Glycosyltransferase mutants and their application in the enzymatic preparation of rare ginsenosides

Abstract

This invention relates to glycosyltransferase mutants and their application in the enzymatic preparation of rare ginsenosides. (The text then abruptly shifts to a discussion of glycosyltransferase UGT.) BL 1. Site-directed mutagenesis was performed, and the resulting mutants can be used to efficiently and directionally prepare rare ginsenoside Rh1. The regioselectivity of the enzymatic preparation of rare ginsenoside Rh1 reached 96.26% (conversion rate 95.56%). Glycosyltransferase UGT BL 1. Some other mutants can also be used to directionally synthesize non-natural ginsenoside protopanaxadiol-3- O -β-glucan (3- O -β-Glc-PPT), enzymatic preparation of non-natural ginsenosides 3- O The regioselectivity of -β-Glc-PPT reached 84.83% (conversion rate 98.13%). This invention utilizes the glycosyltransferase UGT. BL The mutant of 1 and sucrose synthase At The SuSy dual-enzyme coupled catalytic system was used to prepare Rh1 or 3- O -β-Glc-PPT, and significantly increased the levels of rare ginsenosides Rh1 and 3- by using a fed-batch method. O The product concentration level of -β-Glc-PPT simplifies the preparation process; the product is relatively homogeneous, which helps reduce subsequent separation steps and streamlines the product purification process. 3- O -β-Glc-PPT can be used to prepare drugs for the treatment of diseases such as tumors, and has broad prospects for new drug development.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to glycosyltransferase mutants and their application in the enzymatic preparation of rare ginsenosides. Background Technology

[0002] Ginseng (Panax ginseng CA Mayer) is a perennial herb belonging to the Araliaceae family and is a traditional and precious Chinese medicinal herb. Ginseng possesses a wide range of pharmacological activities, including anti-cancer, anti-fatigue, immune-boosting, and cardiovascular-protective effects. The main medicinal component of ginseng—ginsenosides—is a class of tetracyclic triterpenoid compounds. To date, 180 ginsenosides have been identified from 17 species of Panax ginseng, most of which have a tetracyclic dammarane structure. Dammarane-type ginsenosides can be further classified into types such as protopanaxadiol (PPD) and protopanaxtriol (PPT). In plants, the glycosylation sites of PPT are usually the hydroxyl groups at C-6 and C-20, forming ginsenosides Re, Rf, Rg1, Rg2, Rh1, F1, etc. The products obtained after glycosylation modification at the C-3 position of PPT are called non-natural ginsenosides. The structural formulas of rare ginsenoside Rh1 (formula (1)) and non-natural ginsenoside 3-O-β-Glc-PPT (formula (2)) are as follows:

[0003]

[0004] Ginsenosides are a class of triterpenoid compounds and the main medicinal components of ginseng. PPT-type ginsenosides generally possess anti-cancer, anti-stress, and antioxidant effects, and can also be used as adjunctive treatments for diabetes, cardiovascular diseases, immune disorders, and especially neurological diseases. The position and number of glycosyl linkages in PPT-type ginsenoside compounds significantly affect their biological activity. For example, ginsenoside Re can inhibit the central nervous system and promote DNA and RNA synthesis. Ginsenoside Rh1 (6-O-β-Glc-PPT), the C6-OH glycosylated product of PPT, exhibits diverse pharmacological activities. Studies have shown that compared to PPD-type ginsenosides Rh2 and Rg3, ginsenoside Rh1 has excellent activity against neurodegenerative diseases, enhancing neuronal activity, improving behavioral disorders caused by brain injury, significantly inhibiting the proliferation and metabolism of various cancer cells, and regulating the immune system. Therefore, the targeted glycosylation synthesis of PPT-type ginsenoside monomers is of great significance for new drug development.

[0005] Existing methods for obtaining ginsenosides mainly involve plant extraction, plant tissue culture, and chemical synthesis. However, the content of ginsenosides in ginseng and American ginseng is low. The content of some rare ginsenosides that are easily absorbed and exhibit high biological activity (such as ginsenosides Rg3, Rh1, Rh2, and CK) is extremely low, only about one ten-thousandth, making direct extraction very difficult. Furthermore, the content of ginsenosides in artificially cultured plants is often low (at most about one-third that of wild ginseng), making it difficult to meet the growing domestic and international market demand for ginsenosides. For complex natural products, chemical modification methods have drawbacks such as low yield, numerous byproducts, and environmental pollution. To address the problem of scarce medicinal resources, domestic and international research has focused on enzymatic preparation of ginsenosides. Glycosyltransferase-catalyzed glycosylation of ginsenoside aglycones to synthesize ginsenoside products is the final key step in the entire biosynthetic pathway and is crucial for the diversity of biosynthesized ginsenoside products. Researchers have explored relevant synthetic elements in the cDNA database of 13 Ginseng species, selecting open reading frames (ORFs) >1320 bp containing the PSPG motif (a conserved C-terminal sequence of plant secondary metabolite glycosyltransferases used to bind high-energy sugar donors). More than 80 novel UDP-glycosyltransferases (UGTs) were successfully cloned and expressed from mRNA prepared from ginseng callus tissue using RT-PCR. These UGTs were then introduced into yeast chassis cells for functional characterization analysis, identifying five UGTs involved in the synthesis of rare ginsenosides CK, Rh2, Rg3, Rh1, and F1. Based on this, the synthetic pathways of rare ginsenosides CK, F1, Rh1, Rh2, and Rg3 monomers were introduced into yeast chassis cells, successfully achieving one-step de novo synthesis of multiple rare ginsenoside monomers from glucose (yields reaching 2.4 × 10⁻⁶). -4 g / L, 0.042g / L, 0.093g / L, 0.017g / L, 0.049g / L).

[0006] Existing reports indicate that several microbial UGTs, exhibiting only low similarity to plant-derived UGTs, can also catalyze the synthesis of several ginsenoside products. Some microbial glycosyltransferases show promising potential in the biochemical preparation of rare ginsenoside products. For example, BSGT1 from Bacillus subtilis catalyzes the C-3 position of ginsenoside F1, synthesizing the rare ginsenoside Ia (0.20 g / L). UGT109A1 from Bacillus subtilis can glycosylate the C3-OH and C12-OH of PPD and PPT, generating non-natural ginsenoside products.

