Glycosyltransferase mutants and their use in preparing quercetin-3-o-rhamnoside

Abstract

The present application relates to glycosyltransferase mutants and their use in preparing quercetin-3-O-rhamnoside, by replacing the 44 amino acids of the C-terminal PSPG domain of the amino acid sequence of the glucosyltransferase AtUGT78D2 with the 44 amino acids of the C-terminal PSPG domain of the glucosyltransferase AtUGT78D1, obtaining a domain-engineered glycosyltransferase AtUGT78D2-D1PSPG; then performing site-directed mutagenesis on the glycosyltransferase AtUGT78D2-D1PSPG, obtaining a rhamnosyltransferase mutant with good selectivity, good stability and high expression, and the present application also provides a three-enzyme coupling catalytic synthesis reaction system for quercetin-3-O-rhamnoside constructed by the UGT78D2-D1PSPG mutant, which realizes the generation of UDP-rhamnose donor from sucrose as a substrate under the action of UDP-rhamnose synthase, reduces the production cost of UDP-rhamnose donor purchase, and then catalyzes the one-step directional synthesis of quercetin-3-O-rhamnoside from quercetin under the action of glycosyltransferase. At SuSy, UDP-rhamnose synthase At RHM-NRS, glycosyltransferase At UGT78D2-D1PSPG mutant The three-enzyme coupling catalytic synthesis reaction system for quercetin-3-O-rhamnoside constructed by the UGT78D2-D1PSPG mutant realizes the generation of UDP-rhamnose donor from sucrose as a substrate under the action of UDP-rhamnose synthase, reduces the production cost of UDP-rhamnose donor purchase, and then catalyzes the one-step directional synthesis of quercetin-3-O-rhamnoside from quercetin under the action of glycosyltransferase.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology of traditional Chinese medicine, specifically relating to a glycosyltransferase with PSPG domain substitution. At UGT78D2-D1 PSPG mutant and its application in the highly selective preparation of quercetin-3-O-rhamnoside. Background Technology

[0002] Quercetin is a widely distributed flavonol compound in the plant kingdom with various biological activities. It has a benzo(γ)-pyranone structure with a C6-C3-C6 carbon skeleton. Quercetin can regulate many intracellular and extracellular signaling pathways related to disease progression. It is a natural antioxidant with antioxidant, anti-inflammatory, hypoglycemic, anticancer, and cardiovascular disease prevention and treatment effects. However, its poor solubility and low bioavailability limit its clinical application.

[0003] Structure-activity relationship studies show that the presence of glycans or glycosylation modifications can significantly improve the water solubility and stability of compounds, enhance the bioactivity of target compounds, increase their bioavailability, strengthen targeting effects, and reduce toxic side effects. The type of sugar involved in glycosylation modification can also affect the targeting of target compounds; among the diverse types of glycosylation modifications, rhamnylation is a very important one, with various substitution forms. Furthermore, pharmacological studies show that quercetin exhibits significant pharmacological activity after rhamnylation. For example, quercetin-3-O-rhamnoside has a good inhibitory effect on human influenza A / WS / 33 virus; it can also effectively prevent diarrhea in rats, stabilize fluid transport in the rat colon, and minimize damage to the colon; in addition, 12.5~50 μg / mL quercetin-3-O-rhamnoside can alleviate the damage to cardiomyocytes caused by doxorubicin (ADR), exhibiting a significant cardioprotective effect. Research on this type of compound is of great significance.

[0004] The structural formula of quercetin-3-O-rhamnoside is as follows:

[0005]

[0006] Plant glycosylation-related glycosyltransferases mostly belong to the GT1 superfamily, primarily UDP-dependent GT-B folding glycosyltransferases. GT-B glycosyltransferases exhibit highly conserved C-terminal sequences, containing a 44-amino acid-rich PSPG motif rich in glutamate and glycine residues, used for binding glycosyl donors. This conserved sequence has been routinely used for the identification of plant glycoside synthesis-related enzymes. However, the N-terminal sequences possess variable helical and cyclic structures to accommodate a wide variety of acceptor substrates. This uncertainty in the N-terminal sequence contributes to the broad substrate profile of glycosyltransferases, making substrate catalytic function prediction extremely difficult. Theoretically, exchanging N-terminal and C-terminal PSPG domains with different substrate specificities could uncover glycosyltransferases with novel catalytic properties. However, this method is limited to enzyme sequences with high parental similarity; that is, the PSPG domain substitution strategy is not applicable between two glycosyltransferases with low similarity.

