Multi-site mutant of glucosyltransferase and application thereof

Abstract

The invention discloses a multi-site mutant of glucosyltransferase and an application of the multi-site mutant. A GT 1 family O-glycosyl transferase UGT73C33 derived from liquorice is mutated, an excellent multi-site mutant of the O-glycosyl transferase is obtained through screening, the multi-site mutant contains three mutation sites, F148 is mutated into W, F388 is mutated into W, and A389 is mutated into T. The novel O-glycosyl transferase mutant C33-F148W-F388W-A389T is high in specificity, the selectivity of the novel O-glycosyl transferase mutant C33-F148W-F388W-A389T for generating luteolin-4 '-O-glucoside through specific conversion of luteolin reaches 80%, the novel O-glycosyl transferase mutant is high in catalytic activity, and the yield of luteolin-4'-O-glucoside reaches 156.8 mg / L under suitable conditions. The invention provides a unique biocatalyst and a conversion process thereof for the preparation of luteolin-4 '-O-glucoside with high activity and high added value.

Description

Technical Field

[0001] The invention relates to a multi-site mutant of glucosyltransferase and application thereof, belonging to the field of glucosyltransferase. Background Art

[0002] Luteolin is a natural flavonoid found in plants such as olive, paulownia, and cymbidium. It exhibits diverse pharmacological activities, including anti-inflammatory, anti-allergic, uric acid-lowering, anti-tumor, antibacterial, and antiviral properties. Clinically, it is primarily used for cough relief, expectorant, anti-inflammatory, uric acid-lowering, and treatment of cardiovascular disease and hepatitis. However, luteolin's low water solubility and poor oral bioavailability have limited its pharmacological activity and clinical application. Glycosylation modification of luteolin may improve its bioavailability and bioactivity. Luteolin-4'-O-glucoside is a rare glycosylated derivative of luteolin. In addition to its anti-inflammatory, antioxidant, and anti-tumor activities, luteolin-4'-O-glucoside exhibits potent inhibitory activity against interleukin-5 and platelet aggregation. Furthermore, luteolin-4'-O-glucoside is an α-amylase inhibitor and has antibacterial properties, preventing and treating hyperuricemia and acute gouty arthritis. However, the current chemical extraction method can only extract 19.6 mg of luteolin 4'-O-glucoside from each kilogram of Leontodon saxatilis, which seriously hinders the in-depth study of the activity of luteolin 4'-O-glucoside and its development and application.

[0003] One effective approach to preparing rare natural flavonoid glycosides is targeted structural modification of flavonoid aglycones. Compared to chemical methods, enzymatic methods offer advantages such as high specificity, mild reaction conditions, and environmental friendliness. Flavonoid glycosyltransferases possess diverse and specific sugar acceptors and donors, including flavonols, anthocyanidins, flavanones, flavones, isoflavones, and flavanols. Furthermore, depending on the modification site in the glycosylation reaction, flavonoid glycosyltransferases can be divided into flavonoid 3-O-glycosyltransferases, 5-O-glycosyltransferases, 7-O-glycosyltransferases, and other O-glycosyltransferases. Therefore, leveraging these characteristics and advantages of flavonoid O-glycosyltransferases allows for targeted modification of flavonoid compounds. Studies have shown that 3-O-glycosyltransferases are the most abundant of plant O-glycosyltransferases, resulting in relatively high yields of flavonoid 3-O-glycoside synthesis. However, O-glycosyltransferases at specific sites, such as 4'-O-glycosyltransferases, are rarely discovered. Currently, the few 4'-O-glycosyltransferases discovered have poor specificity, capable of recognizing multiple sites and thus producing a variety of byproducts. Their catalytic efficiency is also low, making them difficult to meet the needs of practical applications. With the deepening of research, it has been discovered that site-directed mutagenesis of key amino acids in the active pocket of glycosyltransferases can improve the regioselectivity of glycosyltransferases, providing new possibilities for optimizing the performance of flavonoid glycosyltransferases and expanding their application range. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a multi-site mutant of a novel glucosyltransferase capable of efficiently converting luteolin into luteolin-4'-O-glucoside and its application.

