A glycosyltransferase, a recombinant expression vector, a preparation method and application thereof in preparing ginsenoside Rh2

Abstract

This invention relates to the field of biopharmaceutical technology, specifically disclosing a glycosyltransferase, a recombinant expression vector, a preparation method, and its application in the synthesis of ginsenoside Rh2. The glycosyltransferase is a glycosyltransferase encoded by the GE02773 gene of Bacillus subtilis SL-44, named the GE02773 protein. The nucleotide sequence encoding this protein is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. This invention utilizes the glycosyltransferase GE02773, and in the purification and application process, a magnetic material is used for one-step purification and immobilization of the enzyme. The immobilized glycosyltransferase can efficiently synthesize ginsenoside Rh2.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to a glycosyltransferase, a recombinant expression vector, a preparation method, and its application in the synthesis of ginsenoside Rh2. Background Technology

[0002] Ginseng contains abundant ginsenosides, which possess a wide range of pharmacological activities and promising prospects for new drug development. Based on their aglycone skeleton structure, ginsenosides can be divided into three subclasses: dammarane, octyltriol, and oleanane. Dammarane-type ginsenosides are the most common and can be further divided into protopanaxadiol (PPD) and protopanatriol (PPT) types. Some ginsenosides are produced through the action of intestinal microorganisms after digestion. Ginsenosides Rh2 and CK are partially deglycosylated protopanaxadiols that participate in the regulation of inflammation through IκBα phosphorylation and degradation, while ginsenosides Rg1 and Rb1 possess anti-aging activity. Ginsenosides Rg3 and Rh2 have antitumor activity, and ginsenoside Re has antioxidant and cardioprotective effects. However, the special chemical structure of natural products results in low stability and water solubility, leading to low bioavailability and limited actual human utilization effects.

[0003] Ginsenoside Rh2 (3β-O-Glc-protopanaxadiol) is a trace but distinctive pharmacological component in red ginseng, exhibiting various pharmacological activities such as anti-tumor effects, improvement of cardiac function and fibrosis, anti-inflammatory effects, antibiotic effects, and good medicinal potential. However, due to the extremely low content of ginsenoside Rh2 in ginseng and the complex extraction methods, direct extraction from the plant results in high production costs and unstable yields. Furthermore, the complex structure of Rh2 with multiple active groups requires multiple protection and deprotection processes during chemical synthesis, leading to high synthesis costs and the use of large amounts of organic reagents, increasing the burden on subsequent separation and purification costs and hindering industrial production. Currently, there are reports on constructing cell factories to produce ginsenoside Rh2, but due to the poor performance of UDP glycosyltransferase and the cytotoxicity of ginsenosides to host cells, the biosynthesis of ginsenoside Rh2 remains unfavorable. Cell engineering synthesis of Rh2 is still in the experimental stage, with low yields, complex cellular environments, and complex products, increasing the costs of subsequent classification and purification. Enzymatic synthesis of Rh2 has simple and controllable in vitro catalytic conditions, and the substances in the reaction system are relatively simple and easy to separate and purify in the later stage of the product, making it the most likely method to realize the large-scale industrial preparation of Rh2.

[0004] The preparation methods of ginsenoside Rh2 mainly include chemical and biological methods, primarily through the hydrolysis of glucose by other protopanaxadiol saponins or the condensation of protopanaxadiol and glucose. Examples include: 1) Acid hydrolysis: American ginseng is degraded in a 70℃ water bath for 1 hour under hydrochloric acid conditions to obtain ginsenoside Rh2 (R configuration); 2) Alkaline hydrolysis: PPD-type ginsenosides are hydrolyzed in NaOH solution at 100℃ for 6 hours to obtain ginsenoside Rh2 (S configuration); 3) Microbial transformation: Ginsenoside Rb1 is converted to ginsenoside Rh2 by *Biospora* at 40℃; 4) Enzymatic transformation: α-arabinosidase can hydrolyze ginsenoside Rg3 at 55℃ for 24 hours to convert it into ginsenoside Rh2. Most methods use chemical methods to prepare ginsenoside Rh2, but these methods have drawbacks such as difficulty in controlling the reaction process, high reaction requirements, numerous byproducts, and environmental pollution. Compared with chemical synthesis, enzymatic synthesis has milder reaction conditions, does not change the chemical structure, and has advantages such as substrate specificity and no pollution. It can avoid the harm caused by chemical synthesis. However, enzymatic synthesis also has some drawbacks, such as the easy inactivation of enzymes, which affects the synthesis efficiency.

