Use of glycosyltransferase mutants in the preparation of active ginsenosides

Abstract

The application discloses application of a glycosyltransferase mutant in preparation of active ginsenoside, and the mutant is obtained by mutating the 12th residue in the amino acid sequence of glycosyltransferase Yjic into another amino acid residue; the amino acid sequence of the glycosyltransferase Yjic is shown in SEQ ID NO: 2. With the glycosyltransferase mutant constructed in the application and a sucrose synthase AtSuSy double-enzyme coupling catalytic system, active ginsenoside F12 can be directly prepared in one step with protopanaxadiol as a substrate, the substrate conversion rate is high, the product is relatively single, the subsequent separation step is reduced, and the product purification process is simplified. Ginsenoside F12 has multiple medicinal activities such as inhibiting tumor cells and improving immunity, and therefore has great prospects in development of new drugs.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to a glycosyltransferase Yjic mutant and its application in the preparation of non-natural ginsenosides. Background Technology

[0002] Ginseng (Panax ginseng CA, Mey.) is a precious traditional Chinese medicine and one of the important medicinal herbs used in my country for a long time. The *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) states that ginseng has effects such as tonifying qi, calming the mind, stopping palpitations, and improving eyesight. Modern scientific research shows that ginseng has good auxiliary effects in many aspects, including enhancing immunity, anti-tumor activity, improving cardiovascular health, anti-oxidation, anti-aging, anti-Alzheimer's disease, lowering blood sugar, and whitening the skin, and is hailed as the "King of Herbs." With a few exceptions, ginsenosides in nature generally have a similar basic structure, consisting of a saturated 1,2-cyclopentahydrophenanthrene (sterane or sterane) steroid nucleus. Based on the aglycone skeleton, they are divided into two categories: oleanane-type pentacyclic triterpenoid saponins and dammarane-type tetracyclic triterpenoid saponins. Dammarane-type ginsenosides are more common and have stronger activity. Based on their aglycones, they are divided into two categories: 20(S)-protopanaxadiol (PPD) saponins and 20(S)-protopanaxatriol (PPT) saponins. Common PPD-type ginsenosides include ginsenosides CK, Rb1, Rb2, Rh2, Rd, and Rg3, while common PPT-type ginsenosides include ginsenosides F1, Rh1, Rg1, Rg2, and Re.

[0003] These similar yet different ginsenosides exhibit a wide variety of medicinal activities based on their different parent nuclei, glycosyl groups, glycosylation sites, and degrees of glycosylation modification. Studies have shown that some non-natural ginsenosides have significantly stronger inhibitory effects on lung cancer cells than natural ginsenosides such as Rg3 and Rh2 (Atopkina LN, et al; Planta Medica, 1999, 65(1):30-34). For example, by glycosylating the non-natural glycosylation sites (C-3, C-12) of protopanaxadiol, the synthesized non-natural ginsenoside F12 (3-O-β-D-glucopyranosyl-12-O-β-D-glucopyranosyl-20(S)-protopanaxadiol) possesses unique physiological and pharmacological activities, especially in the preparation of antitumor drugs. It has significant inhibitory effects on colon cancer cells, liver cancer cells, lung cancer cells, and gastric cancer cells, and has even more potential medicinal value.

[0004] Glycosyltransferases specifically transfer active glycosides from glycosyl donors to glycosyl acceptors. They are involved in the synthesis and metabolism of various carbohydrates and glycosides in organisms. Common activated sugar forms are nucleoside diphosphates, including UDP-glucose (uridine diphosphate glucose, UDPG), UDP-galactose, UDP-rhamnose, UDP-xylose, UDP-glucuronic acid, etc. Based on protein sequence, glycosyltransferases can be classified into 96 families, with most families involved in the synthesis of natural glycoside products belonging to family 1. Currently, the folding patterns of glycosyltransferases are mainly categorized into GT-A and GT-B types. GT-A glycosyltransferases consist of two compact Rossmann domains, while GT-B glycosyltransferases consist of two relatively independent domains. Furthermore, the transition state of GT-A glycosyltransferases requires the assistance of divalent metal ions for substrate binding, while the catalytic process of GT-B glycosyltransferases does not require the assistance of divalent metal ions. Summary of the Invention

[0005] The purpose of this invention is to provide a Yjic mutant of glycosyltransferase derived from Bacillus subtilis and its application in the preparation of non-natural ginsenosides.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A glycosyltransferase Yjic mutant, wherein the mutant is based on the amino acid sequence of glycosyltransferase Yjic as shown in SEQ ID NO.2, and the mutation is to mutate the 12th proline (P) in the amino acid sequence of glycosyltransferase Yjic to another amino acid residue.

