Engineered glycosyl transferase and application thereof in efficient preparation of triterpenoid saponin

Abstract

The invention discloses an engineered glycosyl transferase and application thereof in efficient preparation of triterpenoid saponin. The engineered glycosyl transferase is obtained by mutating serine at the 15th site of UGT74AC1 enzyme into alanine, mutating histidine at the 47th site into aspartic acid, mutating leucine at the 48th site into valine, mutating alanine at the 180th site into serine, mutating histidine at the 180th site into aspartic acid, mutating histidine at the 180th site into aspartic acid, mutating histidine at the 180th site into aspartic acid, mutating histidine at the 180th site into aspartic acid The mutant is obtained by mutating serine at the 332 site into proline, phenylalanine at the 367 site into tryptophan and lysine at the 420 site into arginine. According to the invention, the glycosyl transferase with high catalytic activity is obtained by modifying an enzyme engineering technology, and good application of the glycosyl transferase in synthesis of triterpenoid saponin is realized. The glycosyl transferase disclosed by the invention can also be coupled with sucrose synthase, or coupled with N-acetylhexosamine kinase and N-acetylglucosamine-1-uridine phosphate transferase, and acetylglucosamine is used as a glycosyl donor, so that cascade production of triterpenoid saponin with lower cost and higher efficiency is realized.

Description

Technical Field

[0001] The present invention relates to the field of enzyme engineering technology, and in particular to an engineered glycosyltransferase and application thereof in the efficient preparation of triterpenoid saponins. Background Art

[0002] Triterpenes are a class of natural compounds composed of 30 carbon atoms, consisting of six isoprene units. They are synthesized through the mevalonate pathway (MVA pathway) in plants, animals, and fungi and are precursors to steroids and other bioactive compounds. Triterpenes have diverse structures and can be hydrocarbons, alcohols, ketones, aldehydes, or glycosides. Due to their diverse bioactive properties, triterpenes are widely used in the food and pharmaceutical fields. For example, mogroside alcohol has strong free radical scavenging ability, which can protect cells from oxidative damage and reduce the risk of oxidative stress-related diseases, thereby preventing neurodegenerative diseases, cardiovascular diseases, and diseases related to aging. Cucurbitacin can induce cell apoptosis, inhibit angiogenesis, and cancer cell metastasis. It is used as an adjuvant therapy for melanoma, breast cancer, and lung cancer.

[0003] However, triterpenoids face challenges such as poor water solubility, resulting in low bioavailability, complex structural modification processes, and difficulty in efficient synthesis, which remain to be overcome. Glycosylation can significantly improve the water solubility of hydrophobic triterpenes, and glycosylation can enhance or alter the pharmacological properties of triterpenes, such as anticancer, antiviral, or anti-inflammatory activities. It can also reduce the toxicity of triterpenoids and minimize side effects. Glycosylation can improve the metabolic stability of triterpenes, making them more resistant to enzymatic degradation. Glycosides are often involved in plant defense and signal transduction, and glycosylation can affect their biological functions and cellular targeting. Different glycosylation types have different effects on the water solubility of triterpenoids and their interactions with biological targets, thereby affecting binding affinity, specificity, and activity. Therefore, triterpenoid saponins obtained after glycosylation modification are not only novel drug development candidates for the treatment of various diseases, such as cancer, inflammation, and viral infections, but can also be used as components of vaccines to enhance immune responses. Furthermore, triterpenoid saponins may possess specific biomarker properties, allowing for early diagnosis of diseases. In summary, glycosylation modification not only improves the therapeutic efficacy and safety of triterpenes as drugs, but also helps in the development of new treatments and technologies, thus contributing new strategies to human health and social progress.

[0004] At present, the synthesis of triterpenoid saponins is usually carried out by chemical or enzymatic synthesis, but both have certain limitations. Chemical synthesis usually requires tedious protection and deprotection steps, and has low specificity and yield. In the prior art, enzymatic catalysis of triterpenoid glycosylation modification is mainly achieved through UGTs, but most natural UGTs are low in heterologous expression in Escherichia coli, poor in stability, and low in catalytic activity and substrate specificity, making it difficult to meet the requirements of industrial enzymes. In addition, enzymatic synthesis requires expensive glycosyl donors, so the enzyme was further modified and several other enzymes capable of generating glycosyl donors were cascaded, so that different triterpenoid saponins can be prepared on a large scale at low cost and high efficiency. Summary of the Invention

[0005] Purpose of the invention: To address the deficiencies of the prior art, the present invention provides an engineered glycosyltransferase. The present invention utilizes enzyme engineering technology to transform a highly catalytically active glycosyltransferase (mUGT74AC1), and achieves excellent application of the enzyme in the synthesis of triterpenoid saponins. Furthermore, the glycosyltransferase of the present invention can be coupled with sucrose synthase (SuSy) using sucrose as a glycosyl donor, or coupled with N-acetylglucosamine kinase (NahK) or N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) using acetylglucosamine (GlcNAc) as a glycosyl donor, thereby achieving lower-cost and more efficient cascade production of triterpenoid saponins. This effectively addresses the current issues of low activity, poor stability, and a limited substrate spectrum of UGT74AC1. Furthermore, it provides a solution for the 3-position glycosylation of triterpenoid compounds, addressing the high cost, excessive waste, and severe pollution associated with the synthesis of triterpenoid saponins.

[0006] The present invention also provides the use of the engineered glycosyltransferase in the efficient preparation of triterpenoid saponins.

[0007] Technical solution: To achieve the above-mentioned purpose of the invention, the present invention provides an engineered glycosyltransferase, which is a mutant mUGT74AC1 of the UGT74AC1 enzyme. The mutant mUGT74AC1 is a UGT74AC1 enzyme whose amino acid sequence is as shown in SEQ ID NO.3, wherein the serine at position 15 is mutated to alanine, the histidine at position 47 is mutated to aspartic acid, the leucine at position 48 is mutated to valine, the alanine at position 180 is mutated to serine, the serine at position 332 is mutated to proline, the phenylalanine at position 367 is mutated to tryptophan, and the lysine at position 420 is mutated to arginine, to obtain a mutant having mutations S15A / H47D / L48V / A180S / S332P / F367W / K420R.

[0008] The gene encoding the engineered glycosyltransferase of the present invention, the gene sequence of the mutant mUGT74AC1 is shown in SEQ ID NO.2, and its amino acid sequence is shown in SEQ ID NO.4.

[0009] The recombinant vector or recombinant bacterium of the present invention contains the coding gene of the engineered glycosyltransferase.

[0010] The invention relates to the use of the engineered glycosyltransferase, the encoding gene, the recombinant vector or the recombinant bacteria in the efficient preparation of triterpenoid saponins using a bioenzymatic method or a bioenzymatic cascade method.

[0011] Among them, the process of preparing triterpenoid saponins using the bioenzymatic method is to add a glycosyl acceptor terpenoid compound, a glycosyl donor, an engineered glycosyltransferase mUGT74AC1, and salt to a buffer solution, mix them evenly to obtain a mixed solution, and perform an enzyme-catalyzed reaction under stirring to obtain triterpenoid saponins.

[0012] The terpene compounds in the mixed solution include mogroside alcohol, steviol, cucurbitacin, andrographolide, ginseng alcohol, glycyrrhetinic acid, ursolic acid or betulinic acid, and the concentration of the terpene compounds in the mixed solution is 0.01-1mM; the glycosyl donor includes uridine diphosphate glucose, uridine diphosphate galactose, uridine diphosphate glucuronic acid or uridine diphosphate acetylglucosamine, and the concentration of the glycosyl donor in the mixed solution is 0.05-20mM.