[0005] To address the inherent limitations of enzyme reactions, immobilization technology has been introduced. Immobilized enzymes are products in which purified enzyme solutions are fixed in a specific space using appropriate physical and chemical methods, allowing them to retain their inherent properties while enabling continuous reaction and recycling. Immobilized enzymes are easy to recover, allowing for rapid termination of enzyme reactions and repeated use, thus reducing assay costs. Furthermore, the storage stability, pH, and heat resistance of immobilized enzymes are improved, increasing production levels and saving manpower. The flourishing of nanoscience has spurred a strong interest in particle size-dependent properties. For example, due to the advent of superparamagnetism, magnetic nanoparticles exhibit the greatest performance in the typical size range of 10-20 nm. These nanoparticles exhibit huge specific surface areas, high surface area ratios, and are easily separated and transferred with high quality under external magnetic fields, making them perfect supports for catalytic systems. Magnetic Fe3O4 chitosan supports for L-asparaginase immobilization have shown better catalytic efficiency and thermal stability; Yang et al. prepared a shallow porous microsphere support with a core-shell structure to immobilize inulinase, retaining 73% of its initial activity.

[0006] In enzyme immobilization, the adsorption capacity of enzymes on the surface depends on the activation of chemical bonds between nanoparticles and protein surfaces. Magnetite, with its greater saturation magnetization and magnetic susceptibility, is the most widely used magnetic carrier for immobilizing biomolecules / proteins. However, exposed ferromagnetic nanoparticles often exhibit high reactivity and are easily degraded under certain conditions, resulting in poor dispersibility and stability. The unique chemical structure of natural products leads to low stability and water solubility, resulting in low bioavailability and limited actual human benefits. Ginsenoside Rh2 is scarce, and its complex structure makes large-scale synthesis difficult. Limited production has become a key factor restricting the widespread application of triterpenoid saponins Rh2. Currently, most ginsenoside Rh2 preparations are achieved through chemical methods, which are difficult to control, require high reaction conditions, produce numerous byproducts, and pollute the environment. Summary of the Invention

[0007] To obtain a method for efficiently synthesizing ginsenoside Rh2, this invention provides a glycosyltransferase, a recombinant expression vector, a preparation method, and its application in the synthesis of ginsenoside Rh2. This invention utilizes the glycosyltransferase GE02773 and employs a novel one-step enzyme purification and immobilization technology during the enzyme purification process to efficiently synthesize ginsenoside Rh2.

[0008] This invention provides a glycosyltransferase, which is a glycosyltransferase encoded by the GE02773 gene of Bacillus subtilis SL-44, named GE02773 protein, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] The present invention also provides a method for preparing a glycosyltransferase, wherein the glycosyltransferase is obtained by heterologous expression of the GE02773 gene.

[0010] Furthermore, it includes the following steps:

[0011] S1. Obtaining the target fragment of the glycosyltransferase gene: Using Bacillus subtilis SL-44 genomic DNA as a template, the target fragment of the glycosyltransferase gene was obtained by PCR amplification using primers GE02773F (nucleotide sequence as shown in SEQ ID NO.3) and primers (nucleotide sequence as shown in SEQ ID NO.4).

[0012] S2, Construction of recombinant expression vector: The target fragment of the glycosyltransferase gene was cloned into the pUCm-T vector, transformed into E. coli, and amplified by PCR. The amplification product was ligated into the PET-28a(+) plasmid, transformed into E. coli, and the recombinant expression vector was obtained.

[0013] The present invention also provides a recombinant expression vector constructed from a glycosyltransferase.

[0014] The present invention also provides a crude glycosyltransferase enzyme solution prepared from the recombinant expression vector, wherein the preparation process of the crude glycosyltransferase enzyme solution is as follows: the recombinant expression vector is transformed into Escherichia coli and cultured to OD200. 600 Equal to 0.8, after IPTG induction and ultrasonic disruption, the supernatant is collected to obtain the crude glycosyltransferase enzyme solution.

[0015] The present invention also provides the application of the glycosyltransferase, recombinant expression vector or crude glycosyltransferase solution described above in the synthesis of ginsenoside Rh2.

[0016] Furthermore, ginsenoside Rh2 was obtained by conversion of PPD.

[0017] Furthermore, Fe3O4 / PMG / NTA-Ni 2+ Immobilized glycosyltransferases on magnetic microspheres can improve the efficiency of ginsenoside Rh2 synthesis.

[0018] Furthermore, the Fe3O4 / PMG / NTA-Ni 2+ The preparation process of magnetic microspheres is as follows: Fe3O4 is modified by MPS to obtain Fe3O4 / MPS core-shell microspheres. Fe3O4 / MPS core-shell microspheres are dispersed in acetonitrile and subjected to ultrasonic reaction. Then, a mixture of GMA, MBA and AIBN is added to initiate polymerization. After distillation of acetonitrile, Fe3O4 / PMG microspheres are obtained by magnetic separation.

[0019] The triacetic acid and NaOH were dissolved in deionized water and mixed well. Then, Fe3O4 / PMG microspheres were added and stirred vigorously at 75-80℃ for 6-7 hours. Fe3O4 / PMG / NTA microspheres were obtained by magnetic separation.

[0020] Fe3O4 / PMG / NTA microspheres were mixed and reacted with NiCl2 solution to obtain Fe3O4 / PMG / NTA-Ni 2+ Magnetic microspheres.

[0021] Furthermore, the one-step purification and immobilization process is as follows: Fe3O4 / PMG / NTA-Ni 2+ Magnetic microspheres were dispersed in PBS buffer, and then incubated with crude glycosyltransferase solution to obtain immobilized enzyme solution.