[0007] Furthermore, rhamnosyltransferases typically use NAD-rhamnose as a sugar donor to transfer rhamnosyl groups to target receptors, participating in the synthesis of rhamnoside compounds. Currently reported rhamnosyltransferases related to the modification of active small molecule rhamnosyl groups mainly originate from plants, with a small portion originating from microorganisms. Through bioinformatics analysis and various molecular biological methods, a variety of rhamnosyltransferase genes have been identified from plants and microorganisms. For example, Jones et al. (J Biol Chem, 2003, 278(45): 43910-8.) cloned a rhamnosyltransferase gene from Arabidopsis thaliana that can rhamnosyltransferase the 3-OH group of quercetin and kaempferol, respectively, to generate quercetin-3-O-rhamnoside and kaempferol-3-O-rhamnoside. O -UGT78D1 of rhamnoside. Rojas (Plant Mol Biol, 2014, 84(3): 287-300.) cloned a flavonol-3-O-glucoside (1→6) rhamnosyltransferase from soybean. Gm F3G6'R. Casas et al. (Plant Cell, 2016, 28(6): 1297-309.) cloned UGT91L1 from the maize Sm2 locus, which can catalyze the rhamnosylation of isochorin to 2'-O-rhamnosyl isochorin.

[0008] In summary, significant progress has been made in the identification of plant-derived rhamnosyltransferases, but the following problems still exist: (1) Currently, only a small number of rhamnosyltransferases have been identified, so it is not yet possible to summarize the characteristic conserved sequences of rhamnosyltransferases through multiple sequence alignment analysis; (2) Both in vivo and in vitro rhamnosylation reactions require the participation of rhamnosyl donors, such as UDP-rhamnose (UDP-Rha). Due to its very limited sources and complex synthetic routes, this has greatly limited the identification of more rhamnosyltransferases. Summary of the Invention

[0009] The purpose of this invention is to address the problems of limited sources of existing rhamnosyltransferases, poor substrate concentration, poor reaction selectivity, and the need to add exogenous expensive UDP-Rha as a sugar donor during the catalytic process, by providing a glycosyltransferase mutant.

[0010] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0011] By using glucosyltransferase At The 44 amino acids in the C-terminal PSPG domain of the UGT78D2 amino acid sequence are replaced with glycosyltransferase. At The 44 amino acids of the C-terminal PSPG domain of UGT78D1 were used to obtain a domain-engineered glycosyltransferase. At UGT78D2-D1PSPG; then glycosyltransferase At One or more of the amino acid residues at positions 350, 353, 374, 377, 378, and 381 in UGT78D2-D1PSPG are point-mutated to another amino acid residue to obtain the glycosyltransferase mutant.

[0012] Among them, methionine at position 350 was mutated to threonine, asparagine at position 353 was mutated to phenylalanine, glycine at position 374 was mutated to cysteine, isoleucine at position 377 was mutated to phenylalanine, leucine at position 378 was mutated to phenylalanine, and asparagine at position 381 was mutated to glutamine.

[0013] The glycosyltransferase At The amino acid sequence of UGT78D2-D1PSPG is shown in SEQ ID NO: 2.

[0014] In a preferred embodiment, the mutant is a combination mutation of two or three amino acid residues at positions 350, 353, 374, 377, 378, and 381 of the glycosyltransferase AtUGT78D2-D1PSPG.

[0015] In a preferred embodiment, the mutant is M350T / I377F, M350T / N381Q, N353F / G374C, N353F / I377F, N353F / L378F, or N353F / N381Q.

[0016] Another object of the present invention is to provide the use of the above mutant in the preparation of quercetin-3-O-rhamnoside.

[0017] As a preferred embodiment, quercetin-3-O-rhamnoside is synthesized using sucrose as a substrate via a three-enzyme coupled catalytic reaction system.

[0018] The system for the three-enzyme coupled catalytic synthesis of quercetin-3-O-rhamnoside comprises: the glycosyltransferase mutant, and sucrose synthase derived from Arabidopsis thaliana. At SuSy, UDP-rhamnosylsynthase At RHM-NRS, sucrose, uridine diphosphate disodium (UDP), nicotinamide adenine dinucleotide (NAD) + Dimethyl sulfoxide (DMSO), quercetin, and phosphate buffer.

[0019] In a preferred embodiment, the amount of sucrose synthase in the three-enzyme coupled catalytic system is 0.01-0.12 mg / mL, preferably 0.02 mg / mL; the mass ratio of the three enzymes in the reaction system is sucrose synthase: UDP-rhamnose synthase: glycosyltransferase mutant = 1:8:2~8.

[0020] In a preferred embodiment, the concentration of UDP added to the three-enzyme coupled catalytic reaction system is 0.2 mM-3 mM, preferably 1.5 mM; NAD + The added concentration is 0.1 mM-0.9 mM, preferably 0.7 mM.

[0021] In a preferred embodiment, the concentration of sucrose added to the three-enzyme coupled catalytic reaction system is 100 mM-600 mM, preferably 400 mM.

[0022] In a preferred embodiment, the concentration of quercetin added to the three-enzyme coupled catalytic reaction system is 0.5 mM-3 mM, preferably 1.5 mM; the concentration of dimethyl sulfoxide is 5%-30% V / V.

[0023] In a preferred embodiment, the reaction temperature of the three-enzyme coupled catalytic reaction system is 20℃-40℃, preferably 30℃; the pH of the reaction buffer is 6.0-9.0, preferably 7.5.