[0013] The salt includes magnesium salt, calcium salt, iron salt, manganese salt, copper salt, ammonium salt or zinc salt, and the concentration of the salt in the mixed solution is 0.1-10 mM; the pH of the buffer solution is 4.0-9.0; the stirring rate is 100-300 rpm; the temperature of the enzyme-catalyzed reaction is 15-60°C; the time of the enzyme-catalyzed reaction is 1-24 hours; and the enzyme concentration of the enzyme-catalyzed reaction is 0.01-10 g / L.

[0014] Furthermore, the terpene compound in the mixed solution is mogrosanol or cucurbitacin, and its concentration is 0.01-1 mM; preferably, the concentration of the terpene compound in the mixed solution is 0.25 mM.

[0015] Furthermore, the glycosyl donor in the mixed solution is UDP-Glc, UDP-Gal, UDP-GlcA or UDP-GlcNAc, and its concentration is 0.05-20 mM; preferably, the concentration of the glycosyl donor in the mixed solution is 1 mM.

[0016] Furthermore, the salt in the mixed solution includes magnesium salt, calcium salt, iron salt, manganese salt, copper salt, ammonium salt or zinc salt; in the mixed solution, further preferably, the salt is manganese salt or magnesium salt.

[0017] Furthermore, the salt concentration in the mixed solution is 0.1-10 mM, and more preferably, the salt concentration is 1 mM.

[0018] Furthermore, the buffer used in the mixed solution is Acetate, PBS or Tris-HCl; preferably, the buffer is Tris-HCl.

[0019] Furthermore, the pH of the Tris-HCl buffer is 7.5-9.0; further preferably, the pH of the buffer is 8.0,

[0020] Furthermore, the stirring rate is 100-300 rpm; preferably, the stirring rate is 200 rpm.

[0021] Furthermore, the enzyme catalysis temperature is 15-60°C; preferably, the enzyme catalysis temperature is 40°C.

[0022] Furthermore, the enzyme reaction time is 1-24 hours; preferably, the enzyme reaction time is 5 hours.

[0023] Furthermore, the concentration of mUGT74AC1 in the buffer is 0.01-10 g / L; preferably, the enzyme concentration is 0.5 mg / mL.

[0024] In the method provided by the present invention, the mUGT74AC1 used in the glycosylation reaction has an optimum pH of 8.0 and an optimum reaction temperature of 40°C. It is a metal ion-dependent enzyme, Mg 2+ 、Mn 2+ It has a significant promoting effect on its catalysis, among which Mg 2+ The strongest promoting effect.

[0025] In the method provided by the present invention, the optimal reaction conditions of the enzyme are: the final concentrations of various substances in the reaction system are 0.25mM of triterpenoid compounds, 1mM of UDP-Glc or UDP-GlcNAc or UDP-GlcA or UDP-Gal, and 1mM of MgCl2, the reaction temperature is 40°C, the rotation speed is 200rpm, and the reaction time is 5h. Under the optimal reaction conditions, the enzyme catalyzes the conversion of triterpenoid compounds into triterpenoid saponins with the highest conversion rate of up to 99%.

[0026] The reaction is shown below;

[0027]

[0028] Among them, the process of the bio-enzymatic cascade method for efficiently preparing triterpenoid saponins is to add any one or more of a glycosyl acceptor terpenoid compound, a glycosyl donor, uridine-5'-diphosphate disodium salt or adenosine-5'-triphosphate disodium salt or uridine-5'-triphosphate trisodium salt, a cascade multi-enzyme containing an engineered glycosyltransferase mUGT74AC1, and salt to a buffer solution, mix them evenly to obtain a mixed solution, and perform an enzyme-catalyzed reaction under stirring to obtain triterpenoid saponins.

[0029] The glycosyl acceptor terpenoid compound in the mixture includes mogrohol, steviol, cucurbitacin, andrographolide, ginseng alcohol, glycyrrhetinic acid, ursolic acid or betulinic acid, with a concentration of 0.01-1mM; the glycosyl donor or the precursor of the glycosyl donor is sucrose or acetylglucosamine, with a concentration of 0.05-20mM; the concentration of uridine-5'-diphosphate disodium salt is 0.05-20mM; the concentration of adenosine-5'-triphosphate disodium salt is 0.05-20mM. The concentration is 0.1-20 mM; the concentration of the uridine-5′-triphosphate trisodium salt is 0.1-20 mM; the salts include but are not limited to magnesium salts, calcium salts, iron salts, manganese salts, copper salts, ammonium salts, and zinc salts, with a concentration of 0.1-10 mM; the pH of the buffer is 4.0-9.0; the stirring rate is 100-300 rpm, the temperature of the enzyme-catalyzed reaction is 15-60° C., and the time of the enzyme-catalyzed reaction is 1-24 h.

[0030] Wherein, the cascade multi-enzyme is SuSy and mUGT74AC1, wherein the SuSy enzyme concentration is 0.01-10 g / L, wherein the mUGT74AC1 concentration is 0.01-10 g / L; or the cascade multi-enzyme is NahK, GlmU and mUGT74AC1, wherein the NahK enzyme concentration is 0.01-10 g / L, wherein the GlmU concentration is 0.01-10 g / L, wherein the mUGT74AC1 concentration is 0.01-10 g / L.

[0031] Preferably, the present invention provides a second method for efficiently preparing triterpenoid saponins using a bio-enzymatic cascade method, comprising the following steps: adding triterpenoid compounds, sucrose, uridine-5′-diphosphate disodium salt (UDP), mUGT74AC1, SuSy, and salt to a buffer solution, mixing uniformly to obtain a mixed solution, and performing an enzyme-catalyzed reaction under stirring to obtain glucose-glycosylated triterpenoid saponins.

[0032] The reaction looks like this:

[0033]

[0034] Furthermore, in the mixed solution, the triterpenoid compound is mogrosanol or cucurbitacin, and its concentration is 0.01-1 mM; preferably, the concentration of the terpenoid compound in the mixed solution is 0.25 mM.

[0035] Furthermore, in the mixed solution, the concentration of sucrose is 0.05-200 mM; preferably, the concentration of sucrose in the mixed solution is 2 mM.

[0036] Furthermore, in the mixed solution, the concentration of UDP is 0.1-20 mM; preferably, the concentration of UDP is 4 mM.

[0037] Furthermore, the salt in the mixed solution includes magnesium salt, calcium salt, iron salt, manganese salt, copper salt, ammonium salt or zinc salt; in the mixed solution, further preferably, the salt is manganese salt or magnesium salt.

[0038] Furthermore, the salt concentration in the mixed solution is 0.1-10 mM, and more preferably, the salt concentration is 1 mM.

[0039] Furthermore, the mixed solution uses a buffer solution of Acetate, PBS, or Tris-HCl; preferably, the buffer solution is Tris-HCl.

[0040] Furthermore, the pH of the Tris-HCl buffer is 7.5-9.0; more preferably, the pH of the buffer is 8.0.

[0041] Furthermore, the stirring rate is 100-300 rpm; preferably, the stirring rate is 200 rpm.

[0042] Furthermore, the enzyme catalysis temperature is 15-60°C; preferably, the enzyme catalysis temperature is 40°C.

[0043] Furthermore, the enzyme reaction time is 1-24 hours; preferably, the enzyme reaction time is 5 hours.

[0044] Furthermore, the concentration of mUGT74AC1 in the buffer is 0.01-10 g / L; preferably, the enzyme concentration is 1 mg / mL.

[0045] Furthermore, the concentration of SuSy in the buffer is 0.01-10 g / L; preferably, the enzyme concentration is 0.8 mg / mL.