[0022] The immobilized enzyme solution, PPD and UDPG were mixed and catalyzed at 35-37℃ for 4-4.5h. The mixture was then extracted with n-butanol, and the supernatant was freeze-dried to obtain ginsenoside Rh2.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] 1. This invention identifies key enzymes in the biocatalytic synthesis of ginsenoside Rh2 and screens sequence fragments from the Bacillus subtilis SL-44 genome. The results show a high degree of structural similarity to glycosyltransferase family sequences. Potential functional genes that may catalyze the glycosylation of protopanaxadiol to synthesize the rare saponin Rh2 are then screened, and the synthesis is further investigated using Fe3O4 / PMG / NTA-Ni... 2+ A one-step purification and immobilization of glycosyltransferase was proposed to synthesize ginsenoside Rh2 using protopanaxadiol as a substrate, thereby increasing the yield of Rh2. Therefore, it is necessary to obtain a glycosyltransferase gene that can catalyze the synthesis of rare ginsenoside Rh2 from protopanaxadiol, which is an effective way to synthesize ginsenoside Rh2.

[0025] 2. The present invention aims to synthesize rare ginsenoside Rh2 by enzymatic method. A new glycosyltransferase GE02773 required for the synthesis of Rh2 was discovered and screened. A novel one-step enzyme purification and immobilization technology was adopted in the enzyme purification process, which can synthesize ginsenoside Rh2 more effectively.

[0026] 3. This invention modifies Fe3O4 magnetic nanoparticles and then combines them with Ni. 2+ Composite preparation of magnetic Fe3O4 / PMG / NTA-Ni 2+ The microspheres, a composite microsphere, possess higher magnetic susceptibility and excellent binding ability to His-labeled proteins. These microspheres can be cycled multiple times without significantly losing their enzyme binding capacity. This method achieves both one-step purification and immobilization of enzymes and rapid enzyme separation, making it a highly practical immobilization material. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 The results are obtained by HPLC analysis of the crude glycosyltransferase solution in Example 1 of this invention.

[0029] Figure 2 The purification effect of immobilized enzymes.

[0030] Figure 3 Optimization of catalytic conditions for the synthesis of ginsenoside Rh2;

[0031] In the figure, a represents the effect of pH on enzyme-catalyzed conversion;

[0032] b represents the effect of temperature on enzyme-catalyzed conversion;

[0033] c represents the effect of PPD on enzyme-catalyzed conversion;

[0034] d represents the effect of DMSO on enzyme-catalyzed conversion;

[0035] e represents the effect of UDPG on enzyme-catalyzed conversion.

[0036] Figure 4 Analysis of factors influencing immobilized enzymes;

[0037] In the figure, A represents the effect of time on enzyme immobilization;

[0038] B represents the effect of temperature on enzyme immobilization;

[0039] C represents the effect of PBS concentration on enzyme immobilization;

[0040] D represents the effect of free enzyme concentration on enzyme immobilization.

[0041] Figure 5 Functional group analysis for different magnetic materials. Detailed Implementation

[0042] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0043] Example 1: A glycosyltransferase, a recombinant expression vector, its construction method and application.

[0044] To obtain glycosyltransferases capable of catalyzing the glycosylation of triterpenoid saponins, the genomic data of Bacillus subtilis SL-44 obtained previously by our research group was mined. Potential glycosyltransferase gene information (GE02773) capable of catalyzing the synthesis of ginsenoside Rh2 was screened. The triterpenoid saponin glycosyltransferase gene fragment was cloned using PCR. The target fragment was ligated into an expression vector and transformed into recipient cells. Genetic engineering methods were used to construct potential target genes that could be obtained using various PCR techniques or gene synthesis methods. These glycosyltransferases were then heterologously expressed, and then processed using a prepared Fe3O4 / PMG / NTA-Ni... 2+ A magnetic microsphere was used to purify and immobilize glycosyltransferase in one step, and ginsenoside Rh2 was obtained by transformation using ginsenoside PPD as a substrate.

[0045] I. Recombinant Expression Vector of Glycosyltransferase GE02773 Gene, Its Construction Method and Application

[0046] 1. Obtaining the target fragment of the glycosyltransferase gene

[0047] Using Bacillus subtilis SL-44 as the experimental material (Huang Yuanyuan. Study on the disease prevention and growth promotion performance and mechanism of action of Bacillus subtilis SL-44 on pepper [D]. Shihezi University, 2018.), genomic DNA of Bacillus subtilis SL-44 was extracted according to the instructions of the DNA extraction kit. Using the extracted genomic DNA of Bacillus subtilis SL-44 as a template, PCR amplification was performed using primers GE02773F (nucleotide sequence shown in SEQ ID NO.3) and GE02773R (nucleotide sequence shown in SEQ ID NO.4). The amplification system and amplification program are shown below. The target fragment of the glycosyltransferase gene GE02773 was obtained (the nucleotide sequence of the GE02773 gene is shown in SEQ ID NO.1, and the amino acid sequence encoding the protein is shown in SEQ ID NO.2).