[0024] The two Arabidopsis-derived glycosyltransferases used in this invention At UGT78D2 and At The amino acid similarity between UGT78D1 and UGT78D2 is 80.3%. Rhamnosyltransferase UGT78D1 catalyzes the formation of quercetin-3-O-rhamnoside from the 3-OH hydroxyl group of quercetin, while Arabidopsis-derived glucosyltransferase UGT78D2 catalyzes the same formation. By replacing the PSPG domain of UGT78D1 with that of glucosyltransferase UGT78D2, a rhamnosyltransferase specifically accepting rhamnose was obtained. Combined with site-directed mutagenesis, the mutant changed from accepting UDP-glucose donors to accepting UDP-rhamnoside donors, with a significantly improved selectivity for UDP-rhamnoside donors. This achieved a change in the sugar donor selectivity of the glycosyltransferase, ultimately yielding a rhamnosyltransferase mutant with good selectivity, stability, and high expression levels.

[0025] This invention also provides a three-enzyme coupled catalytic reaction system based on this mutant. This system utilizes inexpensive sucrose as a substrate to synthesize UDP-Rha under the action of sucrose synthase AtSuSy and UDP-rhamnose synthase AtRHM-NRS, significantly reducing the preparation cost of UDP-Rha. It achieves a one-pot catalytic synthesis of quercetin-3-O-rhamnoside using quercetin as a substrate and UDP-Rha as a sugar donor. This reaction system not only significantly reduces the preparation cost of rhamnoside products, but also produces a single product through multi-enzyme catalysis, helping to reduce subsequent separation and purification steps and simplifying the product preparation process. Attached Figure Description

[0026] Figure 1 This is a roadmap for the implementation of the present invention.

[0027] Figure 2 SDS-PAGE protein expression of glycosyltransferases and their mutants, sucrose synthase, and rhamnose synthase; where 1: Marker; 2 and 3 are the supernatant and precipitate of pET-28a lysate, respectively; 4 and 5 are the supernatant and precipitate of UGT78D1-D1PSPG lysate, respectively; 6 and 7 are the supernatant and precipitate of UGT78D1-D1PSPG-N353F lysate, respectively; 8 and 9 are the supernatant and precipitate of UGT78D1-D1PSPG-G374C lysate, respectively; 10 and 11 are the supernatant and precipitate of UGT78D1-D1PSPG-N381Q lysate, respectively; 12 and 13 are the supernatant and precipitate of UGT78D1-D1PSPG-N353F / N381Q lysate, respectively; 14 and 15 are Markers; 16 and 17 are sucrose synthases, respectively. At SuSy fragmentation supernatant and precipitate; 18 and 19 are rhamnose synthase, respectively. AtRHM-NRS was used to break down the supernatant and precipitate; 20 was used to break down the supernatant of pET-28a; all the above crude enzyme solutions were at their original concentrations and were not concentrated.

[0028] Figure 3 Sucrose synthase At SuSy and rhamnosyl synthase At The effect of RHM-NRS addition ratio on UDP-Rha synthesis.

[0029] Figure 4 The effect of adding a certain percentage of UDP on the generation of UDP-Rha reactions.

[0030] Figure 5 For NAD + The effect of the addition ratio on the generation of UDP-Rha reaction.

[0031] Figure 6 The effects of different temperatures and pH on the formation of UDP-Rha.

[0032] Figure 7 The effect of reaction time on the generation of UDP-Rha reaction.

[0033] Figure 8 The effect of the ratio of three enzymes added on the conversion rate of the three-enzyme coupled catalytic synthesis system of quercetin-3-O-rhamnoside.

[0034] Figure 9 The effect of DMSO addition on the conversion rate of the three-enzyme coupled catalytic synthesis system of quercetin-3-O-rhamnoside was investigated.

[0035] Figure 10 The effect of substrate quercetin concentration on the conversion rate of the three-enzyme coupled catalytic synthesis system of quercetin-3-O-rhamnoside was investigated.

[0036] Figure 11 It is the result of transformation rate and regioselectivity of the two-site combined mutant.

[0037] Figure 12 These are the results of transformation efficiency and regioselectivity of the three-site combination mutants.

[0038] Figure 13 The liquid chromatography diagrams are for quercetin standard, UGT78D1, and UGT78D2-D1PSPG.

[0039] Figure 14 This is a liquid phase diagram of the three-enzyme reaction of the UGT78D2-D1PSPG mutant. Detailed Implementation

[0040] Example 1: This example illustrates glycosyltransferases At Build of UGT78D2-D1 PSPG

[0041] Search on the NCBI website At UGT78D2 (ID: At 5g17050), At UGT78D1 (ID: At 1g30530).

[0042] Will At The PSPG domain of the UGT78D1 was replaced with... At A domain-engineered glycosyltransferase, UGT78D2-D1 PSPG, was obtained by modifying the domain of UGT78D2. The mutant gene sequence was then constructed into the pET-28a vector, and an insertion was made at the 5' end. Nde I. Restriction site: A BamH I site is inserted at the 3' end. The plasmid vector pET-28a-UGT78D2-D1 PSPG was synthesized in its entirety by Suzhou Genewiz. The plasmid was introduced into E. coli BL21(DE3) competent cells. Positive clones verified by colony PCR were selected as recombinant E. coli expressing the target glycosyltransferase, which is the engineered strain E. coli BL21(DE3) / pET28a-UGT78D2-D1PSPG, and stored at -80℃ for later use.