[0046] As a preferred method, the second bioenzymatic cascade method provided by the present invention is to use sucrose as a glycosyl donor. In the presence of UDP, Susy can cascade catalyze with mUGT74AC1, so that triterpenoid compounds can directly generate triterpenoid saponins. In this method, the optimal pH of the dual enzyme cascade catalytic reaction is 8.0, the optimal reaction temperature is 40 ° C, and the Mg content is 0. 2+ This method significantly improves the performance of the dual-enzyme cascade reaction. The cascade reaction time is 5 hours, the optimal rotation speed is 200 rpm, and the yield can reach 89%. This method utilizes the sucrose synthase SuSy and the mutant glycosyltransferase mUGT74AC1 cascade, allowing the direct use of relatively inexpensive sucrose as a glycosyl donor in production, thereby reducing production costs.

[0047] Preferably, the present invention provides a third method for efficiently preparing triterpenoid saponins using a bio-enzymatic cascade method, comprising the following steps:

[0048] Triterpenoid compounds, GlcNAc, adenosine-5'-triphosphate disodium salt (ATP), uridine-5'-triphosphate trisodium salt (UTP), mUGT74AC1, NahK, GlmU, and salt are added to a buffer solution, mixed evenly to obtain a mixed solution, and an enzyme-catalyzed reaction is carried out under stirring to obtain GlcNAc-glycosylated triterpenoid saponins.

[0049] The reaction looks like this:

[0050]

[0051] Furthermore, in the mixed solution, the triterpenoid compound is mogrosanol or cucurbitacin, and its concentration is 0.01-1 mM; preferably, the concentration of the terpenoid compound in the mixed solution is 0.25 mM.

[0052] Furthermore, in the mixed solution, the concentration of GlcNAc is 0.05-200 mM; preferably, the concentration of GlcNAc in the mixed solution is 2 mM.

[0053] Furthermore, in the mixed solution, the concentration of ATP is 0.1-20 mM; preferably, the concentration of ATP is 4 mM.

[0054] Furthermore, in the mixed solution, the concentration of UTP is 0.1-20 mM; preferably, the concentration of UTP is 4 mM.

[0055] Furthermore, the salt in the mixed solution includes magnesium salt, calcium salt, iron salt, manganese salt, copper salt, ammonium salt or zinc salt; in the mixed solution, further preferably, the salt is manganese salt or magnesium salt.

[0056] Furthermore, the salt concentration in the mixed solution is 0.1-10 mM, and more preferably, the salt concentration is 1 mM.

[0057] Furthermore, the mixed solution uses a buffer solution of Acetate, PBS or Tris-HCl; preferably, the buffer solution is Tris-HCl.

[0058] Furthermore, the pH of the Tris-HCl buffer is 7.5-9.0; more preferably, the pH of the buffer is 8.0.

[0059] Furthermore, the stirring rate is 100-300 rpm; preferably, the stirring rate is 200 rpm.

[0060] Furthermore, the enzyme catalysis temperature is 15-60°C; preferably, the enzyme catalysis temperature is 35°C.

[0061] Furthermore, the enzyme reaction time is 1-24 hours; preferably, the enzyme reaction time is 10 hours.

[0062] Furthermore, the concentration of mUGT74AC1 in the buffer is 0.01-10 g / L; preferably, the enzyme concentration is 1 mg / mL.

[0063] Furthermore, the concentration of NahK in the buffer is 0.01-10 g / L; preferably, the enzyme concentration is 0.8 mg / mL.

[0064] Furthermore, the concentration of GlmU in the buffer is 0.01-10 g / L; preferably, the enzyme concentration is 0.5 mg / mL.

[0065] As a preferred method, the third bioenzymatic cascade method provided by the present invention is to use GlcNAc as a glycosyl donor. In the presence of UTP and ATP, NahK and GlmU can carry out a three-enzyme cascade catalysis with mUGT74AC1, so that triterpenoid compounds can directly generate GlcNAc-glycosylated triterpenoid saponins. In this method, the optimal pH of the dual enzyme cascade catalysis reaction is 8.0, the optimal reaction temperature is 35°C, and the Mg content is 1.5. 2+ This method significantly improves the performance of the dual-enzyme cascade reaction. The cascade reaction time is 10 hours, the optimal rotation speed is 200 rpm, and the yield can reach 65%. This method utilizes the NahK, GlmU, and mUGT74AC1 cascade, allowing the relatively inexpensive GlcNAc glycosyl donor to be directly utilized in production, thereby reducing production costs.

[0066] The present invention uses uridine diphosphate glucose and uridine diphosphate acetylglucosamine as glycosyl donors, and triterpenoid compounds mogroside alcohol, steviol, cucurbitacin, andrographolide, ginseng alcohol, glycyrrhetinic acid, ursolic acid, and betulinic acid as glycosyl acceptors. Under the catalysis of glycosyltransferase (mUGT74AC1), the glycosyl group is transferred to the triterpenoid compound to synthesize triterpenoid saponins. In addition, the glycosyltransferase can also be coupled with sucrose synthase (SuSy) to synthesize triterpenoid saponins using sucrose as the glycosyl donor; it can also be coupled with N-acetylhexosamine kinase (NahK) and N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) to synthesize triterpenoid saponins using acetylglucosamine as the glycosyl donor. This method is a method for producing different triterpenoid saponins with easily available raw materials, low production cost and high efficiency. The glycosyltransferase is a high-catalytic activity enzyme obtained by enzyme engineering technology. It can efficiently convert glycosyl donors to triterpenoid compounds and has the ability to recognize different triterpenoid substrates and different sugar donors, and has great application potential.

[0067] The present invention provides a method for efficiently preparing different triterpenoid saponins using a bio-enzymatic cascade reaction. The method comprises constructing a glycosyltransferase, then constructing an engineered bacterium for protein expression, and then catalyzing the in vitro glycosylation reaction using triterpenes as sugar acceptors to synthesize triterpenoid saponins in a single step. The highly catalytic enzyme, obtained through enzyme engineering technology, can efficiently utilize various sugar donors to convert triterpenoid compounds. This synthesis method has high catalytic efficiency, short reaction time, and a single, easily separable product, promising broad industrial application prospects.

[0068] The present invention also provides a method for efficiently preparing different triterpenoid saponins using a bio-enzymatic cascade reaction. Compared with traditional triterpenoid saponin synthesis methods, this method has the advantages of readily available raw materials, low production costs, higher catalytic efficiency, shorter reaction times, and a single, easily separable product. Furthermore, it can produce triterpenoid saponins with different glycosylation profiles, thus offering great industrial application prospects and laying a solid foundation for research on the application of triterpenoid saponins. This method is of great significance to the development of glycoscience and promotes the practical application of glycosylation modification of terpenoid compounds.

[0069] This invention optimizes the structure of the UGT74AC1 protein to improve its stability and expression efficiency in heterologous expression systems, increase enzyme expression in E. coli, and enhance enzyme stability. While ensuring that the protein's function remains unchanged, the invention selects the optimal design for experimental verification. The optimized UGT74AC1 demonstrates significant improvements in expression levels and thermal stability, laying a good foundation for subsequent functional research and applications. The invention mutates multiple sites in the active center of the UGT74AC1 protein to optimize its substrate compatibility. The mutated enzyme exhibits higher catalytic efficiency across a variety of substrate conditions, demonstrating a broader substrate spectrum and superior enzymatic performance. This optimization significantly enhances the potential of UGT74AC1 in practical applications and lays a foundation for subsequent biosynthesis. To address the application needs of UGT74AC1, the invention designs and constructs an efficient reaction system to synthesize the required glycosyl donors, UDP-Glc and UDP-GlcNAc, through a cascade of enzymatic reactions. This system integrates the synergistic catalytic action of multiple enzymes, achieving a one-pot synthesis of triterpenoid saponins from simple substrates, effectively improving reaction efficiency and product yield. This method provides a new efficient and economical strategy for the synthesis of complex glycosylation products.

