for 12 h. IPTG was added to a final concentration of 0.5 mM to induce expression of the target protein. The culture was then carried out at 25°C and shaken at 180 rpm for 24 h. The fermentation broth in the test tubes was centrifuged, the supernatant was discarded, and the precipitate was collected to obtain *E. coli* cells. An appropriate amount of deionized water was added, and the cells were sonicated and centrifuged again to collect the supernatant, which yielded the target enzyme solution.

[0064] (5) Directed evolution—multi-round high-throughput screening

[0065] Following the steps described above, a random mutant library was constructed starting with pET-11a-UGT708B8, yielding a total of 2730 mutant strains. Each mutant strain was induced to express the enzyme, and the cells were collected and sonicated to obtain the target enzyme solution. The crude extract of *Lysimachia christinae* was refined using the aforementioned screening method for high-throughput screening. The activity of the mutant enzymes was characterized by detecting the weight of the precipitate. The α1426 mutant enzyme produced the least amount of precipitate after the reaction, weighing 192 mg. HPLC analysis revealed that the content of isothiopicrin was 47.1 mg, and the content of Nothofagin was 35.8 mg.

[0066] Therefore, mutant α1426 was selected as the optimal mutant obtained in the first round of directed evolution screening. Using α1426 as a template, the next round of random mutant library iterations was conducted. Following the aforementioned directed evolution-mutant library construction method, the pET-11a-UGT708B8 plasmid was replaced with plasmid α1426, and random mutant libraries were constructed using error-prone PCR and MEGAWHOP PCR. High-throughput screening was then performed. A total of 2050 mutant strains were obtained. By analyzing the precipitate weight, β1066 was found to have the lowest precipitate weight at 101 mg. HPLC analysis revealed that the content of isothiopicrin was 58.3 mg and the content of Nothofagin was 40.1 mg.

[0067] Therefore, mutant β1066 was selected as the optimal mutant obtained in the second round of directed evolution screening. Using β1066 as a template, the next round of random mutant library iterations was conducted. Following the aforementioned directed evolution-mutant library construction method, plasmid pET-11a-UGT708B8 was replaced with plasmid β1066, and random mutant libraries were constructed using error-prone PCR and MEGAWHOP PCR. High-throughput screening was then performed. A total of 1500 mutant strains were obtained. By analyzing the precipitate weight, mutant γ0878 was found to have the lowest precipitate weight at 42 mg. HPLC analysis revealed that the content of isothiopicrin was 66.5 mg, and the content of Nothofagin was 41.9 mg.

[0068] Therefore, mutant γ0878 was selected as the optimal mutant obtained in the third round of directed evolution screening. Using γ0878 as a template, the next round of random mutant library iterations was conducted. Following the aforementioned directed evolution-mutant library construction method, the pET-11a-UGT708B8 plasmid was replaced with plasmid γ0878, and random mutant libraries were constructed using error-prone PCR and MEGAWHOP PCR. High-throughput screening was then performed. A total of 1380 mutant strains were obtained. By analyzing the precipitate weight, δ0614 was found to have the lowest precipitate weight, at 4 mg. HPLC analysis revealed that the content of isothiopicrin was 72.8 mg, and the content of Nothofagin was 42.4 mg.

[0069] Therefore, δ0614 was selected as the optimal mutant obtained in the fourth round of directed evolution screening, and γ0878 was used as a template for the next round of random mutant library iteration. Following the aforementioned directed evolution-mutant library construction method, the pET-11a-UGT708B8 plasmid was replaced with δ0614 plasmid, and the random mutant library was constructed using error-prone PCR and MEGAWHOP PCR. High-throughput screening was then performed. Because the precipitate from δ0614 was almost completely eliminated in the previous round of directed evolution, the evolutionary selection pressure was increased in this screening process, reducing the reaction time from 120 min to 90 min. A total of 1030 mutant strains were obtained. Precipitate weight analysis revealed that ε0197 did not produce any precipitate. HPLC analysis showed that the content of isophoric acid was 75.3 mg, and the content of Nothofagin was 44.8 mg.