[0043] Example 2: This experiment demonstrates the site-directed mutagenesis of glycosyltransferase UGT78D2-D1 PSPG.

[0044] The plasmid of engineered E. coli BL21(DE3) / pET28a-UGT78D2-D1 PSPG was extracted according to the experimental instructions in the Axygen plasmid extraction kit. Using the wild-type UGT78D2-D1 PSPG gene as a template, its nucleic acid sequence is shown in SEQ ID NO: 1. Site-directed mutagenesis primers were designed using the Agilent Quick Change online mutagenesis primer design website. Using the extracted pET28a-UGT78D2-D1 PSPG plasmid as a template, the full plasmid was amplified by high-fidelity DNA polymerase 2×PhantaMastermix directional PCR. The mutant was introduced into the plasmid. The main mutagenesis primers are shown below:

[0045] M350T-R 5'-ACATTCACACCCGTTGCTTCGTGTTTCAGCAGTTCC-3' M350T-F 5'-GGAACTGCTGAAACACGAAGCAACGGGTGTGAATGT-3' N353F-R 5'-CATCCACAATGCGTCACAAACACACCCATTGCTTCGTG-3' N353F-F 5'-CACGAAGCAATGGGTGTGTTTTGTGACGCATTGTGGATG-3' G374C-R 5'-AATTGGCCTGCAAATCATCGGTACACCAGCCGA-3' G374C-F 5'-TCGGCTGGTGTACCGATGATTTGCAGGCCAATT-3' I377F-R 5'-TCTGTTTATCCGCAAGAAATGGCCTGCCAATCATCG-3' I377F-F 5'-CGATGATTGGCAGGCCATTTCTTGCGGATAACAGA-3' L378F-R 5'-CTGTTATCCGCAAAAATTGGCCTGCCAATCATCGG-3' L378F-F 5'-CCGATGATTGGCAGGCCAATTTTTGCGGATAACAG-3' N381Q-R 5'-CTCTTCCGTTCAATCTCTGATCCGCAAGAATTGGCCTGC-3' N381Q-F 5'-GCAGGCCAATTCTTGCGGATCAGAGATTGAACGGAAGAG-3'

[0046] The PCR system for amplifying the full plasmid of the mutant is shown below:

[0047] Template plasmid 1µL Primer-R 2µL Primer-F 2µL <![CDATA[ddH2O]]> 20µL Phanta high-fidelity enzyme 25µL

[0048] The PCR reaction system for amplifying the full plasmid of the mutant is shown below:

[0049] 1 Preheat: 95℃, 3 min 2 Denaturation: 95℃, 30s 3 Annealing: 60℃, 30s 4 Extension: 72℃, 5min 5 Repeat steps 2-4 30 times 6 Complete extension: 72℃, 10min 7 Hold at 4℃

[0050] Template digestion: Add 1 μL DpnI and 5 μL Buffer to 50 μL PCR amplification system, incubate at 37℃ for 45-60 min, then cool to 4℃ and store. Verify by agarose gel electrophoresis.

[0051] The mutant PCR product was transformed into *E. coli* BL21(DE3) competent cells. Positive recombinants were screened on kanamycin-containing resistant plates, and single clones were selected. Colony PCR verification was performed using universal T7 primers (T7: 5'-GCTAGTTATTGCTCAGCGG-3', T7-Term: 5'-TAATACGACTCACTATAGGG-3'). Positive clones were sent to the company for DNA sequencing. Successfully sequenced positive colonies were inoculated into fresh LB medium containing kanamycin and cultured for 12 hours, then stored at -80°C for later use.

[0052] Example 3: This example illustrates the induced expression of glycosyltransferase UGT78D2-D1 PSPG and its mutants.

[0053] The mutants obtained in Examples 1 and 2 were inoculated into LB medium containing kanamycin and cultured at 37°C with shaking at 180 rpm for 12 h to obtain seed culture. Seed culture was then inoculated into fresh LB medium containing kanamycin at a 2% (v:v) inoculation rate and cultured at 37°C with shaking at 180 rpm for 1–2 h. When the OD of the medium… 600 When the concentration was approximately 0.6, IPTG was added to a final concentration of 0.1 mmol / L for induction, and the mixture was incubated at 20°C for 24 h.

[0054] The fermentation broth was centrifuged to collect the cells, which were then washed twice with phosphate buffer (pH 7.5) and resuspended. The cells were then sonicated, centrifuged again, and the supernatant was collected to obtain the crude enzyme solution. The supernatant was used as the sample for intracellular soluble proteins; the remaining fraction after removing the supernatant was resuspended in 1 mL of phosphate buffer and used as the sample for intracellular insoluble proteins. Protein expression was analyzed by SDS-PAGE gel electrophoresis.

