Application of constructed efficient glycosyl transferase cascade catalytic system in synthesis of novel triterpenoid glycoside

Abstract

The invention relates to an application of constructing an efficient glycosyl transferase cascade catalytic system in synthesis of novel triterpenoid glycoside. Specifically, the invention provides a glycosyl transferase mutant, a sucrose synthase SUS mutant and a cascade catalytic system containing the glycosyl transferase mutant and the sucrose synthase SUS mutant, the invention firstly discovers that the glycosyl transferase mutant UGT109A3m and the sucrose synthase SUSm are combined according to a certain proportion, and the sucrose synthase SUSm can be used for preparing the sucrose synthase. Synergistic cascade catalysis can be achieved to obtain high yield tripterine glycoside derivatives.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of constructing an efficient glycosyltransferase cascade catalytic system in synthesizing novel triterpenoid glycosides. Background Art

[0002] Glycosylation modification is a post-modification reaction that is widely present in natural products. It usually occurs in the terminal pathway of biosynthesis and helps to increase the diversity and complexity of natural product structures. Glycosyltransferase-mediated enzymatic glycosylation is an important strategy for the glycosylation of natural products. The glycosylation modification reaction of plant secondary metabolites is mainly catalyzed by uridine diphosphate (UDP)-glycosyltransferase (UGT). UDP-glycosyltransferase transfers sugar molecules from a donor to an acceptor and forms a glycosidic bond at the same time. The glycosylation modification of natural products can change the water solubility, stability, and recognition of cell receptors of compounds, ultimately determining their bioavailability and pharmacodynamic mechanism.

[0003] Celastrol is a quinone methylated triterpenoid compound. It is an active ingredient extracted from the root of the Chinese medicinal material "Thunder God Vine" and appears as red needle-shaped crystals. Celastrol has a wide range of pharmacological activities such as anti-cancer, anti-fungal, anti-fibrosis, anti-inflammatory and antioxidant, as well as anti-neurodegenerative disease activities and other biological activities. In recent years, more and more studies have shown that Celastrol has good anti-tumor activity and can inhibit breast cancer, lung cancer, colorectal cancer, liver cancer, etc. through multiple signaling pathways. It is expected to become one of the most promising drugs for the treatment of tumors and is a natural active product with anti-tumor activity that is worthy of attention. Although Celastrol has good pharmacological effects, its poor water solubility and high toxicity have greatly limited its clinical application potential. At present, using natural drugs as lead compounds and modifying their structures to obtain high-efficiency and low-toxic drugs is one of the important ways to develop new drugs. Therefore, it is very necessary to improve the solubility and stability of Celastrol by modifying its structure and reduce its toxicity by changing its structure. After the sugar modification, the compound has more hydrophilic hydroxyl groups in the structure due to the introduction of sugar chains. Because of the enhanced hydrophilicity, it usually has an important impact on the bioavailability, mechanism of action and pharmaceutical activity of the compound molecule. Therefore, the preparation of tripterygium wilfordii glycoside compounds is an important source of innovative drugs. In addition, the synthesis of glycoside compounds by sugar enzyme catalysis has the advantages of low raw material cost and mild reaction conditions.

[0004] Natural glycoside compounds are an important source of innovative drugs. Glycosylation has an important impact on the efficacy, bioavailability and mechanism of action of drugs. Glycosyltransferase-mediated enzymatic glycosylation is an important strategy for the glycosylation of natural products. Studies have found that some UGT glycosyltransferases have promiscuity, that is, they can not only recognize structurally diverse substrates (glycosyl acceptors and donors), but also catalyze the formation of different glycosidic bonds and have reversibility of reactions, etc., which is an effective means for the preparation of diverse active natural product glycoside derivatives and new drug discovery.

[0005] UGT109A3 is a novel enzyme isolated from Bacillus subtilis. UGT109A3, a glycosyltransferase with strong substrate promiscuity, has great potential in heterologous synthesis of plant triterpenoid saponins.

[0006] Sucrose synthase can catalyze the reversible reaction of sucrose synthesis under specific conditions, mediating the reversible conversion of sucrose and UDP into fructose and UDP-glucose.

[0007] Currently, the synthesis of triptolide glycoside mainly relies on chemical methods. However, the chemical synthesis method has low yield and poor stereoselectivity, and the products are mostly mixtures. The subsequent separation and purification steps are cumbersome and costly. Therefore, it is urgent to develop a new method for preparing high-purity triptolide glycoside with low cost and low pollution.

[0008] Therefore, there is an urgent need in the art to develop a new method that can effectively realize the recycling of UDP-glucose in a cascade catalytic system and improve the structural diversity and water solubility of celastrol triterpenoids. Summary of the invention

[0009] The purpose of the present invention is to provide a new method that can effectively realize the recycling of UDP-glucose in a cascade catalytic system and improve the structural diversity and water solubility of tripterygium wilfordii triterpenoids.

[0010] Another object of the present invention is to provide a glycosyltransferase variant enzyme to construct a cascade catalytic system of glycosyltransferase, which is applied to glycosylation modification of the structure of tripterygium wilfordii and efficient preparation of novel tripterygium wilfordii glycosides.

[0011] In order to achieve the above object, the present invention provides an in vitro glycosylation method, comprising the steps of: transferring the glycosyl of a glycosyl donor to the C-3 / C-29 hydroxyl group of celastrol in the presence of a glycosyltransferase, thereby synthesizing a novel celastrol glycoside;

[0012] The glycosyltransferase is a glycosyltransferase variant enzyme UGT109A3m, and the amino acid sequence is shown in SEQ ID NO. 3. In the present invention, the method further comprises sucrose synthase, wherein the sucrose synthase is a sucrose synthase variant enzyme SUSm, and the amino acid sequence is shown in SEQ ID NO.4.