[0070] Through five rounds of iterative directed evolution, mutant ε0197 was obtained. Wild-type UGT708B8 and the *E. coli* corresponding to mutant ε0197 were cultured in 300ml Erlenmeyer flasks using the aforementioned method to obtain a larger quantity of enzyme extract. Using the aforementioned screening method, a 100g reaction system was used for fine extraction, with a reaction time of 120min. The fine extract treated with glucosyltransferase UGT708B8 produced a precipitate weighing 2584mg. The fine extract treated with glucosyltransferase of mutant ε0197 did not produce a precipitate.

[0071] HPLC analysis revealed that the fine extract treated with glucosyltransferase of mutant ε0197 contained 778.3 mg of isophoric acid and 451.2 mg of nothofagin. Figure 2 As shown.

[0072] Ultimately, mutant ε0197 was selected as the endpoint for directed evolution and named. The target enzyme was named glucosyltransferase UGT197CG, the corresponding E. coli was named BL21(DE3)-UGT197CG, and the plasmid was named pET-11a-UGT197CG.

[0073] The glucosyltransferase UGT197CG, whose amino acid sequence is shown in SEQ ID NO.4, has undergone a combination of P13T, S49R, F148S, S155R, K333N and S422C mutations compared with the wild-type glucosyltransferase UGT708B8.

[0074] SEQ ID NO.4:

[0075] MSEVPPIHVALLTSAGMGHLTPFLRLAAMLVHHNCHVTLITTHPTVSKRESDQISKFHSSFPQVNQLHFHLLPPSDASATKADPFFLRFEAIRSSSHLLPSLLSSVSPPLSSFV YDMTLISPLLPVADSLGVPHYILFTSSATMLSFSSYFPTVRASLPTLNDVEIPGVSSIPRSSIPPMLLVPNSLFGNIFNEDGPKLTKLHGVLINTFEELEAQSLEALNGGKMVKE LPPVYAVGPFVPGEFEKEDQRGAPLKWLDDKAKGSVVYVTFGSRTAMGRDQMREIGEGLVRSGSMFLWVVKGKKVDREEEEEGLEGLLGLELVEKIKERGLVVNEWVDQREILDH EAVGGFVSHCGWNSVVEAAWYGVPIMGWPLGGDQKINAEVVSNKGWGVWNKDWGWEGENVVKGEEIGEAIREMMNDECLRIKAAEVKEAARKAISVGGRGEVTLQKLIEKWNKF;

[0076] The nucleotide sequence encoding the glucosyltransferase UGT197CG is shown in SEQ ID NO.3;

[0077] SEQ ID NO.3:

[0078]

[0079] (6) Preparation of glucosyltransferase UGT197CG enzyme solution

[0080] The BL21(DE3)-UGT197CG strain seed culture was mixed with TB culture medium at a volume ratio of bacterial culture to culture medium of 1:100 and placed in a 5L fermenter. The mixture was then incubated at 37°C until the OD reached [a certain value]. 600 =0.6, lactose was added to a final concentration of 5 mmol / L, and the mixture was cultured at 25°C for 24 h to induce protein expression. After sonication and centrifugation, the supernatant was collected to obtain glucosyltransferase UGT197CG enzyme solution. This enzyme solution can be used to prepare plant extracts.

[0081] Example 1

[0082] Preparation of European red pine bud extract using glucosyltransferase

[0083] Buds of European red pine (Pinus sylvestris) were washed with water and dried in an oven at 40°C until no moisture remained on the surface. The buds were then pulverized using a low-temperature pulverizer and passed through a 10-mesh sieve. 100g of the sieved buds and 8g of a compound extraction enzyme were taken. The compound extraction enzyme consisted of β-glucanase, β-galactosidase, and laccase in a mass ratio of 2:1:1. 3g of glucosyltransferase UGT197CG, 3g of sucrose synthase solution, 0.5g of sucrose, and 0.1g of UDP were added, and water was added to make a 600g reaction system. Extraction was assisted by ultrasound (400W) for 20 minutes. The mixture was then stirred in a reaction vessel for 180 minutes at 25°C. Finally, the mixture was stirred at 60°C for 10 minutes to inactivate the enzymes. The inactivated extract was transferred to a filtration device, first filtered through a 0.5 μm membrane, and then filtered a second time through a 0.22 μm membrane to remove microorganisms. Butylene glycol was added to the extract to make up to 1000 g, and the extract was aliquoted and sealed in a sterile room to obtain the European red pine bud extract.