[0005] Technical solution: To solve the above technical problems, the present invention provides a mutant of O-glucosyltransferase, which is obtained by mutation of wild-type UGT73C33 at position 389, 388 or 148; the alanine at position 389 is mutated to threonine, the phenylalanine at position 388 is mutated to tryptophan, and the phenylalanine at position 148 is mutated to tryptophan; the amino acid sequence of the wild-type UGT73C33 is shown in SEQ ID NO.1.

[0006] Among them, when the mutation site is 389, its amino acid sequence is shown as SEQ ID NO.6.

[0007] Among them, when the mutation sites are positions 388 and 389, the amino acid sequence is shown as SEQ ID NO.7.

[0008] Among them, when the mutation sites are positions 388, 389 and 148, the amino acid sequence is shown in SEQ ID NO.8.

[0009] The present invention also provides a gene encoding the mutant.

[0010] Wherein, its nucleotide sequence is shown in any one of SEQ ID NO.3~5.

[0011] The present invention also provides a method for constructing the mutant, comprising the following steps: (1) Using reverse PCR to reconstruct plasmid pET-28a-C33 As a template, A389T was mutated to obtain the recombinant plasmid pET-28a- C33-A389T ; C33-A389T The nucleotide sequence is shown in SEQ ID NO.3; the recombinant plasmid pET-28a-C33 Synthesized from wild-type glycosyltransferase gene C33 in pET28a Bam H I and Xho I cloning site obtained between; (2) Using recombinant plasmid pET-28a- C33-A389T As a template, F388W was mutated by inverse PCR and the recombinant plasmid pET-28a- C33-F388W-A389T , C33-F388W-A389T The nucleotide sequence is shown in SEQ ID NO.4.

[0012] (3) Using recombinant plasmid pET-28a- C33-F388W-A389T As a template, F148W was mutated by inverse PCR and the recombinant plasmid pET-28a- C33-F148W-F388W-A389T , C33-F148W-F388W-A389T The nucleotide sequence is shown in SEQ ID NO.5.

[0013] The nucleotide sequences of the reverse PCR primers are shown in SEQ ID NOs. 9 to 14.

[0014] The present invention also provides a recombinant plasmid, a recombinant cell or a recombinant bacterium containing the gene.

[0015] Wherein, the host bacteria include Escherichia coli, Bacillus subtilis, Pichia pastoris or Saccharomyces cerevisiae.

[0016] Preferably, the host bacteria is Escherichia coli.

[0017] The present invention also provides the use of the mutant, gene or recombinant plasmid, recombinant cell or recombinant bacteria in the production of luteolin-4'-O-glucoside.

[0018] The present invention also provides the use of the mutant, gene or recombinant plasmid, recombinant cell or recombinant bacteria in improving the conversion rate of luteolin into luteolin-4'-O-glucoside.

[0019] The present invention also provides a method for preparing luteolin-4'-O-glucoside, comprising the following steps: reacting the mutant, gene or recombinant plasmid, recombinant cell or recombinant bacteria in a system; the system also comprises a buffer solution, a sugar donor UDP-glucose and a substrate luteolin.

[0020] Wherein, the buffer solution includes KH2PO4-Na2HPO4 and Gly-NaOH buffer solution.

[0021] Wherein, the concentration of luteolin is 0.2~1.0 mM.

[0022] Furthermore, the concentration of the luteolin is 0.4-1.0 mM.

[0023] Furthermore, the concentration of the luteolin is 0.5-1.0 mM.

[0024] Furthermore, the concentration of the luteolin is 0.6-1.0 mM.

[0025] Furthermore, the concentration of the luteolin is 0.6-0.8 mM.

[0026] Wherein, the final concentration of the sugar donor is 0.5~1.5 mM.