[0007] Most glycosyltransferases have "substrate generality," making it difficult to use their glycosylation reactions for targeted biosynthesis of specific ginsenoside products. The Bs-YjiC glycosyltransferase derived from Bacillus subtilis can catalyze the synthesis of two monosaccharide-containing products: ginsenoside Rh1 and 3-β-O-Glc-PPT. This enzyme can further glycosylate ginsenoside Rh1 or 3-β-O-Glc-PPT as substrates to obtain four non-natural PPT-type ginsenosides (all disaccharide-containing products), indicating that the selectivity of natural glycosyltransferases in the biosynthesis of non-natural ginsenoside products is unsatisfactory (Sun et al., CN 109796516 A; Dai LH et al, J. Agric. Food Chem. 2018, 66, 943-949). Limited by the selectivity of the enzyme's own catalytic reaction, it is difficult to directionally prepare monosaccharide-containing products of PPT or their single products. Furthermore, it only catalyzes the synthesis of trace amounts of the product with low selectivity in the glycosylation region, making it difficult to obtain high-purity rare ginsenoside Rh1 products.

[0008] Glycosyltransferases require high-energy activated sugars as glycosyl donors to catalyze reactions that synthesize corresponding glycoside products. To achieve high substrate glycosylation conversion rates, an excess of activated sugar donors is often required; however, high-energy activated sugars are expensive, resulting in high reaction costs and hindering large-scale application. In vitro, a glycosyltransferase-sucrose synthase-coupled catalytic system is constructed by introducing sucrose synthase. Using uridine diphosphate disodium (UDP) and sucrose as substrates, sucrose synthase regenerates UDP-G (uridine diphosphate glucose) in the reaction system. UDP-G is then used by the glycosyltransferase as a glycosyl donor to catalyze glycosylation reactions, simultaneously releasing UDP. Therefore, the dual-enzyme coupled catalytic reaction not only achieves the cyclic regeneration of UDP-G, but the minimal amount of UDP required in the recycling system also eliminates the inhibition of glycosyltransferase activity caused by the large amount of byproduct UDP generated during glycosyltransferase catalysis, thus enabling low-cost and efficient synthesis of glycoside products. Rare ginsenosides have broad application prospects in the pharmaceutical field, and there is a strong demand for high-purity rare ginsenoside products in pharmaceutical research and development. Therefore, the selective preparation of rare ginsenosides has significant implications for pharmaceutical development. Summary of the Invention

[0009] The primary objective of this invention is to provide a glycosyltransferase mutant. This glycosyltransferase mutant exhibits good regional selectivity, stability, high expression levels, and higher enzyme activity. It can selectively and directionally catalyze the glycosylation modification of the C-3 or C-6 hydroxyl groups using protopanaxadiol (PPT) as a substrate, thereby obtaining higher concentrations of rare ginsenoside Rh1 and non-natural ginsenoside 3-O-β-Glc-PPT.

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

[0011] A glycosyltransferase mutant, which introduces the glycosyltransferase UGT BL The amino acid sequence 1 is obtained by mutating one or more amino acid residues at positions 62, 66, 320, and 321 to another amino acid residue.

[0012] Or glycosyltransferase UGT BL The amino acid sequence of 1 is obtained by performing a four-site combination mutation with an elastic mutation site at amino acid residues at positions 62, 320, and 321, and the other elastic mutation sites are at position 64 or 170.

[0013] The glycosyltransferase UGT BL The amino acid sequence of 1 is shown in SEQ ID NO: 2;

[0014] The glycosyltransferase UGT BL The 62nd amino acid residue of 1 is mutated to any one of tyrosine, histidine, arginine, cysteine, valine, proline, lysine, alanine, or glutamic acid.

[0015] The 66th amino acid residue is mutated to any one of histidine, arginine, valine, cysteine, leucine, phenylalanine, glycine, serine, or tyrosine.

[0016] The 320th amino acid residue is mutated to any one of isoleucine, tyrosine, arginine, tryptophan, leucine, histidine, or glutamine.

[0017] The 321st amino acid residue is mutated to any one of glycine, isoleucine, tyrosine, arginine, leucine, histidine, or cysteine.

[0018] A second objective of this invention is to provide a recombinant vector, expression cassette, or recombinant bacteria containing the gene encoding the glycosyltransferase mutant. The recombinant bacteria are obtained by inserting the glycosyltransferase mutant gene into pET-28a and transforming it into *Escherichia coli* E. coli-BL21(DE3), or by using various conventional vectors containing the gene and various conventional host strains containing the recombinant vector.

[0019] A third objective of this invention is to provide the application of the above-mentioned mutant in the enzymatic synthesis of rare ginsenoside Rh1. Preferably, the mutant is a single mutation at position 62, 320, or 321; a combination mutation at positions 62 and 321; a combination mutation at positions 62, 320, and 321; or a combination mutation at positions 62, 320, 321, and 64 / 170.

[0020] A single mutation at position 62 will reduce the glycosyltransferase UGT BL The isoleucine (I) at position 62 of enzyme 1 is mutated to any one of histidine (H), tyrosine (Y), arginine (R), glutamic acid (E), cysteine ​​(C), valine (V), alanine (A), or proline (P) to obtain the corresponding mutant. Preferred enzyme mutants are I62H, I62Y, I62R, and I62E.

[0021] A single mutation at position 320 will reduce the glycosyltransferase UGT BL The methionine (M) at position 320 of enzyme 1 is mutated to any one of glycine (G), isoleucine (I), arginine (R), tyrosine (Y), leucine (L), or histidine (H) to obtain the corresponding mutant. Preferred enzyme mutants are M320Y, M320R, M320L, and M320H.

[0022] A single mutation at position 321 will reduce the glycosyltransferase UGT BL The proline (P) at position 321 of enzyme 1 is mutated to any one of the following: tyrosine (Y), valine (V), arginine (R), aspartic acid (D), isoleucine (I), glutamine (Q), tryptophan (W), phenylalanine (F), methionine (M), or alanine (A), to obtain the corresponding mutant. Preferred enzyme mutants are P321W, P321I, P321Y, and P321F.

[0023] When the two sites at positions 62 and 321 are mutated in combination, preferably, the glycosyltransferase UGT is mutated. BL The isoleucine (I) at position 62 of enzyme 1 is mutated to any one of histidine (H), tyrosine (Y), arginine (R), or glutamic acid (E), while the proline (P) at position 321 is mutated to any one of tryptophan (W), isoleucine (I), tyrosine (Y), or phenylalanine (F), to obtain the corresponding mutant. Preferably, the enzyme mutants are I62H / P321I, I62H / P321Y, I62R / P321Y, or I62E / P321I.