[0070] This study proposes the first comprehensive modification of UGT74AC1. By optimizing its structure, it increases its expression and stability in heterologous E. coli BL21 (DE3). By modifying its active center, it adapts to a wider range of substrates. This study also proposes a cascade multi-enzyme system containing the engineered glycosyltransferase mUGT74AC1, enabling the cost-effective preparation of triterpenoid saponins.

[0071] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0072] (1) The present invention optimizes the overall structure of the UGT74AC1 enzyme by modifying it, thereby increasing the heterologous expression level of the enzyme in E. coli and the stability of the expressed protein, and greatly reducing production costs and difficulty.

[0073] (2) The present invention uses the modified UGT74AC1 enzyme mUGT74AC1 to synthesize triterpenoid saponins, which greatly improves the catalytic activity of the enzyme and enables it to accept different triterpenoid compounds as substrates and different glycosyl donors, thereby obtaining triterpenoid saponins with different glycosylation, which has great application prospects.

[0074] (3) In the method provided by the present invention, through single-enzyme, double-enzyme, and triple-enzyme cascade catalysis, cheap sucrose and GlcNAc can be used as glycosyl donors, which greatly reduces production costs and simplifies the manufacturing process.

[0075] (4) In the method provided by the present invention, the reaction product has a low impurity content and is easy to separate, which is conducive to subsequent separation and purification in industrial-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 This is the map of the mUGT74AC recombinant plasmid.

[0077] Figure 2 This is the SDS-PAGE gel image of mUGT74AC1.

[0078] Figure 3 The standard curve of Bradford protein quantification method.

[0079] Figure 4 This is the pH optimization diagram for the mUGT74AC1 reaction.

[0080] Figure 5 This is the reaction temperature optimization diagram for mUGT74AC1.

[0081] Figure 6 Optimization diagram of salt ion species for the mUGT74AC1 reaction.

[0082] Figure 7 This is the ESI-MS spectrum of Mogrol-3-O-β-D-Glucose.

[0083] Figure 8 This is the HPLC chart of Mogrol-3-O-β-D-Glucose catalyzed by UGT74AC1 before transformation.

[0084] Figure 9 HPLC chart of the synthesis of Mogrol-3-O-β-D-Glucose catalyzed by mUGT74AC1.

[0085] Figure 10 This is the H NMR spectrum of the product Mogrol-3-O-β-D-Glucose 1 H NMR (400 MHz, Methanol-d4).

[0086] Figure 11 This is the C NMR spectrum of the product Mogrol-3-O-β-D-Glucose 13 C NMR (101 MHz, Methanol-d4).

[0087] Figure 12 This is the ESI-MS spectrum of Mogrol-3-O-β-D-GlcNAc.

[0088] Figure 13This is the HPLC chart of Mogrol-3-O-β-D-GlcNAc catalyzed by UGT74AC1 before transformation.

[0089] Figure 14 HPLC chart of the synthesis of Mogrol-3-O-β-D-GlcNAc catalyzed by mUGT74AC1.

[0090] Figure 15 This is the ESI-MS spectrum of Mogrol-3-O-β-D-Gal.

[0091] Figure 16 This is the HPLC chart of Mogrol-3-O-β-D-Gal catalyzed by UGT74AC1 before transformation.

[0092] Figure 17 HPLC chart of the synthesis of Mogrol-3-O-β-D-Gal catalyzed by mUGT74AC1.

[0093] Figure 18 This is the AESI-MS spectrum of Mogrol-3-O-β-D-Glc.

[0094] Figure 19 This is the HPLC chart of Mogrol-3-O-β-D-GlcA catalyzed by UGT74AC1 before transformation.

[0095] Figure 20 HPLC chart of the synthesis of Mogrol-3-O-β-D-GlcA catalyzed by mUGT74AC1.

[0096] Figure 21 This is the ESI-MS spectrum of Cucurbitacin F-25-acetate-2-O-β-D-Glucose.

[0097] Figure 22 HPLC chart of the synthesis of Cucurbitacin F-25-acetate-2-O-β-D-Glucose catalyzed by mUGT74AC1. DETAILED DESCRIPTION

[0098] The present invention will be further described below with reference to the accompanying drawings and examples.

[0099] Unless otherwise specified, the reagents used in the examples of the present invention can be obtained from commercial sources. Experimental methods without specific conditions in the examples are generally performed under conventional conditions or the conditions recommended by the manufacturer.

[0100] Experimental operations not described in detail in the present invention can be carried out according to conventional experimental operations in this technical field.

[0101] MgCl2: purchased from Sigma, product number: M8266, CAS: 7786-30-3

[0102] Tris: purchased from Sigma, product number: 648311, CAS: 77-86-1

[0103]

[0104] UDP-GlcNAc: purchased from Sigma, product number: U4375, CAS: 91183-98-1,

[0105]

[0106] UDP-Glc: purchased from Sigma, product number: U4625, CAS number: 28053-08-9

[0107]

[0108] Uridine-5′-diphosphoglucuronic acid trisodium salt (UDP-GlcA): purchased from Sigma, product number: U6751, CAS: 63700-19-6

[0109]

[0110] UDP-Gal: purchased from Maclean, product number: U980872, CAS: 2956-16-3

[0111]

[0112] Sucrose: purchased from Maclean, product number: S818046-500g, CAS: 57-50-1

[0113]

[0114] Uridine-5′-diphosphate disodium salt (UDP): purchased from Sigma, product number: 94330, CAS: 27821-45-0

[0115]

[0116] Adenosine 5'-triphosphate disodium salt (ATP): purchased from Sigma, product number: A1852, CAS: 34369-07-8

[0117]

[0118] Uridine-5′-triphosphate trisodium salt (UTP): purchased from Sigma, product number: 94370, CAS: 116295-90-0

[0119]

[0120] Mogroside alcohol: purchased from Shanghai Yuanye, product number: B28255, CAS: 88930-15-8

[0121]

[0122] Cucurbitacin F-25-acetate: purchased from MCE, product number: HY-N8137, CAS: 99530-82-2

[0123]

[0124] Sucrose synthase SuSy (WP_011381564.1) was artificially synthesized according to the sequence on NCBI and obtained by fermentation according to the literature (Liting Zhao, Zhongbao Ma, Qiong Wang, Manfeng Hu, Jingxiang Zhang, Lei Chen, Guiyang Shi, and Zhongyang Ding Journal of Agricultural and Food Chemistry 2023 71(8), 3832-3841).

[0125] N-acetylglucosamine-1-phosphate uridine transferase GlmU (NC_000962.3) was artificially synthesized according to the sequence in NCBI and obtained by fermentation according to the literature (Zhou, Y., Yu, W., Zheng, Q. et al. Identification of aminoacids involved in catalytic process of M. tuberculosis GlmUacetyltransferase. Glycoconj J 29, 297–303 (2012).).

[0126] N-acetylhexosamine kinase NahK (ANC68241.1) was artificially synthesized according to the sequence on NCBI and obtained by fermentation according to the literature (Zheng, J.; Xu, H.; Li, BZ; Sohail, M.; Bi, JJ; Zhang, FM; Linhardt, RJ; Huang, H.; Zhang, X. Spatially Segregated MOF Bioreactor Enables Versatile Modular Glycoenzyme Assembly for Hierarchical Glycan Library Construction. ACS Appl. Mater. Interfaces 2023, 15(16): 19807-19816.).

[0127] Culture medium:

[0128] LB medium (liquid): 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0129] TB medium (liquid): 13.33 g / L peptone, 26.67 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, 5.61 g / L glycerol.