[0008] The glycosyltransferase Yjic described in this invention can be isolated from Bacillus subtilis, isolated from the expression transformant body that recombinantly expresses the protein, or synthesized artificially.

[0009] Furthermore, the amino acid residues are selected from alanine (A), glycine (G), serine (S), asparagine (N), or tryptophan (W).

[0010] Furthermore, the mutation is P12A.

[0011] An expression gene that encodes a mutant of the aforementioned glycosyltransferase Yjic.

[0012] A recombinant plasmid containing the aforementioned expressed gene.

[0013] Application of the above-mentioned Yjic mutant glycosyltransferase in the preparation of non-natural ginsenosides.

[0014] Furthermore, the non-natural ginsenoside is 3-O-β-D-glucopyranosyl-12-O-β-D-glucopyranosyl-20(S)protopanaxadiol.

[0015] A method for preparing non-natural ginsenosides using the above-mentioned glycosyltransferase Yjic mutant, wherein the method utilizes a dual-enzyme coupled catalytic system to synthesize non-natural ginsenosides;

[0016] The dual-enzyme coupled catalytic system includes a glycosyltransferase Yjic mutant, sucrose synthase AtSuSy, protopanaxadiol, sucrose, and uridine diphosphate disodium (UDP).

[0017] In a preferred embodiment, the amount of the glycosyltransferase Yjic mutant in the dual-enzyme coupled catalytic system is 40 mU / mL-320 mU / mL, preferably 40 mU / mL; the amount of sucrose synthase AtSuSy is 10 mU / mL-60 mU / mL, preferably 20 mU / mL.

[0018] In a preferred embodiment, the concentration of protopanaxadiol in the dual-enzyme coupled catalytic system is 0.1 mM-30 mM, preferably 6 mM; the concentration of sucrose is 50 mM-2000 mM, preferably 400 mM; and the concentration of uridine diphosphate disodium is 0.2 mM-1.6 mM, preferably 1.2 mM.

[0019] In a preferred embodiment, dimethyl methacrylate (DMSO) and Tween 80 are also added to the dual-enzyme coupled catalytic system; the concentration of DMSO is 0-20% (V / V), preferably 10% (V / V); and the concentration of Tween 80 is 0-5% (V / V), preferably 2%.

[0020] In a preferred embodiment, the reaction temperature of the dual-enzyme coupled catalytic system is 20–45°C, preferably 35°C; the initial pH is 6.5–10.5, preferably 8.0.

[0021] This invention utilizes the Yjic gene of glycosyltransferase derived from Bacillus subtilis. Through semi-rational enzyme engineering techniques such as alanine scanning, saturation mutagenesis, and iterative saturation mutagenesis, the Yjic gene is mutated to enable one-step targeted synthesis of the non-natural ginsenoside 3-O-β-D-Glc-12-O-β-D-Glc-20(S)PPD using protopanaxadiol (PPD) as a substrate. Simultaneously, the glycosyltransferase mutant coupled with the sucrose synthase AtSuSy dual-enzyme catalytic system described in this invention, using protopanaxadiol as a substrate, can achieve one-step direct targeted synthesis of the non-natural ginsenoside F12 with high substrate conversion rate and a relatively singular product. This helps reduce subsequent separation steps and streamlines the product purification process, thus laying the foundation for the pharmaceutical application of the non-natural ginsenoside. Attached Figure Description

[0022] Figure 1 This is an SDS-PAGE electrophoresis diagram of the glycosyltransferase Yjic and its fusion expression.