[0084] The extract of European red pine buds obtained using this method showed no precipitation after 24 hours of standing.

[0085] Comparative Example 1

[0086] The buds of European red pine were collected, washed with clean water, and dried in an oven at 40°C until no moisture remained on the surface. They were then pulverized using a low-temperature pulverizer, and the pulverized buds were passed through a 10-mesh sieve. 100g of the sieved buds and 8g of a compound extraction enzyme were taken. The compound extraction enzyme consisted of β-glucanase, β-galactosidase, and laccase in a mass ratio of 2:1:1. Water was added to make up to 600g of the reaction system, and ultrasonic extraction (400W) was performed for 20 minutes. The mixture was then stirred in a reaction vessel for 180 minutes at 25°C. Finally, it was stirred at 60°C for 10 minutes to inactivate the enzymes. The inactivated extract was transferred to a filtration device, first filtered through a 0.5μm membrane, and then filtered again through a 0.22μm membrane to remove microorganisms. Butylene glycol was added to the extract to make up to 1000g, and the mixture was aliquoted and sealed in a sterile room to obtain the European red pine bud extract.

[0087] During the extraction process, no glucosyltransferase UGT197CG was used. The resulting extract of European red pine buds produced a precipitate after standing for 20 minutes.

[0088] Example 2

[0089] Preparation of saffron extract using glucosyltransferase

[0090] Saffron (Crocus sativus), also known as Tibetan saffron. Take saffron flowers, wash them with water, and dry them in an oven at 40℃ until the surface is dry. Grind them using a low-temperature grinder, and then pass the ground saffron flowers through a 10-mesh sieve. Take 100g of the sieved saffron flowers and 8g of a compound extraction enzyme. The compound extraction enzyme consists of β-glucanase, β-galactosidase, and laccase in a mass ratio of 2:1:1. Add 3g of glucosyltransferase UGT197CG, 3g of sucrose synthase solution, 0.5g of sucrose, and 0.1g of UDP. Make up the reaction volume to 850g with water, and use ultrasound (400W) for assisted extraction for 20 minutes. Then, stir in the reaction vessel for 180 minutes, maintaining the temperature at 25℃. Finally, stir at 60℃ for 10 minutes to inactivate the enzymes in the system. The inactivated extract was transferred to a filtration device, first filtered through a 0.5 μm membrane, and then filtered a second time through a 0.22 μm membrane to remove microorganisms. 1,3-propanediol was added to the extract to bring the total to 1000 g, and the extract was aliquoted and sealed in a sterile room to obtain the saffron extract.

[0091] The saffron extract obtained using this method showed no precipitation after 24 hours of standing.

[0092] Comparative Example 2

[0093] Saffron flowers were collected, washed with clean water, and dried in an oven at 40℃ until no moisture remained on the surface. The flowers were then pulverized using a low-temperature grinder and passed through a 10-mesh sieve. 100g of the sieved saffron flowers and 8g of a compound extraction enzyme were taken. The compound extraction enzyme consisted of β-glucanase, β-galactosidase, and laccase in a mass ratio of 2:1:1. Water was added to make up to 850g of the reaction system, and ultrasonic extraction (400W) was performed for 20 minutes. The mixture was then stirred in a reaction vessel for 180 minutes at 25℃. Finally, it was stirred at 60℃ for 10 minutes to inactivate the enzymes. The inactivated extract was transferred to a filtration device, first filtered through a 0.5μm membrane, and then filtered again through a 0.22μm membrane to remove microorganisms. 1,3-propanediol was added to the extract to make up to 1000g, and the mixture was aliquoted and sealed in a sterile room to obtain the saffron extract.

[0094] During the extraction process, no glucosyltransferase UGT197CG was used, and the saffron extract obtained produced a precipitate after standing for 20 minutes.