[0130] In the following examples, the HPLC detection method used used an Agilent C18 column and an Agilent 1260-LC high performance liquid chromatograph. The ultraviolet absorption of each component at 262 nm and 310 nm was detected after the above-mentioned heparin skeleton synthase catalyzed product was separated by the chromatographic column.

[0131] Example 1

[0132] Construction of mutants

[0133] The codon-optimized UGT74AC1 gene (as shown in SEQ ID NO. 1) was synthesized and ligated between the restriction sites BamHI (GGATCC) and XhoI (CTCGAG) of the expression vector pET21a(+). The gene was then transformed into E. coli DH5α. After culture on LB plates, single clones were picked and verified by Qingke Biotechnology to obtain positive transformants. Plasmids were extracted from the positive transformants, namely pET21a(+)-UGT74AC1.

[0134] First, using the wild-type plasmid pET21a(+)-UGT74AC1 WT as a template and the mutant sequences designed into the primers, PCR was performed to amplify the DNA fragments with the mutated base sequences. The corresponding primer sequences for the different mutant plasmids are shown in Table 1. Whole-plasmid PCR was used to construct the single-point mutant plasmid pET21a(+)-S15A. Subsequently, the mutant plasmids pET21a(+)-S15A-H47D-L48V were constructed using the single-point mutant plasmids as templates. The above process was repeated, using the mutants from the previous round as templates and the sequences in Table 1 as primers to construct the mutant pET21a(+)-mUGT74AC1 (S15A / H47D / L48V / A180S / S332P / F367W / K420R). The amplification system is shown in Table 2, and the PCR amplification reaction conditions are shown in Table 3.

[0135] After the mutation is completed, the mutant plasmid is introduced into E. coli BL21 (DE3) (for heterologous expression of recombinase protein) competent cells to obtain the corresponding recombinant E. coli containing the target gene. The sequencing results of the obtained plasmid are correct, and the correct plasmid map is as follows Figure 1 As shown, the mutant gene inserted after sequencing is shown as SEQ ID NO.2.

[0136] The primers used for mutation are as follows:

[0137] Table 1 Primer sequences

[0138]

[0139] Table 2 PCR amplification system

[0140]

[0141] Table 3 PCR reaction conditions

[0142]

[0143] Example 2

[0144] Expression of recombinase

[0145] 1) Activation culture: Inoculate solid bacteria stored at -80°C into 5 mL of LB liquid medium containing Kan resistance (final concentration of 50 μg / mL) and incubate overnight in a shaker at 37°C and 220 rpm (incubation time: 14 hours).

[0146] 2) Scale-up: Pipette 10 mL of the activated bacterial solution into a 2-L Erlenmeyer flask containing 1 L of TB liquid medium (containing Kan resistance). Incubate at 37°C and 220 rpm in a shaker until the OD value reaches 0.6-0.8.

[0147] 3) Induction of expression: When the OD value of the bacterial solution reaches 0.6-0.8, stop the 37°C culture and add 500 μL of IPTG (1 M) inducer to the conical flask. Change the temperature to 16°C and the rotation speed to 180 r / min. Continue to culture at low temperature in a constant temperature shaking incubator for 24 h to induce overexpression of the recombinase.

[0148] 4) Harvest the Bacteria by Centrifugation: Centrifuge the recombinant enzyme solution from step 3) after low-temperature induction of expression in a floor-standing centrifuge at 4°C, 6000 rpm, and 10 minutes. After centrifugation, discard the supernatant and collect the bacterial pellet. Resuspend and wash the solution in 50 mM PBS buffer, pH 8.0, to a volume of 40 ml. Centrifuge at 4°C, 8000 rpm for 10 minutes. Discard the supernatant and collect the bacterial pellet.

[0149] 5) Bacterial disruption: After washing, add 50 mL of 50 mM PBS buffer (pH 8.0) to the cell pellet, resuspend it, and then disrupt the cells using a cell disruptor. Centrifuge at 15,000 rpm for 30 minutes, and collect the supernatant as the crude enzyme solution.

[0150] 6) Purify the target protein by affinity chromatography: Add the crude enzyme solution filtered through a 0.45 μm membrane to a nickel column, incubate on ice for 20 min, and perform gradient elution using 30 mM, 150 mM, and 250 mM imidazole, respectively. Elution with 30 mM equates to impurities, while elution with 150 mM equates to the target protein.

[0151] 7) Concentration: The obtained enzyme solution was transferred to an ultrafiltration tube and centrifuged at 5000 rpm for 15 min to obtain the concentrated enzyme solution. The protein size was determined by running an SDS-PAGE gel. Figure 2 shown.

[0152] 8) Enzyme solution protein concentration measurement: After the concentrated enzyme solution was diluted 15 times with storage buffer, the total protein content in the enzyme solution was measured using a Bradford protein quantification kit in a multifunctional microplate reader. The standard curve is as follows: Figure 3 As shown, the absorbance of the diluted enzyme solution at 595 nm was measured to be 1.10, so the concentration of the concentrated enzyme solution was calculated to be 11.7 mg / mL.

[0153] The amino acid sequence of the recombinant enzyme (mUGT74AC1) constructed in Example 2 of the present invention is shown in SEQ ID NO.4, and its encoding gene is shown in SEQ ID NO.2.

[0154] Example 3

[0155] Univariate factor analysis of mUGT74AC1

[0156] A univariate factor exploration experiment on the enzyme catalytic reaction conditions of mUGT74AC1 was conducted to identify its enzyme properties.

[0157] 1) To study the effect of reaction system pH on the catalytic efficiency of mUGT74AC1, three buffer systems were used at 40°C: acetate buffer (50mM, pH buffer range 4.0-5.5), phosphate buffer (50mM, pH buffer range 6.0-8.0), and tris(hydroxymethyl)aminomethane hydrochloride buffer (50mM, pH buffer range 7.5-9.0). Three corresponding reaction systems were then prepared. A final concentration of 0.25mM UDP-Glc, 0.25mM Mogrol (mogrol), 1mM MgCl2, and 0.1mg / mL mUGT74AC1 were added to the reaction system. The reaction was carried out at 200rpm for 5h, and the changes in enzyme activity within the pH range of 4.0-8.5 were detected. Figure 4 As shown, the optimum pH of the enzyme is 8.0 (Tris hydrochloride buffer), and when the pH is equal to 7.0, the catalytic efficiency can be maintained at 60%, indicating that the enzyme has certain potential for biocatalytic large-scale production of triterpenoid saponins.

[0158] 2) To investigate the effect of temperature on the catalytic efficiency of mUGT74AC1, a 50 mM Tris-HCl solution (pH 8.0) was added to the reaction system with a final concentration of 0.25 mM UDP-Glc, 0.25 mM Mogrol (mogrol), 1 mM MgCl2, and 0.1 mg / mL mUGT74AC1. The reaction was run at 200 rpm for 5 h. The catalytic efficiency of the enzyme was measured at 16°C, 25°C, 35°C, 37°C, 40°C, 45°C, 55°C, and 65°C. The catalytic efficiency of the glycosyltransferase decreased significantly at temperatures above 45°C, but exhibited higher catalytic activity within the range of 37°C to 45°C (e.g., Figure 5 The yield of triterpenoid saponins was the highest at 40°C, so the enzyme had the highest catalytic efficiency at 40°C.