[0048] SEQ ID NO.1:

[0049]

[0050] SEQ ID NO.2:

[0051] MKNVLFINFPAEGHVNPTLGMTKAFADRGDHVHYISTEKYKDRLEGVGATVHLHQDWLRTVPVRTGSPDGILSFLKIHIKTSLDILNIVKELSKSIQF DFVYYDKFGAGELVRDYLNIRGISSSASFLFDEDHLKILPLHPDSEVPLQLDKECEDLLLEMKQNYGVSPENMVQFMNNKGELNVVYTSRYFQPESHRF GNEFLFIGPSFPKRAEKTDFPIEKLKDEKVIYISMGTVLDETEEFFNLCIDAFSEFKGKVVIASGEKADLTRLKQAPENFIIASYVPQLEVLEQSDVFITHGGMNSVNEAIHFNVPLVVMPHDKDQPMVAQRLTELQAGYTVSKDHVTAQSLKHAVEEVLNNDRYKEGIQKINESFQECNMNMKEVMEWIDQFIDQRK*

[0052] SEQ ID NO.3:ATGAAAAATGTATTATTTATAAATT

[0053] SEQ ID NO.4:TTATTTTCTCTGATCTATAAATTGA

[0054] Amplification system: 34.75 μL double-distilled water, 15 μL 10×PCR Buffer, 4 μL dNTP (2.5 mMeach), 2 μL template DNA, 4 μL mixed upstream and downstream primers (2 μL each), 0.25 μL Taq DNA Polymerase, total volume 50 μL.

[0055] Amplification program: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 75 s, 30 cycles, 72℃ final extension for 10 min.

[0056] 2. Construction of recombinant expression vectors

[0057] The product obtained from the previous PCR amplification step was cloned into the pUCm-T vector, transformed into Escherichia coli DH5α, and positive clones were screened. The cloning system used upstream enzyme digestion primers as shown in SEQ ID NO.5 and downstream enzyme digestion primers as shown in SEQ ID NO.6 as amplification primers, with restriction sites of BamH1 and Sal1. Secondary products were obtained by PCR amplification.

[0058] SEQ ID NO.5: CGGGATCCATGAAAAATGTATTATTTATAAATT,

[0059] SEQ ID NO.6: GCGTCGACTTATTTTCTCTGATCTATAAATTGA;

[0060] The secondary product and the PET-28a(+) plasmid were double-digested and ligated, transformed into Escherichia coli DH5α, and positive clones were screened to obtain the recombinant expression vector.

[0061] 3. Preparation of crude enzyme solution

[0062] The obtained recombinant expression vector was transformed into Escherichia coli BL21(DE3) for gene expression. The recombinant E. coli culture was then cultured to OD200. 600 When the concentration is equal to 0.8, add 1 mM IPTG, induce at a low temperature of 16℃ for 21 h, and collect the supernatant after ultrasonic disruption to obtain crude glycosyltransferase solution.

[0063] The obtained crude glycosyltransferase enzyme solution was directly used for in vitro activity detection. 1 mg of PPD (protopanaxadiol) was used as the substrate, 10 mg of uridine diphosphate glucose (UDPG) sugar donor and 1 mL of crude enzyme solution were added, and the mixture was reacted at 35℃ for 4 h to obtain the reaction mixture. The reaction mixture was then analyzed by HPLC, and the results are as follows: Figure 1 As shown, PPD can be synthesized into ginsenoside Rh2 by the GE02773 enzymatic method.

[0064] II. Fe3O4 / PMG / NTA-Ni 2+ Preparation of magnetic microspheres

[0065] 1. Modification of Fe3O4 by MPS

[0066] Fe3O4 microspheres were modified with γ-methacryloxypropyltrimethoxysilane (MPS) to form abundant double bonds on their surface. The modification steps are as follows:

[0067] 40 mL of ethanol, 10 mL of deionized water, 1.5 mL of NH3·H2O and 0.6 g of MPS were mixed with 300 mg of Fe3O4. The mixture was then stirred vigorously at 60 °C for 4 hours. The product was separated by a magnet and washed with ethanol to remove excess MPS, thus obtaining Fe3O4 / MPS core-shell microspheres.

[0068] 2. Synthesis of Fe3O4 / PMG microspheres by distillation precipitation polymerization (DPP method)

[0069] First, 50 mg of Fe3O4 / MPS core-shell microspheres were dispersed in 40 mL of acetonitrile and ultrasonically dispersed for 3 min in a dry 100 mL single-necked flask. Then, a mixture of GMA (glycidyl methacrylate), MBA (N,N-methylenebisacrylamide), and AIBN (azobisisobutyronitrile) was added to the flask to initiate polymerization. The specific amounts are shown in Table 1 (the magnetic materials obtained from the polymerization reaction with different amounts are designated as Fe3O4 / PMG-1, Fe3O4 / PMG-2, Fe3O4 / PMG-3, Fe3O4 / PMG-4, and Fe3O4 / PMG-5).