[0027] Furthermore, the final concentration of the sugar donor is 0.75-1.5 mM.

[0028] Furthermore, the final concentration of the sugar donor is 1.0-1.5 mM.

[0029] Furthermore, the final concentration of the sugar donor is 1.0-1.25 mM.

[0030] The reaction temperature is 20~60°C, the pH is 5.0~9.0, and the reaction time is 10~50 min.

[0031] Furthermore, the reaction temperature is 25-55°C, the pH is 5.5-9.0, and the reaction time is 20-50 min.

[0032] Furthermore, the reaction temperature is 30-50°C, the pH is 6.0-9.0, and the reaction time is 30-50 min.

[0033] Furthermore, the reaction temperature is 35-50°C, the pH is 6.5-8.5, and the reaction time is 30-40 min.

[0034] Furthermore, the reaction temperature is 40-50°C and the pH is 7.0-8.5.

[0035] Furthermore, the reaction temperature is 40-45°C and the pH is 7.0-8.0.

[0036] The present invention is from Licorice ( Glycyrrhiza uralensis. ) cloned a GT1 family glycosyltransferase gene, UGT73C33, from a Chinese genomics firm. Key site mutations were performed on this glycosyltransferase, resulting in a novel multi-site mutant, C33-F148W-F388W-A389T, which exhibits 80% selectivity for luteolin-4'-O-glucoside. This mutant, C33-F148W-F388W-A389T, exhibits high specificity and, under suitable conditions, can efficiently and specifically convert luteolin to luteolin-4'-O-glucoside, demonstrating high catalytic activity.

[0037] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. Glycyrrhiza uralensis ) A glycosyltransferase gene UGT73C33 of the GT1 family was cloned from a human embryo, and key site mutations were performed on the glycosyltransferase to obtain a novel glycosyltransferase multi-site mutant C33-F148W-F388W-A389T. The novel glycosyltransferase multi-site mutant C33-F148W-F388W-A389T has strong specificity and can efficiently and specifically convert luteolin into luteolin-4'-O-glucoside with a selectivity of up to 80%; 2. The novel glycosyltransferase multi-site mutant C33-F148W-F388W-A389T obtained by the present invention has an optimum temperature of 40°C and an optimum pH of 8.0, good enzymatic properties, and great application value; 3. A technical process for the efficient and green preparation of luteolin-4'-O-glucoside with significant biological activity has been established. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is the SDS-PAGE image of glycosyltransferase C33-F148W-F388W-A389T; Figure 2 The enzymatic properties of glycosyltransferase C33-F148W-F388W-A389T are shown (A. Optimum temperature; B. Optimum pH; C. Temperature stability; D. pH stability); Figure 3 Schematic diagram of the enzymatic conversion of luteolin to luteolin-4'-O-glucoside; Figure 4 The figure is a liquid phase analysis diagram of luteolin conversion by glycosyltransferase C33 and its mutants; Figure 5 Optimization of conditions for luteolin conversion by the glycosyltransferase C33 mutant (A. optimal temperature; B. optimal pH; C. UDPG concentration; D. luteolin concentration; E. time). DETAILED DESCRIPTION

[0039] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0040] Unless otherwise specified, the raw materials used in the examples were purchased commercially.

[0041] The DNA polymerase (Prime STAR HS DNA Polymerase), deoxyribonucleic acid (dNTP), ligase (T4 DNA ligase), phosphorylase (T4 Polynucleotide Kinase) and related buffers used in the examples were purchased from TaKaRa Biotechnology (Dalian) Co., Ltd. (TaKaRa).

[0042] Example 1. Cloning, expression and enzyme activity determination of the glycosyltransferase gene of the present invention Example 1. Cloning, expression and enzyme activity determination of the glycosyltransferase gene of the present invention 1. Cloning of glycosyltransferase genes and plasmid construction Glycosyltransferase gene C33 (GenBank accession no. QJC58364.1) was optimized according to the codons of Escherichia coli and synthesized by Bioengineering (Shanghai) in pET28a. The cloning site is Bam H I and Xho I, obtained recombinant plasmid pET28a-C33.