[0159] 3) This experiment investigated the effects of various metal ions and ammonium ions on the catalytic efficiency of mUGT74AC1. A 50 mM Tris-HCl solution (pH 8.0) was added to the reaction system with a final concentration of 0.25 mM UDP-Glc, 0.25 mM Mogrol, 1 mM metal ions, and 0.1 mg / mL mUGT74AC1. The reaction was carried out at 40°C and 200 rpm for 5 h. Figure 6 As shown, Mg 2+ , Ca 2+ 、Mn 2+ and Fe 3+ The catalytic efficiency of the enzyme is significantly promoted, among which Mg 2+ The promoting effect of Ca was the strongest. Under the same conditions, the yield of triterpenoid saponins could reach 68.7%, which was about 6 times higher than that of the blank control group (blank group without metal ions). 2+ and Mn 2+ The conversion rate is about 59% under the condition of the presence of NH4 + It also has a strong promoting effect on the enzyme, with a conversion rate of about 53%. 2+ 、Zn 2+ and Ni 2+ It has a significant inhibitory effect on the catalytic efficiency of the enzyme, among which Cu 2+ Ions have the strongest inhibitory effect on the enzyme. In the presence of copper ions, the enzyme conversion rate is about 4%, and Zn 2+ The catalytic efficiency of the enzyme was only half of that of the blank control group when Mg was present. The above results indicate that the enzyme is a metal-dependent enzyme, in which Mg 2+ The most effective way to promote the enzyme is Cu 2+ Ions have the strongest inhibitory effect on the enzyme, significantly reducing its activity.

[0160] 4) To investigate the effects of rotational speed and reaction time on the catalytic efficiency of mUGT74AC1, a final concentration of 0.25 mM UDP-Glc, 0.25 mM Mogrol, 1 mM MgCl2, and 0.1 mg / mL mUGT74AC1 were added to the reaction system in a 50 mM Tris-HCl solution (pH 8.0) at 40°C. The reaction was carried out at 100 rpm, 150 rpm, 200 rpm, and 300 rpm for 24 h. Samples were taken every 2 h to monitor the catalytic efficiency of the enzyme. The glycosyltransferase achieved the highest conversion rate of approximately 80% at a rotational speed of 200 rpm and a reaction time of 5 h.

[0161] Example 4

[0162] Enzymatic synthesis of Mogrol-3-O-β-D-Glucose from Mogrol using UDP-Glc as a glycosyl donor

[0163] 1) Catalytic reaction system

[0164] 1mM UDP-Glc (uridine diphosphate glucose), mUGT74AC1 (0.5mg / mL), 0.25mM Mogrol (mogrol), and 1mM MgCl2 were added to the reaction system, and Tris-HCl (50mM) buffer with a pH of 8.0 was used as the solvent. The reaction was carried out at 40°C and 200rpm for 5h. The sample was placed in a 100°C water bath and heated for 10min. The sample was centrifuged at 12000rpm at room temperature for 1min. The supernatant was filtered through a 0.22μm sterile filter membrane into a liquid phase vial and analyzed by HPLC liquid chromatography and ESI mass spectrometry. The wild type (UGT74AC1), intermediate mutant UGT74AC1(S15A), intermediate mutant UGT74AC1(S15A / H47D), and intermediate mutant UGT74AC1(S15A / H47D / L48V) were used as controls to compare the differences in catalytic activity between the mutant mUGT74AC1 and the wild type and intermediate mutant. The catalytic system of the control group used the wild type UGT74AC1 enzyme before modification and the intermediate mutant (0.5 mg / mL), and the other reaction conditions were the same.

[0165] 2) HPLC detection method

[0166] High-performance liquid chromatography (HPLC) was used to determine the substrate and product contents in the post-enzyme reaction system. The column used was an Ultimate XB-C18 column, 4.6 mm × 250 mm, 5 μm, Welch, Shanghai, China. The mobile phases consisted of A (H2O and 0.1% formic acid) and B (acetonitrile plus 0.1% formic acid), with a flow rate of 1 mL / min. The mobile phase gradient was 25% to 85% B over 0–25 min. UV detection was performed at 205 nm.

[0167] 3) Results

[0168] like Figure 7 As shown, the ESI mass spectrum is [M+Na + ]661.2. According to the HPLC test results, Figure 8As shown, the conversion rate of wild-type UGT74AC1 to Mogrol-3-O-β-D-Glucose (mogroside I E1) was 31%, the conversion rate of intermediate mutant UGT74AC1 (S15A) was 39%, the conversion rate of intermediate mutant UGT74AC1 (S15A / H47D) was 58%, and the conversion rate of intermediate mutant UGT74AC1 (S15A / H47D / L48V) was 71%. In contrast, the mutant mUGT74AC1 enzyme catalyzed the conversion of mogrol to Mogrol-3-O-β-D-Glucose with a conversion rate of up to 99% (as shown in Figure 2). Figure 9 As shown), the enzyme yield was significantly improved compared with the wild type and other intermediate mutants, and no by-products were generated. 1 HNMR spectrum Figure 10 As shown, nuclear magnetic 13 C NMR spectrum Figure 11 shown.

[0169] 1 H NMR (400MHz, Methanol-d4) δ5.50(d,J=5.1Hz,1H),4.28(d,J=7.7Hz,1H),3.84(dd,J=17.8,7.9Hz,2H),3.65(dd,J=11 .8,5.3Hz,1H),3.42(s,1H),3.24–3.16(m,3H),2.48(d,J=11.9Hz,1H),2.39(dd,J=18.9,7.1Hz,1H),2.22(d,J=9.6Hz, 1H),1.96(d,J=8.0Hz,3H),1.88–1.76(m,3H),1.67(d,J=7.3Hz,1H),1.64–1.57(m,1H),1.54–1.47(m,3H),1.36–1.29 (m,3H),1.19(s,2H),1.16(s,3H),1.12(d,J=5.0Hz,5H),1.07(s,3H),0.97(d,J=6.1Hz,3H),0.92(s,3H),0.87(s,3H). 13C NMR (101MHz, Methanol-d4) δ143.52,118.26,105.20,87.16,78.32,77.95,76.85,76.27,74.19,72.47,70.27,50.40,49.18,43.26,41.4 9,39.65,39.48,35.89,35.65,33.92,33.06,28.18,27.67,27.37,26 .42,25.83,24.91,24.79,24.33,23.68,23.49,18.39,17.71,15.70.

[0170] Example 5

[0171] Enzymatic synthesis of Mogrol-3-O-β-D-GlcNAc from Mogrol using UDP-GlcNAc as a glycosyl donor

[0172] 1) Catalytic reaction system

[0173] A reaction system containing 1 mM UDP-GlcNAc, mUGT74AC1 (0.5 mg / mL), 0.25 mM Mogrol, and 1 mM MgCl2 was added, using Tris-HCl (50 mM) buffer (pH 8.0) as the solvent. The reaction was incubated at 40°C, 200 rpm, for 5 h. Samples were then heated in a 100°C water bath for 10 min and centrifuged at 12,000 rpm for 1 min at room temperature. The supernatant was filtered through a 0.22 μm sterile filter membrane into a liquid phase vial and analyzed by HPLC and ESI mass spectrometry. The wild type (UGT74AC1), intermediate mutant UGT74AC1(S15A), intermediate mutant UGT74AC1(S15A / H47D), and intermediate mutant UGT74AC1(S15A / H47D / L48V) were used as controls to compare the differences in catalytic activity between the mutant mUGT74AC1 and the wild type and intermediate mutant. The catalytic system of the control group used UGT74AC1 before modification and the intermediate mutant enzyme (0.5 mg / mL), and the other reaction conditions were the same.

[0174] 2) HPLC detection method

[0175] High-performance liquid chromatography (HPLC) was used to determine the substrate and product contents in the post-enzyme reaction system. The column used was an Ultimate XB-C18 column, 4.6 mm × 250 mm, 5 μm, Welch, Shanghai, China. The mobile phases consisted of A (H2O and 0.1% formic acid) and B (acetonitrile plus 0.1% formic acid), with a flow rate of 1 mL / min. The mobile phase gradient was 25% to 85% B over 0–40 min. UV detection was performed at 205 nm.