[0024] When mutating the three sites at positions 62, 320, and 321 in combination, preferably, the glycosyltransferase UGT is mutated. BLThe isoleucine (I) at position 62 is mutated to any one of histidine (H), arginine (R), or glutamic acid (E); the proline (P) at position 321 is mutated to any one of tyrosine (Y) or isoleucine (I); and the methionine (M) at position 320 is mutated to any one of tyrosine (Y), arginine (R), leucine (L), or histidine (H); thus obtaining the corresponding mutants. Preferred mutants are I62R / M320H / P321Y, I62R / M320Y / P321Y, I62H / M320H / P321Y, and I62E / M320Y / P321I.

[0025] When combining mutations at four sites, the preferred method is to mutate the glycosyltransferase UGT. BL The following mutations were performed: isoleucine (I) at position 62 was mutated to arginine (R); methionine (M) at position 320 was mutated to histidine (H); proline (P) at position 321 was mutated to tyrosine (Y); alanine (A) at position 63, 142, 150, or 168 was mutated; or proline (P) at position 64 was mutated to any one of the other 19 amino acids; or asparagine at position 170 was mutated to any one of the other 19 amino acids; and the corresponding mutants were obtained. The preferred mutants are I62R / M320H / P321Y / P64A, I62R / M320H / P321Y / P64W, I62R / M320H / P321Y / P64D, I62R / M320H / P321Y / P64G, I62R / M320H / P321Y / P64C, I62R / M320H / P321Y / P64R, I62R / M320H / P321Y / N170A, I62R / M320H / P321Y / N170Y, I62R / M320H / P321Y / N170P, and I62R / M320H / P321Y / N170E.

[0026] As a preferred embodiment, the non-natural ginsenosides are synthesized using a dual-enzyme coupled catalysis system; the dual-enzyme coupled catalysis system includes the glycosyltransferase mutant, Arabidopsis-derived sucrose synthase AtSuSy, protopanaxadiol, sucrose, and uridine diphosphate disodium (UDP).

[0027] In a preferred embodiment, the amount of glycosyltransferase in the dual-enzyme coupled catalytic system is 40 mU / mL–160 mU / mL; the amount of sucrose synthase is 50 mU / mL–200 mU / mL; the concentration of protopanaxadiol is 0.1 mM–0.8 mM; the concentration of added sucrose is 100 mM–800 mM; and the concentration of uridine diphosphate is 0.1 mM–12 mM.

[0028] 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 0-50% (V / V), preferably 10% (V / V); and the concentration of Tween 80 is <5% (V / V), preferably 2% (V / V).

[0029] In a preferred embodiment, the reaction temperature of the dual-enzyme coupled catalytic system is 20–50°C, preferably 30°C; the initial pH is 5.0–10.0, preferably 7.5.

[0030] A fourth objective of this invention is to provide the application of the above-mentioned mutant in the enzymatic synthesis of non-natural ginsenoside 3-O-β-Glc-PPT. The mutant is a single-site mutation at position 66, 320, or 321, or a combination mutation at positions 66 and 320.

[0031] When a single site mutation occurs, the preferred enzyme mutants are Q66A, M320W, and M320Q.

[0032] When the two sites at positions 66 and 320 are mutated in combination, preferably, the glycosyltransferase UGT is mutated. BL The methionine (M) at position 320 of 1 is mutated to tryptophan (W), and the glutamine (Q) at position 66 is mutated to any one of histidine (H), arginine (R), valine (V), cysteine ​​(C), leucine (L), phenylalanine (F), glycine (G), serine (S), and tyrosine (Y). Preferably, the mutated position is histidine (H), phenylalanine (F), or glycine (G).

[0033] As a preferred embodiment, the non-natural ginsenosides are synthesized using a dual-enzyme coupled catalysis system; the dual-enzyme coupled catalysis system includes the glycosyltransferase mutant, Arabidopsis-derived sucrose synthase AtSuSy, protopanaxadiol, sucrose, and uridine diphosphate disodium (UDP).

[0034] In a preferred embodiment, the amount of glycosyltransferase in the dual-enzyme coupled catalytic system is 40 mU / mL–160 mU / mL; the amount of sucrose synthase is 50 mU / mL–200 mU / mL; the concentration of protopanaxadiol is 0.1 mM–0.8 mM; the concentration of added sucrose is 100 mM–800 mM; and the concentration of uridine diphosphate is 0.1 mM–12 mM.

[0035] 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 0-50% (V / V), preferably 10% (V / V); and the concentration of Tween 80 is <5% (V / V), preferably 2% (V / V).

[0036] In a preferred embodiment, the reaction temperature of the dual-enzyme coupled catalytic system is 20–50°C, preferably 30°C; the initial pH is 5.0–10.0, preferably 7.5.

[0037] This invention also provides a method for separating and preparing non-natural ginsenoside 3-O-β-Glc-PPT, comprising removing proteins from the reaction solution after the catalytic reaction, adsorbing the mixture of 3-O-β-Glc-PPT, PPT and other substances in the reaction solution using silica gel particle column packing, eluting with dichloromethane:methanol = 8.5:1.5 solvent, and then rotary evaporating or freeze-drying the collected eluent to obtain a high-purity non-natural ginsenoside 3-O-β-Glc-PPT product in powder or crystalline form.

[0038] Another object of the present invention is to provide the application of the above-mentioned non-natural ginsenoside 3-O-β-Glc-PPT in the preparation of tumor-inhibiting drugs. The tumor is preferably melanoma.

[0039] This invention utilizes the glycosyltransferase UGT of Bacillus licheniformis ZSP01. BL Starting with gene 1, we explored the effects of site-directed saturation mutagenesis and multi-site combined mutagenesis on the glycosyltransferase UGT. BL 1. Mutation modification yielded several effective mutants capable of efficiently and directionally synthesizing rare ginsenoside Rh1 and non-natural ginsenoside 3-O-β-Glc-PPT products using protopanaxadiol (PPT) as a substrate. These mutants exhibit advantages such as high reaction selectivity, high reactivity, good stability, and a simple preparation method. The dual-enzyme coupled catalysis combined with batch-feed substrate addition method described in this invention for preparing rare ginsenoside Rh1 and non-natural ginsenoside 3-O-β-Glc-PPT products can significantly improve production efficiency and product concentration levels. This invention utilizes the glycosyltransferase UGT. BL The mutant is coupled with sucrose synthase, using inexpensive sucrose as a glycosyl donor. This not only significantly reduces preparation costs but also substantially reduces the inhibitory effect of excessively high substrate concentrations on enzyme activity, thereby increasing the cumulative concentration of the target product in the reaction system. The dual-enzyme catalyzed glycosylation reaction exhibits high regioselectivity and a relatively singular product, which helps reduce subsequent separation steps and simplify the product purification process. Furthermore, the non-natural ginsenoside 3-O-β-Glc-PPT has a certain inhibitory effect on melanoma proliferation, making it a promising new drug. Attached Figure Description

[0040] Figure 1 SDS-PAGE analysis of the expression and purification of glycosyltransferase UGTBL1 and its mutant proteins.