[0095] Example 3

[0096] Preparation of drug from marshmallow flower extract using glucosyltransferase

[0097] The flowers of *Althaea officinalis* were collected, washed with clean water, and dried in an oven at 40°C until no moisture remained on the surface. The flowers were then pulverized using a low-temperature pulverizer and passed through a 10-mesh sieve. 100g of the sieved flowers and 8g of a compound extraction enzyme were taken. The compound extraction enzyme consisted of β-glucanase, β-galactosidase, and laccase in a mass ratio of 2:1:1. 3g of glucosyltransferase UGT197CG, 3g of sucrose synthase solution, 0.5g of sucrose, and 0.1g of UDP were added, and water was added to make a reaction system of 520g. Extraction was assisted by ultrasound (400W) for 20 minutes. The mixture was then stirred in a reaction vessel for 180 minutes at 25°C. Finally, the mixture was stirred at 60°C for 10 minutes to inactivate the enzymes. The inactivated extract was transferred to a filtration device, first filtered through a 0.5 μm membrane, and then filtered a second time through a 0.22 μm membrane to remove microorganisms. Glycerin was added to the extract to make up to 1000 g, and the extract was aliquoted and sealed in a sterile room to obtain the marshmallow flower extract.

[0098] The marshmallow flower extract obtained using this method showed no precipitation after 24 hours of standing.

[0099] Comparative Example 3

[0100] Take the flowers of the medicinal marshmallow, wash them with clean water, and dry them in an oven at 40℃ until the surface is free of moisture. Grind them using a low-temperature pulverizer, and then pass the pulverized flowers through a 10-mesh sieve. Take 100g of the sieved medicinal marshmallow flowers and 8g of a compound extraction enzyme. The compound extraction enzyme consists of β-glucanase, β-galactosidase, and laccase in a mass ratio of 2:1:1. Make up the reaction system with water to a final volume of 520g, and perform ultrasonic extraction (400W) for 20 minutes. Then, stir in a reaction vessel for 180 minutes at a temperature of 25℃. Next, stir at 60℃ for 10 minutes to inactivate the enzymes. Transfer the inactivated extract to a filtration device, first filtering with a 0.5μm membrane, then filtering a second time with a 0.22μm membrane to remove microorganisms. Add glycerol to the extract to a final volume of 1000g, and aliquot and seal in a sterile room to obtain the medicinal marshmallow flower extract.

[0101] During the extraction process, no glucosyltransferase UGT197CG was used. The resulting marshmallow flower extract produced a precipitate after standing for 20 minutes.

[0102] Example 4

[0103] Preparation of European beech bud extract using glucosyltransferase

[0104] Take the buds of European beech (Fagus sylvatica), wash them with clean water, and dry them in an oven at 40℃ until the surface is free of moisture. Grind them using a low-temperature grinder, and then pass the ground European beech buds through a 10-mesh sieve. Take 100g of the sieved European beech buds and 8g of a compound extraction enzyme. The compound extraction enzyme consists of β-glucanase, β-galactosidase, and laccase in a mass ratio of 2:1:1. Add 3g of glucosyltransferase UGT197CG, 3g of sucrose synthase solution, 0.5g of sucrose, and 0.1g of UDP. Make up the reaction volume to 520g with water, and perform ultrasonic extraction (400W) for 20 minutes. Then, stir in the reaction vessel for 180 minutes, maintaining the temperature at 25℃. Finally, stir at 60℃ for 10 minutes to inactivate the enzymes in the system. The inactivated extract was transferred to a filtration device, first filtered through a 0.5 μm membrane, and then filtered a second time through a 0.22 μm membrane to remove microorganisms. Glycerin was added to the extract to make up to 1000 g, and the extract was aliquoted and sealed in a sterile room to obtain the European beech bud extract.

[0105] The European beech bud extract obtained using this method showed no precipitation after 24 hours of standing.