[0070] Functional groups in magnetic materials were analyzed using FT-IR at 1721 cm⁻¹. -1 and 1528cm -1 The peak values ​​are attributed to the tensile vibrations of the C=O bonds in the ester group of GMA and the bending vibrations of NH in MBA. The tensile strength of C=O increases with the amount of GMA used, and the C=O peak is enhanced. The Fe3O4 / PMG-4 magnetic material is the strongest. As expected, Ni-MNPs-4 contains more Ni than the other two samples. 2+ This is attributed to the richer epoxy groups on the magnetic core. Therefore, Fe3O4 / PMG-4 magnetic material was chosen to further characterize its composition and magnetism, with the expectation that the sample could incorporate the most Ni on its surface. 2+ .

[0071] Therefore, the Fe3O4 / PMG-4 reagent mixture was chosen for subsequent experiments.

[0072] A flask immersed in a heated oil bath was connected to a fractionating column, a Liebig condenser, and a receiver. The Fe3O4 / PMG-4 magnetic material was heated from room temperature to boiling over 30 minutes, and the reaction was completed by distilling 20 mL of acetonitrile from the reaction mixture over 1 hour. The obtained Fe3O4 / PMG microspheres were collected by magnetic separation and repeatedly washed with ethanol and water for later use.

[0073] MBA is a crosslinking agent, and AIBN is an initiator.

[0074] Table 1. Usage of GMA, MBA, and AIBN

[0075]

[0076]

[0077] 3. Preparation of Fe3O4 / PMG / NTA microspheres

[0078] 0.33 g NTA (nitrotriacetic acid) and 0.2 g NaOH were dissolved in 20 mL of deionized water and stirred to obtain a solution. The pH of the solution was adjusted to 11 using NaOH (2 M). Then, 50 mg of Fe3O4 / PMG microspheres were added to the solution to obtain a mixed solution, which was then vigorously stirred at 80 °C for 6 h. The Fe3O4 / PMG / NTA microspheres were separated by magnetic separation and washed with ethanol and water for later use.

[0079] In the above reaction, NTA (nitrotriacetic acid) was used to open the epoxy ring of GMA on the surface of the composite microspheres.

[0080] 4. Fe3O4 / PMG / NTA-Ni 2+ Preparation of magnetic microspheres

[0081] 50 mg of Fe3O4 / PMG / NTA microspheres were added to 10 mL of NiCl2 solution (0.1 M) and stirred at room temperature (25 °C) for 2 h. The product was separated from the solution and washed several times with water to obtain Fe3O4 / PMG / NTA-Ni 2+ Magnetic microspheres. The resulting Fe3O4 / PMG / NTA-Ni... 2+ The magnetic microspheres were dried in a vacuum oven at 40°C.

[0082] III. Utilizing Fe3O4 / PMG / NTA-Ni 2+ One-step purification and immobilization of GE02773 protein (glycosyltransferase) using magnetic microspheres to convert ginsenoside PPD to ginsenoside Rh2.

[0083] 10mg Fe3O4 / PMG / NTA-Ni 2+ Magnetic microspheres (100 μL, 2 mg / mL) were dispersed in 5 mL of PBS buffer (25 mM) to obtain a magnetic microsphere suspension. Then, 2 mL of crude glycosyltransferase solution (1 mg / mL) was mixed with 100 μL of magnetic microsphere suspension (2 mg / mL) and incubated at 4 °C for 3 h to obtain an immobilized enzyme solution.

[0084] Take 1 mL of the immobilized enzyme solution obtained above and place it in a 2 mL centrifuge tube. Add 500 μL of 0.4 mM protopanaxadiol (PPD) and 500 μL of 0.4 M uridine diphosphate glucose (UDPG). Catalyze at 35 °C for 4 h. First, extract with 2 mL of n-butanol. Take the supernatant, freeze-dry it, and then dissolve it in 1 mL of methanol. Finally, take 10 μL of the methanol solution for HPLC analysis. The HPLC detection conditions are: XB-C18 column (250 mm × 4.6 mm, 5 μm), column temperature 30 °C, detection wavelength 203 nm, injection volume 10 μL, flow rate 1.0 mL / min, time 20 min. Mobile phase: 10% ultrapure water, 90% acetonitrile. HPLC analysis shows that the conversion rate of protopanaxadiol is 84%.

[0085] Both protopanaxadiol (PPD) and uridine diphosphate glucose (UDPG) were dissolved in PBS buffer.

[0086] The results are as follows Figure 2 As shown, the results indicate that Fe3O4 / PMG / NTA-Ni 2+ Purification performance of magnetic microspheres on His-labeled glycosyltransferases expressed in Escherichia coli. His-labeled GE02773 protein was isolated from cell lysates using only a small amount of nonspecifically binding protein.