[0055] Example 4: This example illustrates the optimization process of a dual-enzyme coupling system for the efficient regeneration of the UDP-rhamnose system.

[0056] (1) Sucrose synthase At SuSy and rhamnosyl synthase At Heterologous expression of RHM-NRS

[0057] To address the issue of expensive UDP-Rha sources and further increase substrate concentration, this experiment established a UDP-Rha synthase. At RHM-NRS and sucrose synthase At The SuSy dual-enzyme coupling system was used for the synthesis of UDP-Rha. Since the E. coli expression system has a well-established background and is relatively simple to operate, this study chose to express the UDP-Rha synthase gene in E. coli using pET-28a as a vector. At RHM-NRS and sucrose synthase At SuSy.

[0058] This study used E. coli BL21(DE3) / pET28a- previously constructed in our laboratory. At SuSy heterologous expression of Arabidopsis-derived sucrose synthase At SuSy (see CN114836398A). E. coli were picked and placed on solid LB agar plates containing 1 / 1000 kanamycin stock solution, and incubated at 37°C for 12 h for activation. Single bacteria were then picked and placed in liquid LB agar containing kanamycin, and incubated overnight at 37°C and 180 rpm for 12 h. Seed culture was added to 40 mL of liquid medium at a 1% (v / v) inoculation rate, and incubated at 37°C and 180 rpm until the bacterial growth rate reached OD. 600 When the bacterial culture concentration reaches 0.6-0.8, add 0.02 mM IPTG inducer and induce at 16℃ for 24 h. Centrifuge at 12000 rpm for 10 min at 4℃ and discard the supernatant. Add phosphate buffer (pH 7.5) to suspend the bacterial cells, sonicate to disrupt, and centrifuge at 12000 rpm for 10 min to obtain the supernatant crude enzyme solution.

[0059] Similarly, the Arabidopsis-derived UDP-Rha synthase E. coli BL21(DE3) / pET28a- At RHM-NRS was induced at 20°C for 24 h with the addition of 0.1 mM IPTG inducer. The cells were then centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was discarded. The cells were then suspended in a phosphate buffer solution at pH 7.5, sonicated, and centrifuged at 12000 rpm for 10 min to obtain the crude enzyme supernatant.

[0060] SDS-PAGE analysis was performed on the supernatant and precipitate of sucrose synthase and UDP-rhamnose synthase. At SuSy has a relative molecular weight of approximately 96 kDa. UDP-Rha synthase AtThe relative molecular weight of RHM-NRS is approximately 66.4 kDa. Both enzymes were expressed in E. coli and were expressed in a relatively large amount of soluble form intracellularly, producing fewer inclusion bodies. Therefore, crude enzyme solutions will be used for subsequent experiments.

[0061] (2) Sucrose synthase At SuSy and rhamnosyl synthase At The effect of RHM-NRS addition ratio on UDP-Rha synthesis.

[0062] Studies have shown that the enzyme ratio in a two-enzyme system has a significant impact on the overall conversion rate and the types of products. First, bovine serum albumin (BSA) solution was used as the standard protein to create a standard protein concentration curve. 20 µL of a standard protein solution of a specific concentration was added to the microplate, followed by 200 µL of BCA working solution. The plate was incubated at 37°C for 30 min, allowed to cool to room temperature, and the A concentration in each well was measured using a microplate reader. 562 Value, based on A of each hole 562 The values ​​are plotted on the ordinate and the corresponding protein concentration on the abscissa to construct a standard protein curve. The target protein is diluted to an appropriate level, and A is measured using the same method. 562 The value is calculated based on the standard curve. At RHM-NRS and At The protein concentration corresponding to the crude SuSy enzyme.

[0063] Sucrose synthase needs to be added to the UDP-Rha donor generation reaction system. At SuSy, rhamnosyl synthase At RHM-NRS, UDP, NAD + Sucrose and phosphate buffer solution. Preliminary determination of NAD. + The addition amounts were 1 mM of methyl methacrylate (MDA), 2 mM of UDP, and 400 mM of sucrose. The phosphate buffer solution used was pH 7.5. The ratio of the two enzymes was changed. At SuSy: At RHM-NRS ratio: 1:1 to 1:8. The reaction conditions were 30℃, followed by boiling to inactivate the enzyme after 5 hours, cooling, and then adding an equal volume of methanol to terminate the reaction. The supernatant was collected by centrifugation, filtered through a membrane, and analyzed by HPLC.

[0064] HPLC detection and analysis procedure for UDP-Rha: Instrument: Dionex P680 high performance liquid chromatograph; Analytical column: Kromasil C18 column (250mm × 4.6mm, 3μm); Detection wavelength: 260nm; Injection: 20µL; Detection temperature: 30℃; Mobile phase: 5% methanol aqueous solution containing 50mM ammonium formate (pH=4.5); Flow rate: 0.5mL / min.