[0106] Comparative Example 4

[0107] European beech buds were collected, washed with water, and dried in an oven at 40°C until no surface moisture remained. The buds were then pulverized using a low-temperature pulverizer and passed through a 10-mesh sieve. 100g of the sieved European beech buds and 8g of a compound extraction enzyme were taken. The compound extraction enzyme consisted of β-glucanase, β-galactosidase, and laccase in a mass ratio of 2:1:1. Water was added to make up a 520g reaction system, and ultrasonic extraction (400W) was performed for 20 minutes. The mixture was then stirred in a reaction vessel for 180 minutes at 25°C. Finally, it was stirred at 60°C for 10 minutes to inactivate the enzymes. The inactivated extract was transferred to a filtration device, first filtered through a 0.5μm membrane, and then filtered again through a 0.22μm membrane to remove microorganisms. Glycerin was added to the extract to make up to 1000g, and the mixture was aliquoted and sealed in a sterile room to obtain the European beech bud extract.

[0108] During the extraction process, without the use of glucosyltransferase UGT197CG, the obtained European beech bud extract produced a precipitate after standing for 20 minutes.

[0109] Example 5

[0110] Preparation of European sour cherry bud extract using glucosyltransferase

[0111] Take the buds of European sour cherry (Prunus cerasus), wash them with water, and dry them in an oven at 40℃ until the surface is dry. Grind them using a low-temperature grinder, and then pass the ground European sour cherry buds through a 10-mesh sieve. Take 100g of the sieved European sour cherry buds and 8g of a compound extraction enzyme. The compound extraction enzyme consists of β-glucanase, β-galactosidase, and laccase in a mass ratio of 2:1:1. Add 3g of glucosyltransferase UGT197CG, 3g of sucrose synthase solution, 0.5g of sucrose, and 0.1g of UDP. Make up the reaction volume to 300g with water, and use ultrasound (400W) for assisted extraction for 20min. Then, stir in the reaction vessel for 180min, maintaining the temperature at 25℃. Finally, stir at 60℃ for 10min to inactivate the enzymes in the system. The inactivated extract was transferred to a filtration device, first filtered through a 0.5 μm membrane, and then filtered a second time through a 0.22 μm membrane to remove microorganisms. Glycerin was added to the extract to make up to 1000 g, and the extract was aliquoted and sealed in a sterile room to obtain the European sour cherry bud extract.

[0112] The European sour cherry bud extract obtained using this method showed no precipitation after being left to stand for 24 hours.

[0113] Comparative Example 5

[0114] European sour cherry buds were collected, washed with water, and dried in an oven at 40°C until no moisture remained on the surface. The buds were then pulverized using a low-temperature grinder and passed through a 10-mesh sieve. 100g of the sieved European sour cherry buds and 8g of a compound extraction enzyme were taken. The compound extraction enzyme consisted of β-glucanase, β-galactosidase, and laccase in a mass ratio of 2:1:1. Water was added to make up to 300g of the reaction system, and ultrasonic extraction (400W) was performed for 20 minutes. The mixture was then stirred in a reaction vessel for 180 minutes at 25°C. Finally, it was stirred at 60°C for 10 minutes to inactivate the enzymes. The inactivated extract was transferred to a filtration device, first filtered through a 0.5μm membrane, and then filtered again through a 0.22μm membrane to remove microorganisms. Glycerin was added to the extract to make up to 1000g, and the mixture was aliquoted and sealed in a sterile room to obtain the European sour cherry bud extract.

[0115] During the extraction process, no glucosyltransferase UGT197CG was used. The European sour cherry bud extract obtained produced a precipitate after standing for 20 minutes.

[0116] Example 6

[0117] Preparation of Calendula extract using glucosyltransferase

[0118] Take the flowers of Calendula officinalis, wash them with clean water, and dry them in an oven at 40℃ until the surface is free of moisture. Grind them using a low-temperature grinder, and then pass the ground calendula flowers through a 10-mesh sieve. Take 100g of the sieved calendula flowers and 8g of a compound extraction enzyme. The compound extraction enzyme consists of β-glucanase, β-galactosidase, and laccase in a mass ratio of 2:1:1. Add 3g of glucosyltransferase UGT197CG, 3g of sucrose synthase solution, 0.5g of sucrose, and 0.1g of UDP. Make up the reaction system to 700g with water, and use ultrasound (400W) for assisted extraction for 20min. Then, stir in the reaction vessel for 180min, maintaining the temperature at 25℃. Finally, stir at 60℃ for 10min to inactivate the enzymes in the system. The inactivated extract was transferred to a filtration device, first filtered through a 0.5 μm membrane, and then filtered a second time through a 0.22 μm membrane to remove microorganisms. 1,3-propanediol was added to the extract to bring the total to 1000 g, and the extract was aliquoted and sealed in a sterile room to obtain the calendula extract.