[0043] 2. Expression and purification of glycosyltransferase C33 Add 1 μL of the recombinant plasmid pET28a-C33 solution to the Escherichia coli BL21 (DE3) competent cells and mix well. Place on ice for 10 min, incubate in a 42°C water bath for 2 min, and then place on ice for 5 min. Add 1 mL of SOC medium and culture at 37°C for 1 h. Then spread the plate on an LB plate containing kanamycin (final concentration of kanamycin is 50 mg / L) to screen for transformants. Pick a single colony and culture it in LB liquid medium (add kanamycin to a final concentration of 50 mg / L) for 8-10 h. Then, take 1 mL of the bacterial solution for sequencing to confirm the Escherichia coli BL21 (DE3) strain containing the pET28a-C33 recombinant plasmid.

[0044] The recombinant Escherichia coli BL21 (DE3) was inoculated onto LB plates (the final concentration of kanamycin was 50 mg / L) for activation and cultured at 37°C for 12 h. After a single colony grew, it was inoculated into 50 mL LB liquid medium and cultured at 37°C and 180 rpm for 3-4 h until the OD 600=0.4-0.6, IPTG was added to a final concentration of 0.05 mM for induction, and expression was carried out at 28°C for 7-8 h. Escherichia coli BL21(DE3) containing the empty plasmid pET28a was also used as a negative control. 1 mL of bacterial culture was aspirated and centrifuged at 5000 rpm for 3 min to collect the cells. The supernatant was discarded, and the cells were washed twice with 1 mL of PBS buffer. The cells were resuspended in 500 µL of PBS and lysed by sonication using a 3-second cycle with a 5-second interval for 50 cycles. The cells were centrifuged at 12000 rpm for 10 min, and 15 µL of the supernatant was aspirated for later use. The crude enzyme solution was filtered through a 0.22 μm membrane and purified using a Ni affinity column to obtain purified glycosyltransferase C33. The nucleotide sequence of the recombinant glycosyltransferase gene C33 is shown in SEQ ID NO. 2, and the amino acid sequence is shown in SEQ ID NO. 1. Take 15 µL each of the crude enzyme solution expressed by the empty transformant without the C33 gene, the crude enzyme solution expressed by the transformant with the C33 gene, and the glycosyltransferase C33 eluted with 20, 50, 100, 200, and 400 mM imidazole concentrations, add 15 µL of 5×SDS loading buffer and mix well. After boiling for 5 minutes, take 10 µL of each and perform SDS-PAGE electrophoresis to identify the expressed protein. Figure 1 As shown. Using different concentrations of imidazole for elution, when the imidazole concentration is controlled in the range of 100-400 mM, high-purity glycosyltransferase can be obtained, and its electrophoresis pattern shows a single protein band. Figure 1

[0045] 3. Determination of glycosyltransferase C33 activity Luteolin and its glycosylated derivatives were detected using a high-performance liquid chromatography (HPLC) 1260 system (Agilent, USA) using an Xbridge C18 reversed-phase column (5 µm, 250 cm × 4.6 nm). The mobile phases were methanol (A) and ultrapure water containing 0.1% formic acid (B), with an A:B ratio of 55:45. The flow rate was 0.8 mL / min, the detection wavelength was 340 nm, the column temperature was 30°C, and the injection volume was 10 µL.

[0046] The transformant containing the C33 gene was used to express 50 μg of crude enzyme. The reaction was carried out at 40°C and pH 8.0 for 30 min using 0.5 mM luteolin as a substrate. The reaction was terminated with 400 μL of methanol. The product and substrate contents were detected by HPLC at a wavelength of 340 nm, and the enzyme activity was calculated to be 60 mU / mg.

[0047] The enzyme activity assay reaction system was 200 μL, and the proportions of each substance were shown in Table 1.