[0041] Figure 2 Schematic diagram of the reaction between the glycosyltransferase UGTBL1 mutant and the rare ginsenoside Rh1.

[0042] Figure 3 HPLC analysis of rare ginsenoside Rh1 synthesized by glycosyltransferase UGTBL1 mutant.

[0043] Figure 4 The effects of saturation mutations at positions 62, 320, and 321 of glycosyltransferase UGTBL1 on the conversion rate and selectivity of rare ginsenoside Rh1 synthesis.

[0044] Figure 5 The effects of combined mutations at positions 62 and 321, and at position 320, of the glycosyltransferase UGTBL1 on the conversion efficiency and regioselectivity of the synthesis of rare ginsenoside Rh1.

[0045] Figure 6 The effects of the mutant I62R / M320H / P321Y of glycosyltransferase UGTBL1 and the combined mutations at positions 64 and 170 on the conversion rate and regioselectivity of rare ginsenoside Rh1.

[0046] Figure 7 A schematic diagram of the synthesis of non-natural ginsenoside 3-O-β-Glc-PPT by the glycosyltransferase UGTBL1 mutant.

[0047] Figure 8 HPLC analysis of the synthesis of non-natural ginsenoside 3-O-β-Glc-PPT catalyzed by mutant M320W.

[0048] Figure 9 Mass spectrometry analysis of the non-natural ginsenoside 3-O-β-Glc-PPT product.

[0049] Figure 10 NMR chromatogram of non-natural ginsenoside 3-O-β-Glc-PPT product.

[0050] Figure 11 glycosyltransferase UGT BL Schematic diagram of the reaction for the synthesis of rare ginsenoside Rh1 by the dual-enzyme coupling of mutant-sugar synthase AtSuSy.

[0051] Figure 12 Effects of temperature on enzyme activity (A) and stability (B) of WT and mutant I62R / M320H / P321Y / N170A.

[0052] Figure 13 Effects of pH on enzyme activity (A) and stability (B) of WT and mutant I62R / M320H / P321Y / N170A.

[0053] Figure 14 Effect of DMSO concentration on the synthesis of 3-O-β-Glc-20(S)-PPT catalyzed by M320W.

[0054] Figure 15 Rare ginsenoside Rh1 and non-natural ginsenosides were synthesized by a combination of dual-enzyme catalysis and batch-fed reaction.

[0055] Figure 16 Cell survival rate of B16F10 cells after 24 h of treatment with different concentrations of 3-O-β-Glc-PPT. Detailed Implementation

[0056] Example 1: This example illustrates the cloning, expression, and mutation procedures for glycosyltransferases.

[0057] Using the gene for the target glycosyltransferase from the Bacillus licheniformis genome information in the NCBI database as a template, cloning primers were designed. Using the extracted Bacillus licheniformis ZSP01 genome as a template, the target glycosyltransferase UGT was obtained by PCR amplification using the corresponding cloning primers. BL The gene fragment of 1. The target glycosyltransferase UGT obtained by PCR amplification. BL 1. After verification by agarose gel electrophoresis, the target gene fragment was purified and recovered according to the method of the AxyPrep DNA gel recovery kit and stored at -20°C for a short period of time. Glycosyltransferase UGT BL The nucleotide sequence of 1 is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

[0058] Glycosyltransferase UGT BL Perform site-directed saturation mutagenesis on one of the following sites in 1:

[0059] The 62nd, 320th, and 321st positions are mutated to any one of the other 19 amino acids.

[0060] Glycosyltransferase UGT BL Combination mutations were performed on the following two, three, and four sites in section 1:

[0061] 1) Mutate the methionine at position 320 to tryptophan (W) and the glutamine at position 66 to any one of histidine, arginine, valine, cysteine, leucine, phenylalanine, glycine, serine, or tyrosine, performing a two-site combination mutation.

[0062] 2) Mutate isoleucine at position 62 to any one of histidine, tyrosine, arginine, or glutamic acid, and simultaneously mutate proline at position 321 to any one of tryptophan, isoleucine, tyrosine, or phenylalanine, performing a two-site combination mutation.

[0063] 3) Mutate isoleucine at position 62 to any one of histidine, tyrosine, arginine, or glutamic acid, and mutate proline at position 321 to any one of tryptophan, isoleucine, tyrosine, or phenylalanine, and mutate methionine at position 320 to any one of tyrosine, arginine, leucine, or histidine, performing a three-site combination mutation.

[0064] 4) Mutate isoleucine at position 62 to arginine, methionine at position 320 to histidine, and proline at position 321 to tyrosine. Simultaneously, mutate positions 63, 142, 150, or 168 to alanine, performing a combination mutation at four sites.

[0065] 5) Mutate isoleucine at position 62 to arginine, methionine at position 320 to histidine, and proline at position 321 to tyrosine. At the same time, mutate proline at position 64 to any one of the other 19 amino acids. Perform a combination mutation at four sites.

[0066] 6) Mutate isoleucine at position 62 to arginine, methionine at position 320 to histidine, and proline at position 321 to tyrosine. Simultaneously, mutate asparagine at position 170 to any one of the other 19 amino acids; perform a combination mutation at four sites.

[0067] Mutant primers were designed using Primer Premier 5.0 software to encode the glycosyltransferase UGT. BL The gene sequence of 1 (SEQ ID NO: 1) was used as a template, and then PCR mutation was performed.

[0068] Table 1 shows the base sequences of some of the mutation primers.

[0069]

[0070] PCR reaction system:

[0071]

[0072] PCR cycle process:

[0073] 1. Preheat: 94℃, 2 min;

[0074] 2. Denaturation: 94℃, 15s;

[0075] 3. Annealing: 55℃, 30s;

[0076] 4. Extension: 68℃, 6 minutes;

[0077] Repeat cycle 2-4 25 times

[0078] The PCR products were recovered, and agarose gel electrophoresis was used to determine whether site-directed mutagenesis had been completed. Then, Dpn I (1 μL) was added to a 50 μL system, and the mixture was incubated at 37 °C for 3 h to digest the template. The mixture was then cooled to 4 °C and stored.

[0079] The mutated plasmid was transformed into E. coli BL21(DE3) competent cells. Positive recombinants were screened on kanamycin-containing resistant plates. Single clones were selected, and positive clones were verified by colony PCR. After DNA sequencing verification, the corresponding mutants were obtained.

[0080] Example 2: This example illustrates the glycosyltransferase UGT BL 1. Induction of mutant expression and protein purification.