[0176] 3) Results

[0177] like Figure 12 As shown, the ESI mass spectrum is [MH + ]678.4. According to the HPLC test results, Figure 13 As shown, the conversion rate of wild-type UGT74AC1 to Mogrol-3-O-β-D-GlcNAc was less than 1%, the conversion rate of the intermediate mutant UGT74AC1 (S15A) was less than 5%, the conversion rate of the intermediate mutant UGT74AC1 (S15A / H47D) was 26%, and the conversion rate of the intermediate mutant UGT74AC1 (S15A / H47D / L48V) was 46%. Figure 8 As shown, according to the analysis and calculation of the liquid phase detection results, after 5 hours of reaction, the mutant mUGT74AC1 converted mogrol into Mogrol-3-O-β-D-GlcNAc with a conversion rate of up to 70% (as shown in Figure 14 The catalytic yield was significantly improved compared with the wild-type enzyme and other intermediate mutants, and no by-products were generated.

[0178] Example 6

[0179] Enzymatic synthesis of Mogrol-3-O-β-D-Gal from Mogrol using UDP-Gal as a glycosyl donor

[0180] 1) Catalytic reaction system

[0181] A reaction system containing 1 mM UDP-Gal, mUGT74AC1 (0.5 mg / mL), 0.25 mM Mogrol, and 1 mM MgCl2 was added, using Tris-HCl (50 mM) buffer (pH 8.0) as the solvent. The reaction was incubated at 40°C, 200 rpm, and 5 h. Samples were then heated in a 100°C water bath for 10 min and centrifuged at 12,000 rpm for 1 min at room temperature. The supernatant was filtered through a 0.22 μm sterile filter into a liquid phase vial and analyzed by HPLC and ESI mass spectrometry. The catalytic activity of the mutant mUGT74AC1 was compared with that of the wild-type (UGT74AC1) enzyme. The control catalytic system used the unmodified UGT74AC1 enzyme (0.5 mg / mL). All other reaction conditions remained the same.

[0182] 2) HPLC detection method

[0183] High-performance liquid chromatography (HPLC) was used to determine the substrate and product contents in the post-enzyme reaction system. The column used was an Ultimate XB-C18 column, 4.6 mm × 250 mm, 5 μm, Welch, Shanghai, China. The mobile phases consisted of A (H2O and 0.1% formic acid) and B (acetonitrile plus 0.1% formic acid), with a flow rate of 1 mL / min. The mobile phase gradient was 25% to 85% B over 0–40 min. UV detection was performed at 203 nm.

[0184] 3) Results

[0185] like Figure 15 As shown, the ESI mass spectrum is [M+Cl - ]673.1. According to the HPLC test results, Figure 16 As shown, the wild-type UGT74AC1 catalyzed the conversion of Mogrol to Mogrol-3-O-β-D-Gal with a conversion rate of 23%. Figure 17 As shown, according to the analysis and calculation of the liquid phase detection results, after 5 hours of reaction, the mutant mUGT74AC1 converted Mogrol into Mogrol-3-O-β-D-Gal with a conversion rate of up to 99%. The catalytic yield was greatly improved compared with the wild-type enzyme, and no by-products were generated.

[0186] Example 7

[0187] Enzymatic synthesis of Mogrol-3-O-β-D-GlcA from Mogrol using UDP-GlcA as a glycosyl donor

[0188] 1) Catalytic reaction system

[0189] A reaction system containing 1 mM UDP-GlcA, mUGT74AC1 (0.5 mg / mL), 0.25 mM Mogrol, and 1 mM MgCl2 was added, using Tris-HCl (50 mM) buffer (pH 8.0) as the solvent. The reaction was incubated at 40°C, 200 rpm, and 5 h. Samples were then heated in a 100°C water bath for 10 min and centrifuged at 12,000 rpm for 1 min at room temperature. The supernatant was filtered through a 0.22 μm sterile filter into a liquid phase vial and analyzed by HPLC and ESI mass spectrometry. The catalytic activity of the mutant mUGT74AC1 was compared with that of the wild-type (UGT74AC1) enzyme. The control catalytic system used the unmodified UGT74AC1 enzyme (0.5 mg / mL). All other reaction conditions remained the same.

[0190] 2) HPLC detection method

[0191] High-performance liquid chromatography (HPLC) was used to determine the substrate and product contents in the post-enzyme reaction system. The column used was an Ultimate XB-C18 column, 4.6 mm × 250 mm, 5 μm, Welch, Shanghai, China. The mobile phases consisted of A (H2O and 0.1% formic acid) and B (acetonitrile plus 0.1% formic acid), with a flow rate of 1 mL / min. The mobile phase gradient was 25% to 85% B over 0–40 min. UV detection was performed at 210 nm.

[0192] 3) Results

[0193] like Figure 18 As shown, the ESI mass spectrum is [M+Na + ]675.1. According to the HPLC test results, Figure 19 As shown in Figure 2, the conversion rate of wild-type UGT74AC1 to Mogrol-3-O-β-D-GlcA was less than 1%. In contrast, according to the analysis and calculation of the liquid phase detection results, after 5 h of reaction, the mutant mUGT74AC1 converted Mogrol to Mogrol-3-O-β-D-GlcA with a conversion rate of up to 40% (as shown in Figure 2). Figure 20 The catalytic yield was significantly improved compared with the wild-type enzyme, and no by-products were generated.

[0194] Example 8

[0195] Sucrose as glycosyl donor catalyzes the conversion of Mogrol-3-O-β-D-Glucose into

[0196] 1) Catalytic reaction system

[0197] 0.25 mM Mogrol (mogrol), 2 mM sucrose, 4 mM 5-uridine diphosphate disodium salt (UDP), 1 mM MgCl2, 0.8 mg / mL sucrose synthase (SuSy), and 1 mg / mL mUGT74AC1 were added, using Tris-HCl (50 mM) buffer, pH 8.0, as the solvent. The reaction was incubated at 40°C, 200 rpm for 5 h, and the sample was heated in a 100°C water bath for 10 min. The sample was centrifuged at 12,000 rpm for 1 min at room temperature. The supernatant was filtered through a 0.22 μm sterile filter membrane into a liquid phase vial and analyzed by HPLC and ESI mass spectrometry.

[0198] 2) HPLC detection method

[0199] High-performance liquid chromatography (HPLC) was used to determine the substrate and product contents in the post-enzyme reaction system. The column used was an Ultimate XB-C18 column, 4.6 mm × 250 mm, 5 μm, Welch, Shanghai, China. The mobile phases consisted of A (H2O and 0.1% formic acid) and B (acetonitrile plus 0.1% formic acid), with a flow rate of 1 mL / min. The mobile phase gradient was 25% to 85% B over 0–40 min. UV detection was performed at 205 nm.

[0200] 3) Results

[0201] ESI mass spectrum is [M+H + According to the analysis and calculation of the liquid phase detection results, after 7 hours of reaction, mogrol was converted into mogrol-3-O-β-D-Glucose with a conversion rate of up to 89% and no by-products were generated.

[0202] Example 9

[0203] GlcNAc as a glycosyl donor catalyzes the three-enzyme process for the synthesis of Mogrol-3-O-β-D-GlcNAc

[0204] 1) Catalytic reaction system

[0205] 0.25 mM Mogrol, 2 mM GlcNAc, 4 mM UTP, 4 mM ATP, 1 mM MgCl2, 0.8 mg / mL NahK, 0.5 mg / mL GlmU, and 1 mg / mL mUGT74AC1 were added to a 50 mM Tris-HCl buffer (pH 8.0). The reaction was incubated at 35°C, 200 rpm, for 10 h. Samples were then heated in a 100°C water bath for 10 min and centrifuged at 12,000 rpm for 1 min at room temperature. The supernatant was filtered through a 0.22 μm sterile filter membrane into a liquid phase vial and analyzed by HPLC and ESI mass spectrometry.