[0048] Table 1 Enzyme activity assay system

[0049] One unit of enzyme activity (U) is defined as the amount of enzyme required to synthesize 1 μmol of the product luteolin-4'-O-glucoside per minute at the optimal temperature and pH of the enzyme.

[0050] Example 2. Determination of the mutation site of glycosyltransferase C33 Research has shown that the catalytic activity of glycosyltransferases is closely linked to key sites surrounding the active pocket within the protein structure. The three-dimensional structure of glycosyltransferase C33 was determined through homology modeling. Molecular docking was used to screen for mutations. By mutating amino acids within 5 Å to other amino acids, the site C33-A389T (nucleotide sequence shown in SEQ ID NO. 3, amino acid sequence shown in SEQ ID NO. 6) with enhanced regioselectivity was identified. Furthermore, F388 was mutated to generate the mutant C33-F388W-A389T (nucleotide sequence shown in SEQ ID NO. 4, amino acid sequence shown in SEQ ID NO. 7). Furthermore, F148 was mutated to generate the mutant C33-F148W-F388W-A389T (nucleotide sequence shown in SEQ ID NO. 5, amino acid sequence shown in SEQ ID NO. 8).

[0051] Mutation primer design: Using bioinformatics analysis and homology modeling, mutations were designed by altering some amino acids in the glycosyltransferase C33 molecule. Mutation sequences were obtained using inverse PCR, and two forward and reverse oligonucleotide sequences were designed for each mutation site. Mutation sites used codons preferred by E. coli. Mutation primers are listed in Table 2.

[0052] Table 2 Site-directed mutagenesis primers

[0053] Example 3. Construction of a series of recombinant plasmids carrying mutant genes The carrier constructed with Example 1 C33 The recombinant plasmid pET-28a- C33 As a template, reverse PCR was further used to perform mutations, so that C33 The A at position 389 of the amino acid encoded by the gene was mutated to T, and a recombinant plasmid pET-28a- carrying the mutant gene SEQ ID NO: 3 was obtained. C33-A389T. Carrying the recombinant plasmid pET-28a- C33-A389T As a template, reverse PCR was further used to perform mutations, so that C33 The 388th amino acid F of the gene encoding was mutated to W, and the recombinant plasmid pET-28a- carrying the mutant gene SEQ ID NO: 4 was obtained. C33-F388W-A389T. The recombinant plasmid pET-28a- C33-F388W- A389T As a template, reverse PCR was further used to perform mutations, so that C33 The 148th amino acid F encoded by the gene was mutated to W, and the recombinant plasmid pET-28a- carrying the mutant gene SEQ ID NO: 5 was obtained. C33-F148W-F388W-A389T .

[0054] The reaction system and conditions of inverse PCR are shown in Tables 3 and 4.

[0055] Table 3 Preparation of inverse PCR reaction solution (50 μL in total)

[0056] Table 4 Reaction conditions for inverse PCR

[0057] The PCR fragment 5' end phosphorylation reaction was prepared as shown in Table 5 and the reaction was carried out at 37°C for 1 h.

[0058] Table 5 Preparation of phosphorylation reaction solution

[0059] After phosphorylation at 37°C, 1 μL of T4 ligase was added and ligation was carried out at 16°C for 3 h. Plasmid DNA was transformed, and mutants were selected and identified by sequencing.

[0060] The nucleotide sequences of the glycosyltransferase mutant genes are shown in Table 6.

[0061] Table 6 Mutated genes

[0062] A series of recombinant plasmids containing the above nucleotide sequence pET-28a- C33-A389T , pET-28a- C33-F388W- A389T, pET-28a- C33-F148W-F388W-A389T Among them, pET-28a- C3 The cloning sites are Bam H I and Xho I.