[0087] IV. Optimization of Synthetic Conditions for Ginsenoside Rh2

[0088] 1. The effect of temperature on enzyme-catalyzed transformation

[0089] 1 mg PPD, 10 mg UDPG, 50 μL DMSO, and 1 mL of purified enzyme (purified using a conventional nickel column) were weighed into 2 mL centrifuge tubes and incubated at 25℃, 30℃, 35℃, 40℃, 35℃, and 45℃, respectively, with three replicates for each temperature and the average value taken. The centrifuge tubes were placed in a constant-temperature water bath at 150 rpm for 4 h of incubation. After each incubation period, the amount of Rh2 produced at 203 nm was determined using high-performance liquid chromatography (HPLC), and bar charts were plotted to analyze the effect of different temperature conditions on enzyme conversion rate.

[0090] For each group's conversion solution, an equal volume of n-butanol was added for extraction. The supernatant was then lyophilized, dissolved in 1 mL of methanol, and finally 10 μL of the methanol solution was loaded for analysis. The results are as follows: Figure 3 As shown in b, it was found that at 35℃, the conversion rate of the product human saponin Rh2 was 82%.

[0091] Catalytic conversion was performed using GE02773 at five temperatures: 30℃, 35℃, 40℃, 45℃, and 50℃. The resulting methanol mixtures, analyzed by HPLC, showed conversion rates of 62%, 82%, 72%, 74%, and 62%, respectively. Figure 3 As shown in b, the analysis shows that the conversion rate of PPD is the highest and the amount of Rh2 generated is the largest at a temperature of 35℃.

[0092] 2. The effect of pH on enzyme-catalyzed transformation

[0093] 1 mg PPD, 10 mg UDPG, 50 μL DMSO, and 1 mL of purified enzyme solution were weighed into 2 mL centrifuge tubes and converted at pH 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5, respectively, with three replicates for each pH and the average value taken. The centrifuge tubes were placed in a constant temperature water bath at 150 rpm and 40 °C for 4 h of conversion. After each incubation, the amount of Rh2 produced at a wavelength of 203 nm was determined using high-performance liquid chromatography (HPLC), and a bar chart was plotted to analyze the effect of different pH conditions on the enzyme conversion rate.

[0094] For each group's conversion solution, an equal volume of n-butanol was added for extraction. The supernatant was then lyophilized, dissolved in 1 mL of methanol, and finally 10 μL of the methanol solution was loaded for analysis. The results are as follows: Figure 3 As shown in a, at pH 8.0, the conversion rate of the product human saponin Rh2 was 73%.

[0095] Catalytic conversion was performed using GE02773 at pH values ​​of 6, 6.5, 7, 7.5, 8, and 8.5. The resulting methanol mixtures, analyzed by HPLC, showed conversion rates of 59%, 63%, 62%, 67%, 73%, and 57%, respectively. Figure 3 As shown in Figure a, the analysis shows that the conversion rate of PPD is the highest and the amount of Rh2 generated is the largest when the pH is 8.0.

[0096] 3. Effect of PPD on enzyme-catalyzed transformation

[0097] 10 mg UDPG, 50 μL DMSO, and 1 mL of purified enzyme solution were weighed into 2 mL centrifuge tubes and converted at PPD concentrations of 0.4 mM, 0.8 mM, 1.2 mM, 1.6 mM, and 2 mM, respectively. Three replicates were set up for each concentration, and the average value was taken. The centrifuge tubes were placed in a constant temperature water bath at 40 °C and the conversion was carried out for 4 h. After each incubation, the amount of Rh2 generated at a wavelength of 203 nm was determined using high-performance liquid chromatography (HPLC), and a bar chart was plotted to analyze the effect of different PPD concentrations on the enzyme conversion rate.

[0098] For each group's conversion solution, first add an equal volume of n-butanol for extraction, collect the supernatant, freeze-dry it, then dissolve it in 1 mL of methanol, and finally take 10 μL of the methanol solution for analysis. Figure 3 As shown in c, when the PPD concentration is 0.4 mM, the conversion rate of the product human saponin Rh2 is 87%.

[0099] Catalytic conversion was performed using GE02773 at five PPD concentrations of 0.4 mM, 0.8 mM, 1.2 mM, 1.6 mM, and 2 mM, respectively. The resulting methanol mixtures, analyzed by HPLC, showed conversion rates of 87%, 68%, 64%, 61%, and 53%, respectively. Figure 3 As shown in Figure c, the analysis shows that the conversion rate of PPD is the highest and the amount of Rh2 generated is the largest when the PPD concentration is 0.4 mM.

[0100] 4. Effect of DMSO on enzyme-catalyzed conversion

[0101] 1 mg of PPD, 10 mg of UDPG, and 1 mL of purified enzyme solution were weighed into 2 mL centrifuge tubes and converted in 0%, 5%, 10%, 15%, and 20% DMSO, respectively. Three replicates were set up for each concentration to obtain the average. The centrifuge tubes were placed in a constant temperature water bath at 40℃ and the centrifuge tubes were incubated for 4 hours. After each incubation, the amount of Rh2 produced at 203 nm was determined using high-performance liquid chromatography (HPLC), and a bar chart was plotted to analyze the effect of different pH conditions on the enzyme conversion rate.