[0206] 2) HPLC detection method

[0207] High-performance liquid chromatography (HPLC) was used to determine the substrate and product contents in the post-enzyme reaction system. The column used was an Ultimate XB-C18 column, 4.6 mm × 250 mm, 5 μm, Welch, Shanghai, China. The mobile phases consisted of A (H2O and 0.1% formic acid) and B (acetonitrile plus 0.1% formic acid), with a flow rate of 1 mL / min. The mobile phase gradient was 25% to 85% B over 0–40 min. UV detection was performed at 210 nm.

[0208] 3) Results

[0209] ESI mass spectra are [M+Na + According to the analysis and calculation of the liquid phase detection results, after 10 hours of reaction, mogrol was converted into mogrol-3-O-β-D-GlcNAc with a conversion rate of up to 65% and no by-products were generated.

[0210] Example 10

[0211] Cucurbitacin F-25-acetate-2-O-β-D-Glucose is produced by a dual-enzyme cascade using cucurbitacin as a glycosyl acceptor.

[0212] 1) Catalytic reaction system

[0213] 0.25 mM Cucurbitacin F-25-acetate, 2 mM sucrose, 4 mM UDP, 1 mM MgCl2, 0.8 mg / mL sucrose synthase, and 1 mg / mL mUGT74AC1 were added to a 50 mM Tris-HCl buffer (pH 7.4) as the solvent. The reaction was incubated at 30°C, 200 rpm for 6 h, and terminated by the addition of 300 μl of icy methanol. The supernatant was centrifuged at 12,000 rpm for 10 min, and filtered through a 0.22 μm sterile filter into a liquid phase vial. The supernatant was then analyzed by HPLC and ESI mass spectrometry.

[0214] 2) HPLC detection method

[0215] High-performance liquid chromatography (HPLC) was used to determine the substrate and product contents in the post-enzyme reaction system. The column used was an Ultimate XB-C18 column, 4.6 mm × 250 mm, 5 μm, Welch, Shanghai, China. The mobile phases consisted of A (H2O and 0.1% formic acid) and B (acetonitrile plus 0.1% formic acid), with a flow rate of 1 mL / min. The mobile phase gradient was 25% to 85% B over 0–40 min. UV detection was performed at 205 nm.

[0216] 3) Results

[0217] like Figure 21 The ESI mass spectrum shown is [M+H + ]723.38. According to the analysis and calculation of the liquid phase detection results, after 6 hours of reaction, Cucurbitacin F-25-acetate was converted into Cucurbitacin F-25-acetate-2-O-β-D-Glucose, and the conversion rate reached 50% ( Figure 22 ), no by-products are generated.

Claims

1. An engineered glycosyltransferase, characterized in that The engineered glycosyltransferase is a mutant of the UGT74AC1 enzyme, mUGT74AC1. The mutant mUGT74AC1 is obtained by mutating the serine at position 15 of the UGT74AC1 enzyme as shown in SEQ ID NO.3 to alanine, the histidine at position 47 to aspartic acid, the leucine at position 48 to valine, the alanine at position 180 to serine, the serine at position 332 to proline, the phenylalanine at position 367 to tryptophan, and the lysine at position 420 to arginine.

2. A gene encoding the engineered glycosyltransferase according to claim 1, characterized in that: The gene sequence of the mutant mUGT74AC1 is shown in SEQ ID NO.

2.

3. A recombinant vector or recombinant bacterium, characterized in that: It contains the gene encoding the engineered glycosyltransferase according to claim 2.

4. Use of the engineered glycosyltransferase according to claim 1, the encoding gene according to claim 2, or the recombinant vector or recombinant bacterium according to claim 3 in the preparation of triterpenoid saponins using a bioenzymatic method or a bioenzymatic cascade method.

5. The use according to claim 4, characterized in that The process of preparing triterpenoid saponins by using the bioenzymatic method is to add a glycosyl acceptor terpenoid compound, a glycosyl donor, an engineered glycosyltransferase, and a salt into a buffer solution, mix them evenly to obtain a mixed solution, and perform an enzyme-catalyzed reaction under stirring to obtain the triterpenoid saponins.

6. The use according to claim 5, characterized in that The terpene compounds in the mixed solution include mogroside alcohol, steviol, cucurbitacin, andrographolide, ginseng alcohol, glycyrrhetinic acid, ursolic acid or betulinic acid, and the concentration of the terpene compounds in the mixed solution is 0.01-1mM; the glycosyl donor includes uridine diphosphate glucose, uridine diphosphate galactose, uridine diphosphate glucuronic acid or uridine diphosphate acetylglucosamine, and the concentration of the glycosyl donor in the mixed solution is 0.05-20mM.

7. The use according to claim 5, characterized in that The salt includes magnesium salt, calcium salt, iron salt, manganese salt, copper salt, ammonium salt or zinc salt, and the concentration of the salt in the mixed solution is 0.1-10mM; the pH of the buffer solution is 4.0-9.0; the stirring rate is 100-300rpm; the temperature of the enzyme-catalyzed reaction is 15-60°C; the time of the enzyme-catalyzed reaction is 1-24h; and the enzyme concentration of the enzyme-catalyzed reaction is 0.01-10g / L.

8. The use according to claim 4, characterized in that The process of preparing triterpenoid saponins by bioenzymatic cascade is as follows: adding a glycosyl acceptor terpenoid compound, a glycosyl donor, any one or more of uridine-5'-diphosphate disodium salt or adenosine-5'-triphosphate disodium salt or uridine-5'-triphosphate trisodium salt, a cascade multi-enzyme containing an engineered glycosyltransferase, and salt to a buffer solution, mixing them evenly to obtain a mixed solution, and performing an enzyme-catalyzed reaction under stirring to obtain the triterpenoid saponins.

9. The use according to claim 8, characterized in that The glycosyl acceptor terpenoid compound in the mixed solution includes mogrohol, steviol, cucurbitacin, andrographolide, ginseng alcohol, glycyrrhetinic acid, ursolic acid or betulinic acid, with a concentration of 0.01-1mM; the glycosyl donor or the precursor of the glycosyl donor is sucrose or acetylglucosamine, with a concentration of 0.05-20mM; the concentration of uridine-5'-diphosphate disodium salt is 0.05-20mM; the concentration of adenosine-5'-triphosphate disodium salt is The concentration of the trisodium salt of uridine-5′-triphosphate is 0.1-20 mM; the salts include magnesium salts, calcium salts, iron salts, manganese salts, copper salts, ammonium salts, and zinc salts, with a concentration of 0.1-10 mM; the pH of the buffer solution is 4.0-9.0; the stirring rate is 100-300 rpm, the temperature of the enzyme-catalyzed reaction is 15-60° C., and the time of the enzyme-catalyzed reaction is 1-24 h.

10. The use according to claim 8, characterized in that The cascade multi-enzyme is preferably SuSy and mUGT74AC1, wherein the SuSy enzyme concentration is 0.01-10 g / L, wherein the mUGT74AC1 concentration is 0.01-10 g / L; or the cascade multi-enzyme is preferably NahK, GlmU and mUGT74AC1, wherein the NahK enzyme concentration is 0.01-10 g / L, wherein the GlmU concentration is 0.01-10 g / L, wherein the mUGT74AC1 concentration is 0.01-10 g / L.

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