[0063] Example 4. Determination of enzymatic properties of recombinant enzyme C33 mutants 1. Determination of the optimal reaction temperature of the mutant enzyme C33-F148W-F388W-A389T The enzyme activity of the mutant enzyme C33-F148W-F388W-A389T was measured at pH 6.5 in a water bath at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, and 60°C according to the standard enzyme activity assay method. The highest enzyme activity measured was taken as 100%, and the relative enzyme activity of the recombinant enzyme C33 mutant at each reaction temperature was calculated to determine the effect of reaction temperature on the activity of the C33 mutant enzyme. The reaction temperature at which the enzyme activity reached the highest was the optimal reaction temperature of the C33 mutant enzyme ( Figure 2 A). The optimal temperature of the C33 mutant enzyme is 40°C.

[0064] 2. Determination of the optimal reaction pH of the mutant enzyme C33-F148W-F388W-A389T The enzyme activity of the mutant enzyme C33-F148W-F388W-A389T was measured in KH2PO4-Na2HPO4 buffer at pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, and glycine-NaOH at 8.0, 8.5, 9.0, and 9.5, respectively, in a 40°C water bath according to the standard enzyme activity assay method. The highest enzyme activity value measured was taken as 100%, and the relative enzyme activity of the C33 mutant enzyme at each pH was calculated to determine the effect of reaction pH on the activity of the C33 mutant enzyme. The reaction pH at which the enzyme activity reached the highest was the optimal reaction pH of the C33 mutant enzyme ( Figure 2B). Although the C33 mutant enzyme in glycine-NaOH buffer showed higher catalytic activity towards 4'-OH, its selectivity was slightly decreased. Therefore, KH2PO4-Na2HPO4 buffer with a pH of 8.0 was selected as the optimal reaction pH.

[0065] 3. Study on temperature stability of mutant enzyme C33-F148W-F388W-A389T The mutant enzyme C33-F148W-F388W-A389T was incubated at 35°C, 40°C, and 45°C for 0, 30, 60, 90, 120, 150, 180, 210, and 240 min, respectively. The residual enzyme activity of the C33 mutant enzyme was measured at each time point under the three temperature groups. The activity of the recombinant protease without incubation treatment was set as 100%. The relative residual enzyme activity (%) of the C33 mutant enzyme after different incubation times under the three temperature groups was calculated to determine the temperature stability trend of the C33 mutant enzyme ( Figure 2 C). The C33 mutant enzyme has good temperature stability. After incubation at the optimal temperature of 35°C for 4 h, the relative enzyme activity remains above 40%.

[0066] 4. pH stability study of mutant enzyme C33-F148W-F388W-A389T In the absence of substrate, the purified and concentrated C33 mutant enzyme was diluted to 200 μg / mL using KH2PO4-Na2HPO4 buffer at pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, respectively. After each sample was incubated at 25°C for 4 hours, the residual enzyme activity of the enzyme was determined by HPLC. The enzyme activity of the C33 mutant enzyme without incubation in pH buffer was set as 100%, and the relative residual enzyme activity of samples treated with different pH buffers was calculated to determine the pH stability trend of the C33 mutant enzyme ( Figure 2 D). The enzyme remains stable in a wide pH range of 5.5-8.0 and retains more than 60% of its activity after 4 hours of incubation.

[0067] Example 5. Application of recombinase C33 mutant in transforming luteolin The ability of recombinant enzyme C33 and its mutants to transform luteolin was determined (see the schematic diagram of the transformation). Figure 3). During the determination, the concentration of luteolin was 0.5 mM, and the conversion was carried out for 0.5 h under the same catalytic conditions, including the optimal temperature, optimal pH, and the same amount of enzyme used for the recombinant enzyme. The results showed that after 0.5 h of reaction, the conversion rate of luteolin to luteolin-4'-O-glucoside by C33 was about 40%, the conversion rate of luteolin to luteolin-4'-O-glucoside by mutant C33-A389T was greater than 50%, the conversion rate of luteolin to luteolin-4'-O-glucoside by mutant C33-F388W-A389T was greater than 60%, and the conversion rate of luteolin to luteolin-4'-O-glucoside by mutant C33-F148W-F388W-A389T was greater than 70%, as shown in Figure 2. Figure 4 .