[0023] Figure 2 This is a schematic diagram of the reaction for the synthesis of 3-O-β-D-Glc-12-O-β-D-Glc-20(S)PPD by coupling glycosyltransferase Yjic or its mutant with sucrose synthase AtSuSy.

[0024] Figure 3 SDS-PAGE analysis of sucrose synthase AtSuSy expression.

[0025] Figure 4 The effect of Tween 80 concentration on the activity of the two-enzyme system.

[0026] Figure 5 The method for one-pot feeding synthesis of ginsenoside F12.

[0027] Figure 6 This is an HPLC chromatogram of the product from the PPD glycosylation reaction catalyzed by the glycosyltransferase mutant P12A. Detailed Implementation

[0028] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] Example 1

[0032] 1. Extraction of glycosyltransferase Yjic plasmid

[0033] The recombinant glycosyltransferase strain (pET-28a-YjiC) was inoculated into liquid LB medium containing kanamycin and cultured at 37°C and 180 rpm for 10–12 h in a shaker. Plasmids were extracted according to the instructions of the "AxyPrep Plasmid DNA Mini-Extraction Kit". 2 μL of the plasmid sample was analyzed by agarose gel electrophoresis; the presence of a clearly visible bright band indicated its suitability for subsequent PCR amplification. The nucleotide sequence of the glycosyltransferase Yjic is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

[0034] 2. Site-directed mutagenesis of glycosyltransferase Yjic

[0035] A three-dimensional structural model of the glycosyltransferase Yjic was established using the Swiss-model online server. Hotspot scanning prediction was performed using the hotspotwizard online software. Amino acid residues surrounding the predicted active site were mutated to alanine. The mutated sites in Yjic to alanine were P12A, L128A, I11A, E83A, P64A, and G294A. Mutants of Yjic were induced to produce mutants. Non-natural ginsenosides were synthesized using wild-type glycosyltransferase Yjic or its mutants. High-performance liquid chromatography (HPLC) was used to detect the products, and the conversion rate and product selectivity of the substrate catalyzed by the mutants were calculated. Some sites with decreased activity, such as I11, E83, P64, and G294, were removed. Based on the wild-type glycosyltransferase YjiC, mutations were performed on P12 and L128 to improve the regioselectivity of the mutant ginsenoside F12. Considering both transformation rate and selectivity, the mutant P12A, which showed improved transformation rate and selectivity compared to the wild type, was selected for saturation mutation.

[0036] The sites in the saturation mutation sequence of glycosyltransferase Yjic that show improved substrate conversion and selectivity were selected as candidate sites for iterative mutation. Proline (P) at position 12 in the Yjic sequence was mutated to another amino acid residue, selected from alanine (A), glycine (G), serine (S), asparagine (N), and tryptophan (W).

[0037] The specific method is as follows:

[0038] Primers were designed using Agilent's online primer design software QuikChange Primer Design, and plasmids containing Bacillus subtilis-derived glycosyltransferase Yjic or its mutants were used as templates for site-directed mutagenesis via whole-plasmid PCR.

[0039] Table 1 Primers for several mutants

[0040]

[0041] Table 2. PCR system for amplification of the mutant plasmid

[0042]

[0043] Table 3. PCR reaction system for amplification of the mutant plasmid

[0044]

[0045] A 2 μL sample was subjected to agarose gel electrophoresis. The presence of a bright band with a bp of approximately 6500 confirmed that the whole plasmid PCR amplification was successful.

[0046] Template digestion: The template plasmid was removed by digestion with Quick Cut Dpn I digestive enzyme at 37℃ for 30 min.

[0047] Table 4 Quick-cut Dpn I digestive system

[0048]

[0049] The digested PCR products were transformed into competent E. coli BL21(DE3) cells using the heat shock method. After transformation, the cells were plated on Kana-resistant solid LB medium and incubated at 37°C for 12–16 h. Well-grown colonies were then selected for colony PCR verification. The polymerase used in colony PCR was Novizan P112-02 Taq Master Mix (Dye Plus).