[0081] 1. Glycosyltransferase UGT BL 1. Inducible expression of mutants:

[0082] (1) The recombinant transformants obtained in Example 1 were inoculated into LB medium containing 50 mg / L kanamycin (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 6.5) and cultured at 37°C with shaking for 12 h to obtain seed culture.

[0083] (2) Inoculate 1% (v / v) into a 250mL Erlenmeyer flask containing 40mL of LB medium and incubate at 37℃ and 180rpm on a shaker. When the OD of the culture medium... 600 When the concentration reaches 0.6, lactose with a final concentration of 20 g / L is added as an inducer, and the mixture is induced and cultured at 20°C for 24 h.

[0084] (3) Centrifuge the culture medium, collect the cells, and wash them twice with an equal volume of physiological saline to obtain resting cells. Resuspend the obtained resting cells in an equal volume of buffer solution (pH 7.5), sonicate them in an ice bath, centrifuge and collect the supernatant, which is the crude enzyme solution.

[0085] 2. Glycosyltransferase UGT BL Purification procedure for mutant 1

[0086] Protein purification using Ni + Column purification: The collected wild-type glycosyltransferase UGT BL1. The crude enzyme solution of the mutant was filtered and added to a Ni Sepharose 6 Fast Flow (FF) packing material. Impurities were eluted with 10 column volumes of buffer A (10 mM Tris-HCl, pH 8.0, 250 mM NaCl, 5 mM imidazole). The target protein was then eluted with a gradient of buffer B (10 mM Tris-HCl, pH 8.0, 250 mM NaCl, 100 mM imidazole). The collected target proteins were subjected to SDS-PAGE protein electrophoresis. Some results (I62R / M320H / P321Y / N170A, etc.) are shown below. Figure 1 As shown.

[0087] Example 3: This experiment demonstrates the effects of glycosyltransferase UGT BL 1. Application of mutants in the synthesis of rare ginsenoside Rh1.

[0088] The technical approach adopted in this embodiment is as follows: Figure 2 As shown.

[0089] Reaction system: Phosphate buffer (pH 7.5) was used as the reaction medium. The total reaction volume was 200 μL, containing 1 mM PPT, 5 mM UDPG, 2% (v / v) Tween-80, 5% (v / v) DMSO, and glycosyltransferase mutant enzyme solution. The reaction was carried out at 30℃ and 200 rpm for 8 h. The reaction was terminated by adding methanol to the sample. HPLC was used for detection.

[0090] HPLC detection and analysis procedure:

[0091] Instrument: Thermo U3000 high performance liquid chromatograph; Analytical column: Thermo Scientific C18 column (150mm×4.6mm, 5μm); Detection wavelength: 203nm; Injection volume: 20 μL; Detection temperature: 30℃; Mobile phase: methanol-water = 65:35 (v / v), 1mL / min.

[0092] The results show ( Figure 3 ): Glycosyltransferase UGT BL The mutant (I62R / M320H / P321Y / N170A as an example) exhibits a good ability to synthesize rare ginsenoside Rh1. The glucose group of UDPG is transferred to the C6-OH of PPT to obtain the product, rare ginsenoside Rh1. Analysis of the product using LC-MS and NMR confirmed that it is indeed rare ginsenoside Rh1.

[0093] The results show ( Figure 4 Compared to wild-type glycosyltransferase UGT BL1 (Regioselectivity of ginsenoside Rh1 synthesis was 10.98%, conversion rate was 48.14%), glycosyltransferase UGT BL Of the 19 mutants obtained from the saturation mutation at position 62, 8 mutants significantly improved both the conversion rate and regioselectivity of rare ginsenoside Rh1 synthesis. The four best mutants, I62H, I62Y, I62R, and I62E, increased the conversion rate to 77.48%-87.05%, and simultaneously improved the regioselectivity of rare ginsenoside Rh1 synthesis to 40.43%-62.49%, with the glycosyltransferase UGT showing the best results. BL The conversion rate and regioselectivity of the mutant I62H for synthesizing rare ginsenoside Rh1 were 80.30% and 62.49%, respectively.

[0094] glycosyltransferase UGT BL Nineteen mutants were obtained from the saturation mutation at position 321. Ten of these mutants achieved a conversion rate of over 80% for the synthesis of rare ginsenoside Rh1. The four best mutants, P321W, P321I, P321Y, and P321F, achieved conversion rates ranging from 91.11% to 98.53%. Among them, mutant P321W achieved a conversion rate of 98.53% and a regioselectivity of 27.74% for the synthesis of rare ginsenoside Rh1.

[0095] glycosyltransferase UGT BL Of the 19 mutants obtained from the saturation mutation at position 320, 12 mutants showed improved regioselectivity for synthesizing rare ginsenoside Rh1, with 5 showing improved conversion rates. The four best mutants, M320Y, M320R, M320L, and M320H, showed improved conversion rates ranging from 52.64% to 85.13% and improved regioselectivity for synthesizing rare ginsenoside Rh1 ranging from 33.07% to 62.99%. Mutant M320Y showed improved conversion rates and regioselectivity for synthesizing rare ginsenoside Rh1 to 85.13% and 33.07%, respectively, while mutant M320R showed corresponding improvements to 65.78% and 62.99%, respectively.

[0096] The results show ( Figure 5 ): Glycosyltransferase UGT BL Mutants at position 62 (I62H, I62Y, I62R, I62E) and positions 321 (P321W, P321I, P321Y, P321F) were combined to produce 12 mutants with a conversion rate exceeding 90% for rare ginsenoside Rh1, and 6 of these mutants exhibited regioselectivity exceeding 50%. The I62H / P321I mutant achieved a conversion rate of 98.71% and a regioselectivity of 69.56% for rare ginsenoside Rh1 synthesis. (Glycosyltransferase UGT) BLMutants I62H / P321I, I62H / P321Y, I62R / P321Y, and I62E / P321I were combined with four mutants M320Y, M320R, M320L, and M320H at site 320 for combined mutations. All combined mutants showed a regioselectivity >60% for the synthesis of rare ginsenoside Rh1, with eight mutants showing >80%. The best mutant, I62R / M320H / P321Y, achieved a conversion rate of 96.90% and a regioselectivity of 87.31% for the synthesis of rare ginsenoside Rh1.