[0068] The mutant C33-F148W-F388W-A389T was reacted at different temperatures (20-60°C), pH 8.0, UDPG concentration of 1 mM, and luteolin concentration of 0.5 mM for 0.5 h. Figure 5 As shown in A, the optimum temperature is 40℃.

[0069] The mutant C33-F148W-F388W-A389T was reacted at 40°C, different pH values ​​(5.5-9.5), UDPG concentration of 1 mM, and luteolin concentration of 0.5 mM for 0.5 h. Figure 5 As shown in B, the optimum pH is 8.0℃.

[0070] The mutant C33-F148W-F388W-A389T was reacted at 40°C, pH 8.0, different UDPG concentrations (0.5-1.5 mM), and luteolin concentration of 0.5 mM for 0.5 h. Figure 5 As shown in C, the optimal UDPG concentration is 1 mM.

[0071] The mutant C33-F148W-F388W-A389T was reacted at 40°C, pH 8.0, UDPG concentration of 1 mM, and different luteolin concentrations (0.2-1 mM) for 0.5 h. Figure 5 As shown in D, the optimal luteolin concentration is 0.5 mM.

[0072] The mutant C33-F148W-F388W-A389T was reacted at 40°C, pH 8.0, UDPG concentration of 1 mM, and luteolin concentration of 0.5 mM for different time periods (10-50 min). Figure 5 As shown in E, the optimum time is 0.5 hours.

[0073] Under the optimal conditions, luteolin was used as the substrate at a concentration of 0.5 mM, the final concentration of the sugar donor was 1 mM, the reaction temperature was 40°C, the pH was 8.0, and the reaction time was 30 min. Under these conditions, 156.8 mg / L was obtained.

Claims

1. A mutant of O-glucosyltransferase, characterized in that It is obtained by mutating any one or more of positions 389, 388 or 148 of a wild-type glycosyltransferase; the alanine at position 389 is mutated to threonine, the phenylalanine at position 388 is mutated to tryptophan, and the phenylalanine at position 148 is mutated to tryptophan; the amino acid sequence of the wild-type glycosyltransferase is shown in SEQ ID NO.

1.

2. The mutant according to claim 1, characterized in that When the mutation site is 389, the amino acid sequence is shown as SEQ ID NO.

6.

3. The mutant according to claim 1, characterized in that When the mutation sites are positions 388 and 389, the amino acid sequence is shown in SEQ ID NO.

7.

4. The mutant according to claim 1, characterized in that When the mutation sites are positions 388, 389 and 148, the amino acid sequence is shown in SEQ ID NO.

8.

5. A gene encoding the mutant according to any one of claims 2 to 4.

6. The gene according to claim 5, characterized in that The nucleotide sequence thereof is shown in any one of SEQ ID NOs. 3 to 5.

7. A recombinant plasmid, recombinant cell or recombinant bacterium, characterized in that: It contains the gene according to claim 5 or 6.

8. Use of the mutant according to any one of claims 1 to 4, the gene according to claim 5 or 6, or the recombinant plasmid, recombinant cell, or recombinant bacterium according to claim 7 in producing luteolin-4'-O-glucoside or improving the conversion rate of luteolin to luteolin-4'-O-glucoside.

9. A method for preparing luteolin-4'-O-glucoside, characterized in that: The method comprises the following steps: reacting the mutant according to any one of claims 1 to 4, the gene according to claim 5 or 6, or the recombinant plasmid, recombinant cell or recombinant bacteria according to claim 7 in a system; the system comprises a sugar donor UDPG and a substrate luteolin.

10. The method according to claim 9, characterized in that The concentration of luteolin is 0.2-1.0 mM, the final concentration of the sugar donor is 0.5-1.5 mM, the reaction temperature is 20-60°C, the pH is 5.0-9.0, and the reaction time is 10-50 min.

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