[0119] The calendula extract obtained using this method showed no precipitation after being left to stand for 24 hours.

[0120] Comparative Example 6

[0121] Take the flowers of calendula, wash them with clean water, and dry them in an oven at 40℃ until the surface is free of moisture. Grind them using a low-temperature grinder, and then pass the ground calendula flowers through a 10-mesh sieve. Take 100g of the sieved calendula flowers and 8g of a compound extraction enzyme. The compound extraction enzyme consists of β-glucanase, β-galactosidase, and laccase in a mass ratio of 2:1:1. Make up the reaction system with water to a final volume of 700g, and perform ultrasonic extraction (400W) for 20 minutes. Then, stir in a reaction vessel for 180 minutes at 25℃. Next, stir at 60℃ for 10 minutes to inactivate the enzymes. Transfer the inactivated extract to a filtration device, first filtering with a 0.5μm membrane, then filtering a second time with a 0.22μm membrane to remove microorganisms. Add 1,3-propanediol to the extract to a final volume of 1000g, and aliquot and seal in a sterile room to obtain the calendula extract.

[0122] During the extraction process, no glucosyltransferase UGT197CG was used. After standing for 20 minutes, the obtained calendula extract produced a precipitate.

[0123] Example 7

[0124] Preparation of milk thistle extract using glucosyltransferase

[0125] Milk thistle (Silybum marianum) was washed with water and dried in an oven at 40°C until no moisture remained on the surface. It was then pulverized using a low-temperature grinder and passed through a 10-mesh sieve. 100g of the sieved milk thistle and 8g of a compound extraction enzyme were taken. The compound extraction enzyme consisted of β-glucanase, β-galactosidase, and laccase in a mass ratio of 2:1:1. 3g of glucosyltransferase UGT197CG, 3g of sucrose synthase solution, 0.5g of sucrose, and 0.1g of UDP were added, and water was added to make a 300g reaction system. Extraction was assisted by ultrasound (400W) for 20 minutes. The mixture was then stirred in a reaction vessel for 180 minutes at 25°C. Finally, it was stirred at 60°C for 10 minutes to inactivate the enzymes. The inactivated extract was transferred to a filtration device, first filtered through a 0.5μm membrane, and then filtered again through a 0.22μm membrane to remove microorganisms. Glycerin was added to the extract to make up to 1000g, and the mixture was dispensed and sealed in a sterile room to obtain milk thistle extract.

[0126] The milk thistle extract obtained using this method showed no precipitation after 24 hours of standing.

[0127] Comparative Example 7

[0128] Milk thistle was collected, washed with clean water, and dried in an oven at 40°C until no moisture remained on the surface. It was then pulverized using a low-temperature grinder and passed through a 10-mesh sieve. 100g of the sieved milk thistle and 8g of a compound extraction enzyme were taken. The compound extraction enzyme consisted of β-glucanase, β-galactosidase, and laccase in a mass ratio of 2:1:1. Water was added to make up to 300g of the reaction system, and ultrasonic extraction (400W) was performed for 20 minutes. The mixture was then stirred in a reaction vessel for 180 minutes at 25°C. Finally, it was stirred at 60°C for 10 minutes to inactivate the enzymes. The inactivated extract was transferred to a filtration device, first filtered through a 0.5μm membrane, and then filtered again through a 0.22μm membrane to remove microorganisms. Glycerin was added to the extract to make up to 1000g, and the mixture was aliquoted and sealed in a sterile room to obtain the milk thistle extract.

[0129] During the extraction process, no glucosyltransferase UGT197CG was used. The milk thistle extract obtained produced a precipitate after standing for 20 minutes.