[0097] The results show ( Figure 6 ): Glycosyltransferase UGT BL Five mutants of the 1 mutant I62R / M320H / P321Y, combined with mutants at positions 64 / 170, showed regioselectivity >90% for synthesizing the rare ginsenoside Rh1. Among these, the mutants I62R / M320H / P321Y / P64A and I62R / M320H / P321Y / N170A exhibited increased conversion rates of 99.15% and 95.56%, respectively, with regioselectivity increasing to 92.27% and 96.25%, respectively. (Glycosyltransferase UGT) BL The mutant I62R / M320H / P321Y and the saturation mutation at position 64 yielded most mutants with regioselectivity exceeding 80% for the synthesis of rare ginsenoside Rh1. The optimal mutant was I62R / M320H / P321Y / P64G, achieving a conversion rate of 98.53% and a regioselectivity of 92.91%. Glycosyltransferase UGT BL The mutant I62R / M320H / P321Y and the saturation mutation at position 170 yielded most mutants with regioselectivity exceeding 80% for the synthesis of rare ginsenoside Rh1.

[0098] Example 4: This example illustrates the glycosyltransferase UGT BL 1. Application of mutants in the synthesis of non-natural ginsenoside 3-O-β-Glc-PPT.

[0099] The technical solution adopted in this embodiment is as follows: Figure 7 As shown. Glycosyltransferase transfers the glucose group of UDPG to the C3-OH of PPT, yielding the product non-natural ginsenoside 3-O-β-Glc-PPT.

[0100] The reaction system is the same as in Example 3.

[0101] Table 2 Glycosyltransferases (UGT) BL 1. Effects of mutants on the conversion rate and selectivity of non-natural ginsenoside 3-O-β-Glc-PPT synthesis

[0102]

[0103] The results showed (Table 2): glycosyltransferase UGT BL Mutations at sites 320 and 321 of enzyme 1 yielded mutants with excellent ability to synthesize the non-natural ginsenoside 3-O-β-Glc-PPT. Combined mutations of enzyme mutants M320W and Q66 showed that the regioselectivity of mutants M320W / Q66F and M320W / Q66G could be increased to 94.80% and 94.54%, respectively. Glycosyltransferase UGT BL The mutant M320W of 1 Figure 8 The variety of products was significantly reduced, with the main product being non-natural ginsenoside 3-O-β-Glc-PPT, which had a PPT conversion rate of 98.13%, while the regioselectivity of 3-O-β-Glc-20(S)-PPT reached 84.83%.

[0104] Example 5: This example illustrates the preparation, purification, and identification of non-natural ginsenoside 3-O-β-Glc-PPT.

[0105] Reaction system: A pH 7.5 phosphate buffer solution was used as the reaction medium. The total reaction volume was 50 mL, containing 5 mM PPT, 25 mM UDPG, 2% Tween-80, 5% (v / v) DMSO, and an appropriate amount of glycosyltransferase mutant enzyme solution. The reaction was carried out at 30℃, pH 7.5, and a rotation speed of 200 rpm for 24 h, followed by extraction with an equal volume of n-butanol. The organic phase was collected and rotary evaporated until completely eliminated. A small amount of methanol was used to dissolve a mixed sample containing non-natural ginsenoside 3-O-β-Glc-20(S)-PPT and substrate PPT. The sample was obtained by rotary evaporation and a powder was obtained. The sample was weighed and approximately 2.5–3.0 times its volume of 80–100 mesh silica gel was added. This powder was then added to the methanol sample solution and stirred in a water bath until homogeneous. The methanol was then completely evaporated.

[0106] Seal one end of the cylindrical glass column with degreased cotton, then pack the silica gel (separating gel) into the column. Take 20 times the weight of the sample in 300-400 mesh silica gel as the separating gel, mix it with an appropriate amount of dichloromethane, and slowly pour it into the glass column using a funnel. Vacuum the column from below to compact it, then add the prepared sample gel on top. Finally, place a thin layer of degreased cotton on the sample gel, ready for elution.

[0107] Impurities and less polar substrates in the silica gel were eluted with 100% dichloromethane, followed by elution with a dichloromethane:methanol ratio of 8.5:1.5. The results were analyzed by TLC. The collected solvent containing non-natural ginsenoside 3-O-β-Glc-PPT was combined, the solvent was removed by rotary evaporation, and the product was vacuum dried and freeze-dried to obtain a solid sample. The purity of the product was approximately 99% as determined by HPLC.

[0108] HR-MS spectra of the above 3-O-β-Glc-PPT products ( Figure 9 In this context: m / z 661.4295 [M+Na] - , 1 H-NMR and 13 C-NMR chemical shift ( Figure 10 The characteristics of ) are as follows:

[0109] 1 H-NMR (500MHz, DMSO-d6): δ=5.10(1H,H-24),4.1(1H,J=10,H-6),3.98(1H,J=10,H-1') ,3.64(1H,m,H-6'),3.52(1H,m,H-3),3.43(1H,m,H-6'),3.36(1H,br,s,H-3'),3.11(1 H,m,H-3),3.05(1H,m,H-5'),3.03(1H,m,H-4'),2.97(1H,m,H-2'),1.91(1H,m,H-23), 1.90(1H,m,H-23)1.88(1H,m,H-17),1.87(1H,m,H-2),1.66(1H,m,H-16),1.64(1H,m,H -11),1.56(3H,s,H-27),1.53(3H,m,H-13),1.52(2H,m,H-1,2),1.48(1H,m,H-7),1.44 (1H,m,H-15),1.40(1H,m,H-22),1.38(1H,m,H-7),1.35(1H,m,H-9),1.32(3H,s,H-29) ,1.30(1H,m,H-22),1.22(1H,m,H-16),1.03(1H,m,H-11),0.98(3H,s,H-18),0.94(2H, s,H-1,15),0.85(3H,s,H-28),0.84(3H,s,H-19),0.81(3H,s,H-30),0.79(1H,m,H-5).

[0110] 13C-NMR (125MHz, DMSO-d6): δ = 130.6 (C-25), 125.9 (C-24), 106.0 (C-1'), 88.67 (C-3), 77.4 (C-3'), 77.1 (C-5'), 74.0 (C-2') ,72.5(C-20),70.2(C-4'),66.7(C-12),65.5(C-6),61.7(C-7'),60.9(C-5),54.0(C-17),51.3(C-14),49.3(C-9),47.5(C- 13),46.8(C-7),40.9(C-8),40.6(C-4),39.0(C-1),38.5(C-10),34.7(C-22),31.0(C-11),30.9(C-15),30.5(C-29),26.8( C-21),26.4(C-16),26.0(C-2),26.0(C-26),21.9(C-23),18.0(C-27),17.5(C-18),17.4(C-19),17.0(C-30),16.7(C-28).