[0050] Table 5 Colony PCR Reaction System

[0051]

[0052] Table 6 Colony PCR Reaction Conditions

[0053]

[0054] Two μL of each colony PCR product was subjected to agarose gel electrophoresis, and positive strains were sequenced at Anhui General Biotechnology Co., Ltd. Strains with the correct mutations were inoculated into LB medium containing kanamycin and cultured at 37°C and 180 rpm for 10–12 h on a shaker. Then, 800 μL of fresh bacterial culture was transferred to cryovials and stored at -80°C.

[0055] 3. Induced expression of glycosyltransferase Yjic and its mutants

[0056] The mutants obtained above were inoculated into LB liquid medium containing kanamycin resistance (Kana) and cultured on a constant temperature shaker at 37°C and 180 rpm for 10-12 hours to obtain seed culture. Fresh bacterial culture was then transferred at an inoculation rate of 2% (v / v) into fresh LB liquid medium containing kanamycin (Kana) and cultured on a constant temperature shaker at 37°C and 180 rpm for 1.5 hours (dose of bacterial culture was 0.05%). 600 Approximately 0.6), then add IPTG to a final concentration of 0.1 mmol / L, and induce expression at 20℃ and 180 rpm for 10-12 h.

[0057] The induced fermentation broth was collected in centrifuge bottles and centrifuged at 12000 rpm for 20 min in a benchtop centrifuge. After centrifugation and discarding the supernatant, the cells were resuspended in Tris-HCl (pH 8.0) buffer. The cells were then sonicated in an ice bath throughout the process. After sonication, the cells were centrifuged at 12000 rpm for 10 min. A suitable amount of supernatant was used for SDS-PAGE to verify protein expression. The collected supernatant was used as the crude enzyme solution for subsequent experiments.

[0058] 4. Induced expression of sucrose synthase strains

[0059] The sucrose synthase strain (Escherichia coli BL21 containing plasmid pET28a-AtSuSy, Genbank sequence number: AK316826) on the plate was inoculated into LB liquid medium containing kanamycin resistance (Kana) and placed in a constant temperature shaker at 37°C and 180 rpm for 10-12 h. Then, fresh bacterial culture was transferred at a 2% inoculation rate to LB liquid medium containing kanamycin resistance (Kana) and placed in a constant temperature shaker at 37°C and 180 rpm for about 1.5 h (the OD600 of the bacterial culture was about 0.6). Then, IPTG was added to a final concentration of 0.1 mmol / L, and expression was induced at 16°C and 180 rpm for 12-13 h.

[0060] The induced fermentation broth was collected in centrifuge flasks and centrifuged at 12000 rpm for 20 min in a benchtop centrifuge. After centrifugation and discarding the supernatant, the cells were resuspended in Tris-HCl (pH 8.0) buffer. The cells were then sonicated in an ice bath throughout the process. After sonication, the cells were centrifuged at 12000 rpm for 10 min. A suitable amount of supernatant was subjected to polyacrylamide gel electrophoresis (SDS-PAGE) to verify protein expression. The collected supernatant was used as the crude enzyme solution for subsequent experiments.

[0061] 5. Isolation and purification of Yjic mutant glycosyltransferase and sucrose synthase protein

[0062] The Ni column packing material was packed into the chromatography column and compacted by gravity. It was then rinsed with EDTA-deionized water-nickel sulfate solution-deionized water to remove contaminating proteins and re-attach nickel ions. The column was then rinsed with buffer A at a flow rate of 1.0 mL / min until the baseline of 254 nm UV absorbance stabilized. The crude enzyme solution was injected into the column at a flow rate of 0.5 mL / min and rinsed with buffer A at a flow rate of 1.0 mL / min until the baseline of 254 nm UV absorbance stabilized, removing proteins that could not adhere to the nickel column. The column was then rinsed with A:B (94:6) buffer at a flow rate of 1.0 mL / min until the baseline stabilized again, at which point the weakly binding proteins on the nickel column were largely removed. The column was then rinsed with A:B (80:20) buffer at a flow rate of 1.0 mL / min, and the collected eluent contained the target protein. The collected eluent was transferred multiple times into a 10 kDa ultrafiltration tube and concentrated by centrifugation. After centrifuging all the eluent, add Tris-HCl buffer (pH 8.0) and centrifuge again, repeating this process several times. Remove any residual imidazole from the solution before aliquoting. After purification, store the purification instrument and packing material in 20% ethanol.