[0065] The effect of the ratio of two enzyme additions on UDP-Rha production is as follows: Figure 3 As shown in Table 1, the results for the two-enzyme ratio AtSuSy:AtRHM-NRS = 1:8 are as follows:

[0066] Table 1

[0067] SuSy (mg / mL) RHM-NRS (mg / mL) <![CDATA[NAD + (mM)]]> UDP(mM) Sucrose (mM) UDP-Rha generation volume (mM) 0.02 0.16 1 1 400 0.294

[0068] It can be concluded that when At The amount of SuSy added was 0.02 mg / mL. At The highest concentration of UDP-Rha (0.294 mM) was generated in the reaction system when the addition amount of RHM-NRS was 0.16 mg / mL, i.e., the ratio of the two enzymes was 1:8.

[0069] (3) The effect of the UDP addition ratio on the generation of UDP-Rha reaction.

[0070] The initial reaction system is At SuSy 0.02mg / mL At RHM-NRS 0.16 mg / mL, NAD + The concentration was 1 mM, the sucrose concentration was 400 mM, the phosphate buffer solution pH was 7.5, and the UDP addition range was 0.2 mM to 4 mM. The reaction conditions were 30℃, followed by boiling to inactivate the enzyme after 5 hours, cooling, and then adding an equal volume of methanol to terminate the reaction. The supernatant was centrifuged, filtered through a membrane, and UDP-Rha was detected by HPLC. The results are as follows. Figure 4 As shown in Table 2, the results when the UDP addition size is 1.5mM are as follows:

[0071] Table 2

[0072] SuSy (mg / mL) RHM-NRS (mg / mL) <![CDATA[NAD + (mM)]]> UDP(mM) Sucrose (mM) UDP-Rha generation volume (mM) 0.02 0.16 1 1.5 400 0.3401

[0073] It can be concluded that when the UDP concentration is 1.5 mM, the maximum amount of UDP-Rha generated in the reaction system is 0.3401 mM. When the UDP concentration is greater than 3 mM, the amount of UDP-Rha generated in the reaction decreases significantly, indicating that high concentrations of UDP have an inhibitory effect on the enzyme.

[0074] (4) NAD + The effect of the addition ratio on the generation of UDP-Rha reaction.

[0075] The initial reaction system is At SuSy 0.02 mg / mL At RHM-NRS 0.16 mg / mL, UDP concentration 1.5 mM, sucrose concentration 400 mM, phosphate buffer pH 7.5, NAD +The addition range was 0.1 mM to 1.0 mM. The reaction conditions were 30℃, followed by boiling to inactivate the enzyme after 5 hours, cooling, and then adding an equal volume of methanol to terminate the reaction. The supernatant was centrifuged, filtered through a membrane, and UDP-Rha was detected by HPLC. The results are as follows: Figure 5 As shown. It can be concluded that when NAD... + When the added concentration was 0.7 mM, the maximum amount of UDP-Rha generated in the reaction system was 0.946 mM. When NAD... + When the concentration is greater than 0.9 mM, the amount of UDP-Rha generated in the reaction shows a decreasing trend, and NAD... + Too At Potential inhibitor of RHM-NRS activity, high concentrations of NAD + It can inhibit nicotinamide adenine dinucleotide (NADH) as At A cofactor of RHM NRS.

[0076] (5) Effects of different temperatures and pH on the formation of UDP-Rha.

[0077] Effect of temperature: The reaction system is as follows: At SuSy 0.02mg / mL At RHM-NRS 0.16 mg / mL, UDP added at a concentration of 1.5 mM, NAD + The concentration of added sugar was 0.7 mM, the sucrose concentration was 400 mM, and the pH of the phosphate buffer solution was 7.5. The reaction system was subjected to conditions of 20℃, 25℃, 30℃, 35℃, 40℃, and 45℃, respectively, and the amount of UDP-Rha generated was measured.

[0078] The effect of pH: The reaction system is as follows: At SuSy 0.02mg / mL At RHM-NRS 0.16 mg / mL, UDP added at a concentration of 1.5 mM, NAD + The concentration of added 0.7 mM sucrose was 400 mM, and the reaction temperature was 30 °C. The reaction was carried out in buffer solutions with different pH values, and the amount of UDP-Rha generated was measured. The buffer solutions were 50 mM NaH₂PO₄-Na₂HPO₄ buffer (pH 6.0, 6.5, 7.0, 7.5) and 50 mM Tris-HCl buffer (pH 8.0, 8.5, 9.0). The results are as follows: Figure 6 As shown.

[0079] (6) Effect of reaction time on UDP-Rha reaction generation.

[0080] The reaction system is At SuSy 0.02mg / mL AtRHM-NRS 0.16 mg / mL, UDP added at a concentration of 1.5 mM, NAD + The enzyme was added at a concentration of 0.7 mM, sucrose at 400 mM, and phosphate buffer solution at pH 7.5. The reaction conditions were 30℃, with samples taken at intervals. Each 100 µL sample was boiled to inactivate the enzyme, cooled, and then an equal volume of methanol was added to terminate the reaction. The supernatant was centrifuged, filtered through a membrane, and UDP-Rha was detected by HPLC. The results are as follows. Figure 7 As shown.