[0102] For each group of conversion solutions, the same volume of n-butanol was added for extraction, the supernatant was lyophilized, dissolved in 1 mL of methanol, and finally 10 μL of the methanol solution was loaded for analysis, and the conversion rate of the product human saponin Rh2 was 85%.

[0103] Catalytic conversion was performed using GE02773 at DMSO concentrations of 0%, 5%, 10%, 15%, and 20%. The resulting methanol mixtures, analyzed by HPLC, showed conversion rates of 66%, 85%, 49%, 45%, and 35%, respectively. The results are as follows: Figure 3 As shown in d, when DMSO is 5%, the conversion rate of PPD is the highest and the amount of Rh2 generated is the largest.

[0104] 5. Effect of UDPG on enzyme-catalyzed conversion

[0105] 1 mg PPD, 50 μL DMSO, and 1 mL of purified enzyme solution were weighed into 2 mL centrifuge tubes and converted at 0.2 M, 0.4 M, 0.6 M, 0.8 M, and 1 M UDPG, respectively. Three replicates were set up for each concentration, and the average value was taken. The centrifuge tubes were placed in a constant temperature water bath at 40 °C and the conversion was carried out for 4 h. After each incubation, the amount of Rh2 generated at a wavelength of 203 nm was determined using high-performance liquid chromatography (HPLC), and a bar chart was plotted to analyze the effect of different temperature conditions on the enzyme conversion rate.

[0106] For each group's conversion solution, an equal volume of n-butanol was added for extraction. The supernatant was then lyophilized, dissolved in 1 mL of methanol, and finally 10 μL of the methanol solution was loaded for analysis. The results are as follows: Figure 3 As shown in Figure e, the conversion rate of the product ginsenoside Rh2 was 71%.

[0107] Catalytic conversion was performed using GE02773 at UDPG concentrations of 0.2M, 0.4M, 0.6M, 0.8M, and 1M. The resulting methanol mixtures, analyzed by HPLC, showed conversion rates of 26%, 71%, 70%, 61%, and 55%, respectively. Figure 3 As shown in Figure e, the analysis shows that the conversion rate of PPD is the highest and the amount of Rh2 generated is the largest when the concentration of UDPG is 0.4 mM.

[0108] V. Analysis of Factors Affecting Immobilized Enzymes

[0109] 1. Time

[0110] The effect of immobilization time on enzyme immobilization is shown in the figure. With increasing immobilization time, the amount of enzyme immobilized gradually increases, reaching a maximum of 179 mg / g at 3 hours. Further extending the immobilization time leads to a stable immobilization amount with minimal increase. Considering that excessively long immobilization times may decrease enzyme activity, the optimal enzyme immobilization time is 3 hours. Figure 4 (A).

[0111] 2. Temperature

[0112] The effect of immobilization temperature on enzyme immobilization is shown in the figure. The enzyme immobilization amount reaches its maximum of 180 mg / g at 4℃. As the immobilization temperature increases, the enzyme immobilization amount continuously decreases. When the temperature reaches 35℃, the enzyme immobilization amount decreases by half, indicating that high temperature has a certain impact on the immobilization process, thus reducing immobilization efficiency. Furthermore, high temperatures easily inactivate enzymes; the higher the temperature, the lower the enzyme activity or even complete inactivation. Therefore, the optimal temperature for enzyme immobilization is 4℃. Figure 4 (B).

[0113] 3. PBS concentration

[0114] The effect of different PBS concentrations on enzyme immobilization is shown in the figure. As the PBS concentration increases, the amount of enzyme immobilized gradually increases, reaching a maximum of 64 mg / g at a PBS concentration of 25 mM. Further increases in PBS concentration lead to a slight decrease in enzyme immobilization. This is because excessively high PBS concentrations affect the binding of the enzyme to the carrier, resulting in a decrease in immobilization. Therefore, the optimal PBS concentration for enzyme immobilization is 25 mM. Figure 4 (C).

[0115] 4. Free enzyme concentration

[0116] During the immobilization process, the concentration of added free enzyme was varied while other immobilization conditions remained constant. The effect of different free enzyme concentrations on immobilization was studied, and the results are shown in the figure. Within the range of added free enzyme concentration of 0.2-1.4 mg / mL, the amount of enzyme immobilized increased with increasing free enzyme concentration, reaching a maximum of 138 mg / g when the added free enzyme concentration was 1 mg / mL. Further increasing the amount of free enzyme resulted in a decreasing trend in the amount of enzyme immobilized. This may be because excessive free enzyme, due to steric hindrance, makes the binding of the enzyme to the carrier more difficult, and the glycosyltransferases in the binding pores on the carrier surface are already close to saturation. Excessive compression reduces the amount of immobilized enzyme. Therefore, increasing the free enzyme concentration gradually increases the amount of immobilized enzyme until a certain saturation point is reached. Further increasing the enzyme concentration will decrease the amount of immobilized enzyme. Therefore, the optimal enzyme addition amount is 1 mg / mL. Figure 4 (D).