[0063] Buffer solutions A and B are prepared as follows:

[0064] Buffer A: 10mM Na2HPO4·12H2O, 1.8mM KH2PO4, 140mM NaCl, 2.7mM KCl, bring to volume with deionized water, and adjust pH to 8.0 with NaOH.

[0065] Buffer B: 50mM Na2HPO4·2H2O, 300mM NaCl, 500mM imidazole, deionized water to volume, pH adjusted to 8.0 with NaOH.

[0066] 6. Determination of catalytic activity and regioselectivity of mutants

[0067] The effect of mutant glycosylation progenitor ginsenoside diol (PPD) on the reaction was determined by in vitro enzymatic reactions, which were divided into two types: detection using a single enzyme system with UDPG (uridine diphosphate glucose) as a glycosyl donor and detection using a glycosyltransferase coupled with sucrose synthase.

[0068] The single-enzyme reaction system (400 μL) included: 6 mM PPD, 18 mM UDPG, 20 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 10% DMSO, 1% (v / v) Tween 80, and 40 μL of crude glycosyltransferase solution. The reaction was carried out in a constant-temperature shaker at 35 °C and 200 rpm for 4 h or 12 h, and then the reaction was terminated by adding an equal volume (400 μL) of n-butanol and shaking thoroughly.

[0069] The two-enzyme reaction system (400 μL) included: 6 mM PPD, 1.2 mM UDP (uridine diphosphate), 20 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 10% DMSO, 1% (v / v) Tween 80, 400 mM sucrose, 40 μL crude glycosyltransferase solution, and 20 μL crude sucrose synthase solution. The reaction was carried out in a constant temperature shaker at 35 °C and 200 rpm for 4 h or 12 h, and then the reaction was terminated by adding an equal volume (400 μL) of n-butanol and shaking thoroughly.

[0070] 7. Optimization program for the reaction conditions catalyzed by glycosyltransferase Yjic

[0071] (1) Pretreatment of liquid phase samples

[0072] After adding an equal volume of n-butanol to the reaction system and shaking thoroughly, substances such as protopanaxadiol (PPD) and ginsenoside F12 in the reaction were extracted by n-butanol. The mixture was then centrifuged at 20,000 g for 5 min, and the supernatant was collected, filtered through a 0.22 μm organic microporous membrane, and directly analyzed by high-performance liquid chromatography (HPLC).

[0073] (2) HPLC detection and analysis procedure:

[0074] Instrument: Dionex P680 high performance liquid chromatograph; Analytical column: Acclaim 120 C18 column (150mm×4.6mm, 5μm); Detection wavelength: 203nm; Injection volume: 20 μL; Detection temperature: 30℃; Mobile phase: methanol-water = 85:15 (v / v), 1mL / min.

[0075] The results showed that the Yjic mutant glycosyltransferase could catalyze the glycosylation reaction of protopanaxadiol. Analysis of the product using LC-MS and NMR confirmed that the Yjic mutant could synthesize not only ginsenoside Rh2 but also non-natural ginsenoside F12 (3-O-β-D-Glc-12-O-β-D-Glc-20(S)PPD). Under the above detection conditions, the peak elution times of ginsenoside F12 were 10-13 min, ginsenoside Rh2 were 18-20 min, and PPD was 35-37 min.

[0076] The glycosyltransferase Yjic was purified using Ni-NTA metal chelate column chromatography. Based on this, the catalytic reaction conditions of glycosyltransferase Yjic were further studied, laying the foundation for its efficient synthesis in non-natural ginsenosides.

[0077] 8. Application of the Yjic mutant glycosyltransferase and sucrose synthase in the dual-enzyme catalytic synthesis of non-natural ginsenosides.

[0078] This reaction requires sucrose synthase AtSuSy. Based on GenBank accession number AED92895.1, the full-length AtSuSy gene was synthesized and ligated into the cloning vector pET-28a. The ligation product was directly transformed into *E. coli* BL21(DE3) competent cells, and single colonies were selected for PCR verification. The correctly sequenced strain was fermented, and the resulting crude sucrose synthase AtSuSy enzyme solution was obtained after centrifugation. SDS-PAGE analysis of expression was performed, and the results are shown below. Figure 3 As shown.