[0111] Example 6: This example illustrates the glycosyltransferase UGT BL Application of mutant in the dual-enzyme coupled synthesis of rare ginsenoside Rh1 and non-natural ginsenoside 3-O-β-Glc-PPT using 1-molecular coupling with sucrose synthase AtSuSy

[0112] The sucrose synthase AtSuSy used in this embodiment was synthesized according to GenBank accession number AED92895.1. The full-length AtSuSy gene was synthesized and ligated into the vector pET-28a. The ligation product was directly transformed into E. coli BL21(DE3) competent cells, and single colonies were picked for PCR verification. The correctly sequenced strain was fermented and induced to express sucrose synthase AtSuSy. After centrifugation, the crude sucrose synthase AtSuSy enzyme solution was obtained.

[0113] Effects of different temperatures on glycosyltransferase UGT BL 1. Effects of mutant activity and stability ( Figure 12 The reaction system was subjected to conditions of 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, and 50℃, respectively, to determine the glycosyltransferase UGT. BL 1. Enzyme activity and stability of the mutant (1 h). The reaction system contained 1 mM protopanaxadiol (PPT), 5 mM UDP-Glc, 5% (v / v) DMSO, 1% (v / v) Tween-80, 160 mU / mL of purified mutant protein, 50 mM phosphate buffer (pH 7.5), and the reaction time was 2 h.

[0114] Different pH values ​​affect glycosyltransferase UGT BL 1. Effects of mutant activity and stability ( Figure 13 The reactivity and stability of the mutant were measured (1 h) in different pH buffer systems: 50 mM citrate buffer (pH 4.0, 4.5, 5.0, 5.5), 50 mM NaH2PO4-Na2HPO4 buffer (pH 6.0, 6.5, 7.0, 7.5, 8.0), 50 mM Tris-HCl buffer (pH 7.5, 8.0, 8.5, 8.9), and 50 mM Gly-NaOH buffer (pH 8.5, 9.0, 9.5, 10.0). The reaction system contained 1 mM protopanaxadiol (PPT), 5 mM UDP-Glc, 5% (v / v) DMSO, 1% (v / v) Tween 80, and 160 mU / mL purified mutant protein. The reaction was carried out at the optimum temperature for 2 h.

[0115] Dual-enzyme coupled catalytic reaction system ( Figure 11 The total reaction volume of 200 μL system contained 1 mM PPT, 0.5 mM UDP, 5%-20% (v / v) DMSO, 2% (v / v) Tween-80, 400 mM sucrose, 50 mM NaH2PO4-Na2HPO4 buffer (pH 7.5), and an appropriate amount of glycosyltransferase UGT was added. BL 1. The mutant was reacted with sucrose synthase AtSuSy. The reaction was carried out at 30℃ with shaking at 200 rpm for 2 h.

[0116] Effects of enzyme dosages of glycosyltransferase and sucrose synthase on the conversion rate and regioselectivity of ginsenoside synthesis:

[0117] The mutants at concentrations of 40-160 mU / mL and sucrose synthase at concentrations of 50-200 mU / mL were added to the reaction system, and the mixture was shaken at 30°C and 200 rpm for 2 h. The reaction was then terminated by adding an equal volume of methanol, and the reaction was detected by HPLC.

[0118] Table 3. Effects of the ratio of mutant I62R / M320H / P321Y / N170A-AtSuSy on the conversion rate and regioselectivity of Rh1 synthesis.

[0119]

[0120] Table 4. Effects of the ratio of mutant M320W-AtSuSy enzymes on the conversion rate and regioselectivity of 3-O-β-Glc-20(S)-PPT synthesis.

[0121]

[0122] Effects of PPT substrate concentration on the conversion rate and regioselectivity of ginsenoside synthesis catalyzed by two enzymes:

[0123] Based on the optimized dual-enzyme catalytic reaction system, substrate PPT with initial concentrations of 2, 4, 6, 8, 10, and 12 mM was added. The reaction was carried out at 30°C and 200 rpm for 2 hours with shaking. Then, an equal volume of methanol was added to terminate the reaction, and the reaction was detected by HPLC.

[0124] Table 5 Effects of PPT concentration on the conversion and regioselectivity of Rh1 synthesized from I62R / M320H / P321Y / N170A-AtSuSy

[0125]

[0126] Table 6. Effects of substrate PPT concentration on the conversion and regioselectivity of 3-O-β-Glc-20(S)-PPT synthesized from M320W-AtSuSy.

[0127]

[0128] Effects of DMSO content on the conversion rate and regioselectivity of ginsenoside products synthesized by dual-enzyme catalysis ( Figure 14 According to the optimized dual-enzyme catalytic reaction system, 5%, 10%, 15%, 20%, 30%, 40%, and 50% (v / v) DMSO were added. The reaction was carried out at 30℃ and 200 rpm for 2 hours with shaking. Then, an equal volume of methanol was added to terminate the reaction. The reaction was detected by HPLC.

[0129] Synthesis of rare (non-natural) ginsenosides via dual-enzyme catalysis combined with batch-fed reaction: The total reaction volume was 10 mL. The dual-enzyme catalytic reaction system contained: an initial PPT concentration of 12 mM, 0.5 mM UDP, 5%-20% (v / v) DMSO, 2% (v / v) Tween-80, 400 mM sucrose, and 50 mM NaH2PO4-Na2HPO4 buffer (pH 7.5), with optimized dual-enzyme addition amounts. 4 mM PPT was added at 2, 4, 6, 8, 10, and 12 h of reaction.

[0130] The results show ( Figure 15 ): Glycosyltransferase UGT BL The mutant can mainly produce rare ginsenoside Rh1, and the yield continues to increase, eventually producing 20.48 g / L of rare ginsenoside Rh1 with a conversion rate of 93.02%. The regioselectivity of the synthesized rare ginsenoside Rh1 reaches 95.74%, and the maximum number of UDPG regeneration cycles (RcMax) reaches 66.97, which is relatively easy for subsequent separation.

[0131] glycosyltransferase UGT BLThe mutant can efficiently and directionally synthesize the non-natural ginsenoside product 3-O-β-Glc-PPT to a concentration level of 18.04 g / L, with a substrate PPT conversion rate of 92.15%. The regioselectivity of the non-natural ginsenoside product 3-O-β-Glc-PPT is 85.13%, and the RcMax reaches 66.35, which facilitates subsequent separation.

[0132] Example 7: This example illustrates the inhibitory effect of product 3-O-β-Glc-PPT on tumor cell proliferation.

[0133] Cell lines and drugs: Mouse melanoma cells B16F10 were purchased from the cell bank of Shanghai Institute of Cell Biology, Chinese Academy of Sciences; natural rare ginsenoside 3-O-β-Glc-PPT was prepared by the method of this invention.