[0117] The above experiments show that the optimal catalytic conversion conditions without immobilization are: temperature 35℃, pH 8.0, PPD concentration 0.4 mM, DMSO content 5%, and UDPG concentration 0.4 mM. Under these conditions, the maximum conversion rate to ginsenoside Rh2 can reach 80.2%. The immobilized enzyme further increases the conversion rate of the substrate protopanaxadiol to 84%.

[0118] In summary, this invention, utilizing databases such as NCBI and PDB, screened several glycosyltransferases that may catalyze the glycosylation of protopanaxadiol based on principles such as substrate similarity and catalytic reaction type. The screened genes were functionally expressed in an *E. coli* expression system to obtain crude enzyme solutions of recombinant proteins. Experiments demonstrated that the GE02773 glycosyltransferase from *Bacillus subtilis* SL-44 could catalyze the glycosylation of protopanaxadiol. The obtained glycosylated product was singular, and the optimized conversion rate of the substrate protopanaxadiol was 80.2%. This invention also provides a one-step purification and immobilization method for glycosyltransferases, which involves preparing Fe3O4 / PMG / NTA-Ni... 2+Magnetic microspheres are used to specifically adsorb glycosyltransferases onto their surface, followed by reaction with a substrate to synthesize ginsenoside Rh2. This method not only allows for direct enzyme purification but also optimizes the catalytic conditions during the reaction process, significantly improving enzyme stability and reusability. Furthermore, the conversion rate of the immobilized enzyme to the substrate protopanaxadiol is increased to 84%.

[0119] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0120] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A glycosyltransferase for synthesizing ginsenoside Rh2, characterized in that, The glycosyltransferase is derived from Bacillus subtilis SL-44. GE02773 The gene-encoded glycosyltransferase, named GE02773 protein, has the nucleotide sequence shown in SEQ ID NO.1 and the amino acid sequence shown in SEQ ID NO.

2.

2. A method for producing the glycosyltransferase according to claim 1, characterized by, The glycosyltransferase is composed of GE02773 Obtained from heterologous gene expression.

3. The method for preparing glycosyltransferase according to claim 2, characterized in that, Includes the following steps: S1. Obtaining the target fragment of the glycosyltransferase gene: Using Bacillus subtilis SL-44 genomic DNA as a template, the target fragment of the glycosyltransferase gene was obtained by PCR amplification using primers GE02773F (nucleotide sequence as shown in SEQ ID NO.3) and primers (nucleotide sequence as shown in SEQ ID NO.4). S2, Heterologous expression of glycosyltransferase: The target fragment of the glycosyltransferase gene was ligated with the pET-28a(+) plasmid, transformed into E. coli, and expressed by IPTG to obtain crude glycosyltransferase enzyme solution.

4. A recombinant expression vector constructed from the glycosyltransferase of claim 1.

5. A crude glycosyltransferase enzyme solution prepared from the recombinant expression vector of claim 4, characterized in that, The preparation process of crude glycosyltransferase solution is as follows: the recombinant expression vector is transformed into E. coli and cultured to OD200. 600 Equal to 0.8, after IPTG induction and ultrasonic disruption, the supernatant is collected to obtain the crude glycosyltransferase enzyme solution.

6. The use of the glycosyltransferase of claim 1, the recombinant expression vector of claim 4, or the crude enzyme solution of the glycosyltransferase of claim 5 in the synthesis of ginsenoside Rh2.

7. Use according to claim 6, characterized in that, Ginsenoside Rh2 was obtained by conversion of PPD.

8. Use according to claim 7, characterized in that, Using Fe3O4 / PMG / NTA−Ni 2+ Immobilized glycosyltransferases on magnetic microspheres improve the efficiency of ginsenoside Rh2 synthesis.

9. The application according to claim 8, characterized in that, The Fe3O4 / PMG / NTA−Ni 2+ The preparation process of magnetic microspheres is as follows: Fe3O4 is modified by MPS to obtain Fe3O4 / MPS core-shell microspheres. Fe3O4 / MPS core-shell microspheres are dispersed in acetonitrile and subjected to ultrasonic reaction. Then, a mixture of GMA, MBA and AIBN is added to initiate polymerization. After distillation of acetonitrile, Fe3O4 / PMG microspheres are obtained by magnetic separation. The triacetic acid and NaOH were dissolved in deionized water and mixed well. Then, Fe3O4 / PMG microspheres were added and stirred vigorously at 75-80℃ for 6-7 hours. Fe3O4 / PMG / NTA microspheres were obtained by magnetic separation. Fe3O4 / PMG / NTA microspheres were mixed and reacted with NiCl2 solution to obtain Fe3O4 / PMG / NTA-Ni 2+ Magnetic microspheres.

10. The application according to claim 9, characterized in that, The immobilization process is as follows: Fe3O4 / PMG / NTA-Ni 2+ Magnetic microspheres were dispersed in PBS buffer, and then incubated with crude glycosyltransferase solution to obtain immobilized enzyme solution. The immobilized enzyme solution, PPD and UDPG were mixed and catalyzed at 35-37℃ for 4-4.5 h. The mixture was then extracted with n-butanol, and the supernatant was freeze-dried to obtain ginsenoside Rh2.

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