[0081] It can be concluded that when the reaction time is 6 hours, the maximum amount of UDP-Rha generated in the reaction system is 0.987 mM.

[0082] In summary, the optimal conditions for constructing the UDP-Rha cycling system using the two-enzyme method are as follows: At SuSy 0.02mg / mL At RHM-NRS 0.16 mg / mL, UDP added at a concentration of 1.5 mM, NAD + The concentration of added sugar was 0.7 mM, the sucrose concentration was 400 mM, and the pH of the phosphate buffer solution was 7.5. The reaction conditions were 30℃ and the reaction time was 6 h.

[0083] Example 5: This example illustrates the optimization of the three-enzyme (sucrose synthase, UDP-rhamnosyl synthase, and glycosyltransferase) coupled catalytic synthesis system for quercetin-3-O-rhamnoside.

[0084] According to the optimized conditions in Example 4, firstly, a two-enzyme coupling system ( At SuSy 0.02 mg / mL At RHM-NRS 0.16mg / mL, 1.5mM UDP, 0.7mM NAD + The reaction was carried out at 30°C for 6 hours with 400 mM sucrose and phosphate buffer solution (pH 7.5) to generate a certain amount of UDP-Rha sugar donor in the reaction system. Then, crude rhamnosyltransferase enzyme solution, quercetin substrate and a certain amount of DMSO were added to carry out in vitro glycosylation reaction to catalyze the synthesis of quercetin-3-O-rhamnoside.

[0085] The HPLC method for detecting quercetin-3-O-rhamnoside was as follows: Instrument: Dionex P680 high performance liquid chromatograph; Analytical column: Kromasil C18 column (250 mm × 4.6 mm, 3 μm); Detection wavelength: 368 nm; Injection volume: 20 µL; Detection temperature: 30 ℃; Mobile phase: 55% methanol aqueous solution; Flow rate: 0.6 mL / min.

[0086] First, the effect of the addition of the three enzymes (sucrose synthase, UDP-rhamnose synthase, and glycosyltransferase) on the synthesis of quercetin-3-O-rhamnoside was investigated. Following the method in Example 3, crude enzyme solutions of glycosyltransferase UGT78D2-D1 PSPG and its mutants were obtained, and the protein concentration of the crude enzyme solution was determined using the BCA method. The amount of glycosyltransferase added ranged from 0.02 mg / mL to 0.16 mg / mL. The amount of quercetin-3-O-rhamnoside produced was detected by HPLC, and its conversion rate was calculated. The results are as follows: Figure 8 As shown.

[0087] Secondly, the effect of DMSO addition on the synthesis of quercetin-3-O-rhamnoside was investigated. The DMSO addition amount was 5-30% (V:V). HPLC was used to detect quercetin-3- O - The amount of rhamnoside produced was used to calculate its conversion rate. Results are as follows: Figure 9 As shown.

[0088] Finally, the effect of substrate quercetin concentration on the synthesis of quercetin-3-O-rhamnoside was investigated. The concentration of quercetin added ranged from 0.5 mM to 3 mM. HPLC was used to detect quercetin-3-O-rhamnoside. O - The amount of rhamnoside produced was used to calculate its conversion rate. Results are as follows: Figure 10 As shown:

[0089] In summary, the optimized conditions for the three-enzyme coupled catalytic synthesis of quercetin-3-O-rhamnoside are as follows:

[0090] The optimal ratio of the three enzymes is: At SuSy 0.02mg / mL At RHM-NRS 0.16 mg / mL, glycosyltransferase 0.12 mg / mL, i.e., the ratio of the three enzymes added to the reaction system is 1:8:6; the optimal concentration of UDP is 1.5 mM; NAD + The optimal addition concentration was 0.7 mM; the optimal addition ratio of DMSO was 10%; the optimal addition ratio of the substrate quercetin was 1.5 mM; the optimal reaction temperature was 30℃; and the optimal reaction pH was 7.5.

[0091] Example 6: This example illustrates the sugar donor selectivity of glycosyltransferase UGT78D2-D1 PSPG and its mutants.

[0092] The three-enzyme reaction was catalyzed according to the method in Example 5. After the reaction was completed, the enzymes were inactivated by boiling, cooled, and then an equal volume of methanol was added to terminate the reaction. The supernatant was collected by centrifugation and passed through a membrane. HPLC analysis was performed to calculate the conversion and regioselectivity.

[0093] Glycosylation conversion rate = [Total product peak area / (Total product peak area + Substrate quercetin peak area)] × 100%

[0094] Glycosylation regioselectivity = (Peak area of ​​corresponding product / Peak area of ​​total product) × 100%

[0095] Transformation efficiency and regioselectivity of different mutants, such as Figure 11-12 As shown.