[0134] Preparation of experimental drugs and solutions:

[0135] Drug stock solution: Weigh 127.6 mg of product 3-O-β-Glc-PPT, dissolve it in 1 mL of DMSO to prepare 200 mM non-natural rare ginsenosides, filter through a 0.22 μm filter membrane, dispense 200 μL per tube, and store at -20℃. At the same time, filter high concentration DMSO through a 0.22 μm filter membrane for use as a control group.

[0136] Cell culture medium: Take DMEM medium, add 10% (v / v) fetal bovine serum and 1% penicillin-streptomycin, bring the volume to 1L, filter with a 0.22μm filter membrane for sterilization, dispense into 50mL tubes, and store at 4℃.

[0137] PBS (Phosphate Buffered Solution): Weigh 8g of NaCl, 0.2g of KCl, 1.44g of Na2PO4 and 0.24g of KH2PO4, dissolve in 1L of distilled water, adjust the pH of the solution to 7.4 with HCl, autoclave for 20 minutes, and store at 4℃.

[0138] MTT application solution: Weigh 500mg of MTT powder, dissolve it in 100mL of PBS to prepare a 5mg / mL MTT solution. After stirring and dissolving, filter through a 0.22μm filter membrane for sterilization, aliquot and store at 4℃ protected from light for later use.

[0139] Main reagents and instruments: High performance liquid chromatograph (DIONEX P680, Dionex, USA); Microplate reader (Power Wave XS, BIO-TECH, USA); CO2 incubator (Thermo Fisher Scientific, USA); Benchtop low-speed centrifuge (CenLee 4K, Hunan Xiangli Scientific Instruments Co., Ltd.); Constant temperature decolorizing shaker (DHZ 0720, Shanghai Shenneng Biotechnology Co., Ltd.); Inverted microscope (OPTIKA, Italy); Fetal Bovine Serum (Nanjing Shenghang Biotechnology Co., Ltd.); DMEM (Nanjing Shenghang Biotechnology Co., Ltd.); Penicillin-Streptomycin Solution (Nanjing Shenghang Biotechnology Co., Ltd.); Trypsin-EDTA Solution (Nanjing Shenghang Biotechnology Co., Ltd.); MTT (Shanghai Yisheng Biotechnology Co., Ltd.).

[0140] Cell culture: B16F10 cells were cultured in DMEM medium containing 10% (v / v) fetal bovine serum and 1% penicillin-streptomycin at 37°C in a 5% (v / v) CO2 incubator.

[0141] Experimental methods: Cytotoxicity assay: B16F10 cells in logarithmic growth phase were adjusted to a concentration of 2×10⁻⁶. 5 Cells were seeded per well in a 96-well plate, with 100 μL of cell suspension added to each well. Experimental groups were treated with different final concentrations (0.01, 0.1, 0.2, 0.5, 1, 2, 5, 10 mmol / L) of non-natural rare ginsenoside 3-O-β-Glc-PP, with 100 μL of the drug added to each well. A DMSO solvent control group and a blank control group were also included. Each drug concentration was used in 5 replicates. The culture plates were returned to the cell culture incubator, and after 24 hours, 20 μL of MTT (5 mg / mL) solution was added. After incubation for another 4 hours, 200 μL of DMSO was added to each well, and the plates were shaken at low speed for 10 min to completely dissolve the crystals. The absorbance was measured at 570 nm using a microplate reader. Cell viability (%) = (A570) / (200 μL / 200 μL) = 100 μL / 200 μL. 实验组 -A570 空白对照 ) / (A570 对照组- A570 空白对照 )×%.

[0142] Statistical analysis: Correlation analysis and Student T-test were performed using Microsoft Excel 2003 and GraphPad Prism 8 software. Data are presented in charts.

[0143] Table 7. Inhibitory effect of different concentrations of 3-O-β-Glc-PPT on the proliferation of B16F10 cells after 24 h of treatment.

[0144]

[0145] Note: Compared with the control group, *P<0.05, **P<0.01.

[0146] Experimental results show that ( Figure 16 Table 7 shows that the statistical results after MTT assay analysis indicate that after 24 hours of treatment with non-natural rare ginsenoside 3-O-β-Glc-PPT, the number of mouse melanoma cells was significantly reduced. At doses of 5 mmol / L and 10 mmol / L, the inhibition rates were 44.97% and 50.80%, respectively. This indicates that non-natural rare ginsenoside 3-O-β-Glc-PPT can significantly inhibit the proliferation of mouse melanoma cells, and the inhibition rate increases with increasing dose.

Claims

1. The application of glycosyltransferase mutants in the enzymatic synthesis of rare ginsenoside Rh1, characterized in that, The glycosyltransferase mutant is a glycosyltransferase UGT mutant. BL The amino acid sequence of 1 is obtained by mutating the amino acid residues at positions 62, 64, 320, and 321 to another amino acid residue. The glycosyltransferase UGT BL The amino acid sequence of 1 is shown in SEQ ID NO:

2. The glycosyltransferase UGT BL The 62nd amino acid residue of 1 is mutated to arginine; The 64th amino acid residue is mutated to either alanine or glycine; The 320th amino acid residue is mutated to histidine; The 321st amino acid residue is mutated to tyrosine.

2. The application according to claim 1, characterized in that, The non-natural ginsenosides were synthesized using a dual-enzyme coupled catalysis. The dual-enzyme coupled catalytic system includes the glycosyltransferase mutant and sucrose synthase derived from Arabidopsis thaliana. At SuSy, protopanaxadiol, sucrose, and uridine diphosphate disodium (UDP).

3. The application according to claim 2, characterized in that, In the dual-enzyme coupled catalytic system, the amount of the glycosyltransferase mutant is 40 mU / mL–160 mU / mL; the amount of sucrose synthase is 50 mU / mL–200 mU / mL; the concentration of protopanaxadiol is 0.1 mM–0.8 mM; the concentration of added sucrose is 100 mM–800 mM; and the concentration of uridine diphosphate is 0.1 mM–12 mM.

4. The application according to claim 2, characterized in that, The dual-enzyme coupled catalytic system also contains dimethyl methacrylate (DMSO) and Tween 80; the concentration of DMSO is 0-50% (V / V); and the concentration of Tween 80 is <5% (V / V).

5. The application according to claim 4, characterized in that, The concentration of DMSO added was 10% (V / V); the concentration of Tween 80 added was 2% (V / V).

6. The application according to claim 2, characterized in that, The reaction temperature of the dual-enzyme coupled catalytic system is 20~50℃; the initial pH is 5.0~10.

0.

7. The application according to claim 6, characterized in that, The reaction temperature of the dual-enzyme coupled catalytic system is 30℃; the initial pH is 7.5.

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