[0079] The effect of Tween 80 concentration on the two-enzyme system:

[0080] The effect of Tween 80 concentration on substrate solubility and catalytic PPD glycosylation in a two-enzyme system was investigated. The results showed that the optimal molar ratio of UDP to PPD in the two-enzyme coupled reaction system was 0.2. The two-enzyme reaction system was used to investigate the effect of different Tween 80 concentrations (v / v) (0%, 0.5%, 1%, 2%, 3%, 4%, 5%) on the glycosylation reaction at PPD concentrations of 3 mM, 6 mM, and 12 mM. The system also included a UDP to PPD molar ratio of 0.2, 20 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 10% DMSO, 400 mM sucrose, 45 mU / mL crude enzyme solution of mutant P12A, and 60 mU / mL crude enzyme solution of sucrose synthase. The reaction conditions were 35℃ and 200 rpm, and the reaction was terminated by adding an equal amount of n-butanol after 12 h. The results are as follows: Figure 4 As shown.

[0081] The effect of one-pot feeding on the two-enzyme system:

[0082] To efficiently produce ginsenoside F12, this invention establishes a dual-enzyme coupled feed system (UGT-SuSy cascade reaction), which efficiently and directionally produces ginsenoside F12 by utilizing the regioselectivity of the P12A mutant for ginsenoside F12 synthesis.

[0083] The initial reaction system (10 mL) contained: 4 mM PPD, 0.8 mM UDP, 20 mM Tris-HCl (pH 8.0), 400 mM sucrose, 2.4% DMSO, 2% (v / v) Tween 80, crude enzyme solution of 45 mU / mL glycosyltransferase mutant, and crude enzyme solution of 60 mU / mL sucrose synthase. The reaction was carried out at 35 °C and 200 rpm. 50 μL of the reaction sample was taken out every hour and an equal volume of n-butanol was added to terminate the reaction. Subsequent extraction and liquid chromatography analysis were then performed.

[0084] The feeding method was as follows: PPD substrate was dissolved in DMSO to prepare a PPD stock solution with a concentration of 180 mM. Since the glycosylation rate of PPD was relatively fast in the early stages, 1 mM PPD (55.6 μl of PPD stock solution) was added at 1 h and 2 h of reaction. At 4 h, 6 h, 8 h, and 10 h of reaction, 2 mM PPD (111.2 μl of PPD stock solution) was added. At 12 h of reaction, 4 mM PPD (222.4 μl of PPD stock solution) was added. The final DMSO concentration was approximately 10%.

[0085] The enzyme supplementation method is as follows: Enzyme activity gradually decreases as the reaction proceeds. To improve reaction efficiency, enzyme supplementation is necessary midway through the reaction. Fresh crude enzyme solution (45 mU / mL mutant and 60 mU / mL sucrose synthase) is added at 6 h and 12 h of reaction. The mutant SNF is used for the synthesis of ginsenoside Rh2, and the mutant P12A is used for the synthesis of ginsenoside F12.

[0086] A one-pot fed-batch process was used to produce ginsenoside F12. In the first hour of the reaction, PPD converted from the mutant P12A generated 0.74 mM ginsenoside F12 and 0.89 mM ginsenoside Rh2. Ginsenoside Rh2 is a precursor of ginsenoside F12, therefore its concentration initially exceeded that of ginsenoside F12. In subsequent reactions, the concentration of ginsenoside Rh2 increased slightly with each addition of PPD, due to the increased concentration of the substrate PPD. The concentration of ginsenoside Rh2 eventually stabilized throughout the reaction, while ginsenoside F12 accumulated continuously. After 28 hours of continuous feeding, the final DMSO concentration was approximately 10%, resulting in the synthesis of 17.3 mM (13.58 g / L) ginsenoside F12 and 0.61 mM ginsenoside Rh2. The total conversion rate of PPD reached 99.5%, ginsenoside F12 accounted for 96.6% of the total glycoside products, and the UDPG cycle regeneration number (RC value) was approximately 44.4. The results are as follows... Figure 5 As shown.