[0096] like Figure 13 As shown, under these conditions, the peak retention time of quercetin is 14.8 min. Glycosyltransferases are known. At UGT78D1 specifically accepts UDP-Rha as a sugar donor, catalyzing the 3-OH hydroxyl position of quercetin to synthesize quercetin-3-O-rhamnoside. Under these HPLC conditions, the peak retention time of quercetin-3-O-rhamnoside is 10.54 min. Point mutations were performed on the glycosyltransferase mutant UGT78D2-D1 PSPG with a PSPG domain substitution. The catalytic results of glycosyltransferase UGT78D2-D1 PSPG and its mutant are shown in the table below, and the catalytic HPLC chromatogram is shown in the figure below. Figure 14 As shown.

[0097] The results showed that mutants of the glycosyltransferase UGT78D2-D1 PSPG, namely M350T / I377F, M350T / N381Q, N353F / G374C, N353F / I377F, N353F / L378F, or N353F / N381Q, exhibited good ability to synthesize quercetin-3-O-rhamnoside. This indicates that the mutants accept UDP-Rha as a sugar donor with strong selectivity and good catalytic activity. By coupling glycosyltransferase with sucrose synthase and rhamnosyl synthase, a one-step direct preparation of the product from sucrose can be achieved without the need for the addition of high-energy activating sugars, simplifying the preparation process, significantly reducing production costs, and demonstrating broad application prospects in the biopharmaceutical industry.

Claims

1. A glycosyltransferase mutant, characterized in that, by replacing the 44 amino acids of the C-terminal PSPG domain of UGT78D2 with a glycosyltransferase At UGT78D2 amino acid sequence C-terminal PSPG domain At UGT78D1 C-terminal PSPG domain of 44 amino acids, obtaining a domain engineered glycosyltransferase At UGT78D2-D1 PSPG; The glycosyltransferase At The amino acid sequence of UGT78D2-D1PSPG is set forth in SEQ ID NO: 2; A glycosyltransferase At UGT78D2-D1PSPG in which the asparagine at position 353 is mutated to phenylalanine and the glycine at position 374 is mutated to cysteine, resulting in the glycosyltransferase mutant N353F / G374C; or, the glycosyltransferase At UGT78D2-D1PSPG in which the asparagine at position 353 is mutated to phenylalanine and the isoleucine at position 377 is mutated to phenylalanine, resulting in the glycosyltransferase mutant N353F / I377F; or, the glycosyltransferase At UGT78D2-D1PSPG in which the asparagine at position 353 is mutated to phenylalanine and the leucine at position 378 is mutated to phenylalanine, resulting in the glycosyltransferase mutant N353F / L378F; or, a glycosyltransferase At UGT78D2-D1PSPG in which the asparagine at position 353 is mutated to phenylalanine and the asparagine at position 381 is mutated to glutamine, resulting in the glycosyltransferase mutant N353F / N381Q.

2. Use of the mutant of claim 1 in the preparation of quercetin-3-O-rhamnoside.

3. Use according to claim 2, characterized in that, Quercetin-3-O-glucuronide was synthesized by three-enzyme coupling catalytic reaction system with sucrose as substrate O -rhamnoside product; Three-enzyme coupled catalytic synthesis of quercetin-3-O-glucoside O - The system of rhamnose glycosyltransferase includes: said sugar transferase mutant, sucrose synthase from Arabidopsis thaliana At SuSy, UDP-rhamnose synthase At RHM-NRS, sucrose, uridine diphosphate disodium UDP, nicotinamide adenine dinucleotide NAD + , dimethyl sulfoxide DMSO, quercetin and phosphate buffer.

4. Use according to claim 3, characterized in that, Sucrose synthase in a three-enzyme coupled catalytic reaction system At The amount of SuSy used is 0.01 mg / mL-0.12 mg / mL, and the mass ratio of the three enzymes is sucrose synthase:UDP-rhamnose synthase: glycosyltransferase mutant = 1:8:2~8.

5. Use according to claim 3, characterized in that, The concentration of UDP added in the three-enzyme coupled catalytic reaction system is 0.2 mM -3 mM; NAD + The concentration added is 0.1 mM-0.9mM.

6. Use according to claim 5, characterized in that, The concentration of UDP added in the three-enzyme coupled catalytic reaction system was 1.5 mM; NAD + added at a concentration of 0.7 mM.

7. Use according to claim 3, characterized in that, The concentration of sucrose added in the three-enzyme coupling catalytic reaction system is 100 mM-600 mM.

8. Use according to claim 7, characterized in that, The concentration of sucrose added in the three-enzyme coupling catalytic reaction system is 400 mM.

9. Use according to claim 3, characterized in that, The concentration of quercetin added in the three-enzyme coupling catalytic reaction system is 0.5 mM-3 mM; the concentration of dimethyl sulfoxide is 5%-30 % V / V.

10. Use according to claim 3, characterized in that, The concentration of quercetin added in the three-enzyme coupling catalytic reaction system is 1.5 mM.

11. Use according to claim 3, characterized in that, The reaction temperature of the three-enzyme coupling catalytic reaction system is 20℃-40℃; the pH of the buffer of the reaction solution is 6.0-9.

0.

12. Use according to claim 11, characterized in that, The reaction temperature of the three-enzyme coupling catalytic reaction system is 30℃; the pH of the buffer of the reaction solution is 7.5.

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