[0130] Example 8

[0131] Preparation of fig extract using glucosyltransferase

[0132] Take fig leaves, fruits, or buds (Ficus carica), wash them with clean water, and dry them in an oven at 40℃ until the surface is dry. Grind them using a low-temperature grinder, and then pass the ground fig leaves, fruits, or buds through a 10-mesh sieve. Take 100g of the sieved fig leaves, fruits, or buds, and 8g of a compound extraction enzyme. The compound extraction enzyme consists of β-glucanase, β-galactosidase, and laccase in a mass ratio of 2:1:1. Add 3g of glucosyltransferase UGT197CG, 3g of sucrose synthase solution, 0.5g of sucrose, and 0.1g of UDP. Make up the reaction volume to 300g with water, and use ultrasound (400W) for assisted extraction for 20 minutes. Then, stir in the reaction vessel for 180 minutes, maintaining the temperature at 25℃. Finally, stir at 60℃ for 10 minutes to inactivate the enzymes in the system. The inactivated extract was transferred to a filtration device, first filtered through a 0.5 μm membrane, and then filtered a second time through a 0.22 μm membrane to remove microorganisms. Glycerin was added to the extract to make up to 1000 g, and the extract was aliquoted and sealed in a sterile room to obtain fig extract.

[0133] The fig extract obtained using this method showed no precipitation after 24 hours of standing.

[0134] Comparative Example 8

[0135] Take fig leaves, fruits, or buds, wash them with clean water, and dry them in an oven at 40℃ until the surface is free of moisture. Grind them using a low-temperature grinder, and then pass the ground fig leaves, fruits, or buds through a 10-mesh sieve. Take 100g of the sieved fig leaves, fruits, or buds, and 8g of a compound extraction enzyme. The compound extraction enzyme consists of β-glucanase, β-galactosidase, and laccase in a mass ratio of 2:1:1. Make up the reaction system with water to a final volume of 300g, and perform ultrasonic extraction (400W) for 20 minutes. Then, stir in a reaction vessel for 180 minutes, maintaining the temperature at 25℃. Next, stir at 60℃ for 10 minutes to inactivate the enzymes. Transfer the inactivated extract to a filtration device, first filtering with a 0.5μm filter membrane, and then filtering a second time with a 0.22μm filter membrane to remove microorganisms. Glycerin was added to the extract to make up to 1000g, and the mixture was dispensed and sealed in a sterile room to obtain fig extract.

[0136] During the extraction process, no glucosyltransferase UGT197CG was used, and the resulting fig extract produced a precipitate after standing for 20 minutes.

[0137] As can be seen from the above embodiments, the present invention provides a glucosyltransferase obtained by directed evolution and its application. The glucosyltransferase UGT197CG of the present invention can convert poorly soluble flavonoids into flavonoid-C-glucosides and increase their solubility, thereby reducing the amount of precipitation of plant extracts and related products.

[0138] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A glucosyltransferase UGT197CG obtained by directed evolution, characterized in that, The amino acid sequence of the glucosyltransferase UGT197CG is shown in SEQ ID NO.

4.

2. The glucosyltransferase UGT197CG according to claim 1, characterized in that, The glucosyltransferase UGT197CG is a mutation occurring at positions 13, 49, 148, 155, 333, and 422 of the amino acid sequence of the wild-type glucosyltransferase UGT708B8. The amino acid sequence of the wild-type glucosyltransferase UGT708B8 is shown in SEQ ID NO.

2.

3. The glucosyltransferase UGT197CG according to claim 2, characterized in that, The mutations include changing the 13th amino acid from proline to threonine, the 49th amino acid from serine to arginine, the 148th amino acid from phenylalanine to serine, the 155th amino acid from serine to arginine, the 333rd amino acid from lysine to asparagine, and the 422nd amino acid from serine to cysteine.

4. The use of the glucosyltransferase UGT197CG according to any one of claims 1 to 3 in the preparation of plant extracts.

5. A nucleic acid molecule encoding the glucosyltransferase UGT197CG according to any one of claims 1 to 3, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

3.

6. A recombinant plasmid, characterized in that, The recombinant plasmid comprises the nucleic acid molecule as described in claim 5.

7. A recombinant bacterium, characterized in that, The recombinant bacteria include the recombinant plasmid as described in claim 6.

8. The use of the nucleic acid molecule of claim 5, the recombinant plasmid of claim 6, and the recombinant bacteria of claim 7 in the preparation of plant extracts.

Images