[0087] The dual-enzyme reaction system (400 μL) included: 6 mM PPD, 1.2 mM UDP, 20 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 10% DMSO, 1% (v / v) Tween 80, 400 mM sucrose, 40 μL crude glycosyltransferase solution, and 20 μL crude sucrose synthase solution. The reaction was carried out in a constant-temperature shaker at 35 °C and 200 rpm for 4 h or 12 h, and then the reaction was terminated by adding an equal volume (400 μL) of n-butanol and shaking thoroughly. After membrane treatment, the target product was detected by high-performance liquid chromatography (HPLC).

[0088] Table 7. Overview of the synthesis of ginsenoside F12

[0089]

[0090] The results indicate that, using protopanaxadiol (PPD) as a substrate, the mutant of glycosyltransferase Yjic exhibits a good ability to synthesize non-natural ginsenosides. Among them, mutant P12A showed the best synthesis effect. A highly efficient, directed catalytic two-enzyme system for the glycosylation of PPD to ginsenoside F12 was successfully constructed, as shown in Table 7. Utilizing the high activity and regioselectivity of mutant P12A, 17.3 mM (13.58 g / L) ginsenoside F12 and 0.61 mM ginsenoside Rh2 were efficiently synthesized using PPD as a raw material. The total conversion rate of substrate PPD reached 99.5%, and ginsenoside F12 accounted for 96.6% of the total glycoside products. The UDPG cycle regeneration number (RC value) was approximately 44. Among the currently published literature, the yield of F12 synthesized in this study, its proportion in the total glycoside products, and the total conversion rate are all at a high level. This is also the first time that the yield of non-natural ginsenoside F12 has exceeded 10 g / L.

[0091] This indicates that the mutant can specifically produce the disaccharidated product F12(3-O-β-D-Glc-12-O-β-D-Glc-20(S)PPD) with high selectivity. Utilizing a two-enzyme coupling reaction consisting of a glycosyltransferase mutant and sucrose synthase, a one-step direct preparation of the non-natural ginsenoside F12 product can be achieved, simplifying the preparation process and significantly reducing production costs, thus showing broad application prospects in the biopharmaceutical industry.

Claims

1. A glycosyltransferase Yjic mutant, characterized in that, The mutant is a mutant of the glycosyltransferase Yjic amino acid sequence shown as SEQ ID NO. 2, and the mutation is that the proline at position 12 in the glycosyltransferase Yjic amino acid sequence is mutated to alanine.

2. An expression gene, characterized in that, The gene encodes the glycosyltransferase Yjic mutant of claim 1.

3. A recombinant plasmid, characterized in that, The recombinant plasmid contains the expression gene of claim 2.

4. Use of the glycosyltransferase Yjic mutant according to claim 1 for the preparation of non-natural ginsenosides, characterized in that, The non-natural ginsenoside is 3-O-β-D-glucopyranosyl-12-O-β-D-glucopyranosyl-20(S)-protopanaxadiol.

5. A method for preparing unnatural ginsenosides using the glycosyltransferase Yjic mutant of claim 1, characterized in that, The method utilizes a double-enzyme coupling catalytic system to synthesize a non-natural ginsenoside; the non-natural ginsenoside is 3-O-β-D-glucopyranosyl-12-O-β-D-glucopyranosyl-20(S)-protopanaxadiol; and the double-enzyme coupling catalytic system comprises the glycosyltransferase Yjic mutant of claim 1, sucrose synthase AtSuSy, protopanaxadiol, sucrose, and uridine diphosphate UDP.

6. The method of claim 5, wherein, The amount of the glycosyltransferase Yjic mutant in the double-enzyme coupling catalytic system is 40 mU / mL-320 mU / mL, and the amount of sucrose synthase AtSuSy is 10 mU / mL-60 mU / mL.

7. The method of claim 5, wherein, DMSO and Tween 80 are also added to the double-enzyme coupling catalytic system, the concentration of DMSO is 10% V / V, and the concentration of Tween 80 is 2% V / V.

8. The method of claim 5, wherein, The reaction temperature of the double-enzyme coupling catalytic system is 20-45°C, and the initial pH is 6.5-10.5.

Images