[0013] The present invention also provides a recombinant plasmid carrying the above gene. In one embodiment of the present invention, the recombinant plasmid preferably has a pET-28a(+) plasmid.

[0014] The present invention also provides a host cell carrying the above gene or the above recombinant plasmid. In one embodiment of the present invention, the host cell is preferably Escherichia coli BL21 (DE3).

[0015] The present invention also provides a method for glycosylation of tripterygium wilfordii, which comprises constructing a cascade catalytic system of a glycosyltransferase variant enzyme UGT109A3m and a sucrose synthase SUSm, using tripterygium wilfordii as a glycosyl acceptor, adding the above-mentioned glycosyltransfer variant enzyme UGT109A3m and the sucrose synthase variant SUSm in an appropriate proportion to a reaction system in which UDP, sucrose and tripterygium wilfordii are added for reaction; separating the reaction products by high performance liquid chromatography (HPLC), and performing chromatographic analysis by mass spectrometry (LC-MS), which indicates that the catalytic system can effectively obtain a novel tripterygium wilfordii glycoside.

[0016] The invention provides an effective double enzyme cascade catalytic system, which improves the conversion rate of tripterygium wilfordii and simultaneously generates a sugar donor UDP-glucose method. The optimum pH of the reaction system is 7.5 and the optimum temperature is 40°C.

[0017] In the first aspect of the present invention, a glycosyltransferase mutant is provided, wherein the mutant is a non-natural protein, and the mutant has the activity of catalyzing the reaction of tripterygium wilfordii to form tripterygium wilfordii glycoside derivatives, and the mutant has a mutation in the following core amino acids related to the enzyme catalytic activity corresponding to the amino acid sequence of SEQ ID NO.1 of the wild-type glycosyltransferase:

[0018] Alanine (A) at position 45;

[0019] Lysine (K) at position 73;

[0020] Cysteine ​​(C) at position 127; and

[0021] Threonine (T) at position 229.

[0022] In another preferred embodiment, the alanine (A) at position 45 is mutated to one or more selected from the following group: leucine (L), valine (V), isoleucine (I).

[0023] In another preferred embodiment, the lysine (K) at position 73 is mutated to alanine (A).

[0024] In another preferred embodiment, the cysteine ​​(C) at position 127 is mutated to one or more selected from the following group: tyrosine (Y), phenylalanine (F).

[0025] In another preferred embodiment, the 229th threonine (T) is mutated to one or more selected from the following group: isoleucine (I), leucine (L), and valine (V).

[0026] In another preferred embodiment, the mutation is selected from the following group: A45L, K73A, C127Y, T229I, or a combination thereof.

[0027] In another preferred embodiment, the amino acid sequence of the glycosyltransferase mutant is shown in SEQ ID NO. 3. In another preferred embodiment, except for the mutation (such as 45, 73, 127 or 229), the remaining amino acid sequence of the mutant protein is the same or substantially the same as the sequence shown in SEQ ID NO. 1.

[0028] In another preferred embodiment, the basic identity means that there are at most 50 (preferably 1-20, more preferably 1-10, and more preferably 1-5) amino acid differences, wherein the differences include amino acid substitutions, deletions or additions, and the mutant protein still has the activity of catalyzing the reaction of tripterygium wilfordii to form tripterygium wilfordii glycoside derivatives.

[0029] In another preferred embodiment, the amino acid sequence of the glycosyltransferase mutant has a homology of at least 80% with the sequence shown in SEQ ID NO.1, preferably at least 85% or 90%, more preferably at least 95%, and most preferably at least 98%.

[0030] In a second aspect of the present invention, a sucrose synthase SUS mutant is provided, wherein the mutant is a non-natural protein, and the mutant has sucrose synthase activity, and the mutant has a mutation in the following amino acid related to enzyme stability corresponding to SEQ ID NO.2 in the amino acid sequence of the wild-type sucrose synthase SUS enzyme:

[0031] Glycine (G) at position 154;

[0032] Glutamic acid (E) at position 358; and

[0033] Threonine (K) at position 792.

[0034] In another preferred embodiment, the mutant catalyzes the synthesis of UDP-Glucose from sucrose and a UDP substrate.

[0035] In another preferred embodiment, the glycine (G) at position 154 is mutated to alanine (A).

[0036] In another preferred embodiment, the glutamic acid (E) at position 358 is mutated to isoleucine (I).

[0037] In another preferred embodiment, the 792nd threonine (K) is mutated to one or more selected from the following group: tyrosine (Y), phenylalanine (F).

[0038] In another preferred embodiment, the mutation is selected from the following group: G154A, E358I, K792F or a combination thereof.

[0039] In another preferred embodiment, the amino acid sequence of the sucrose synthase mutant is shown in SEQ ID NO. 4. In another preferred embodiment, except for the mutation (such as 154, 358 or 792), the remaining amino acid sequence of the mutant protein is the same or substantially the same as the sequence shown in SEQ ID NO. 2.

[0040] In another preferred embodiment, the substantially identical amino acids are different in at most 50 (preferably 1-20, more preferably 1-10, and more preferably 1-5) amino acids, wherein the differences include substitutions, deletions or additions of amino acids, and the mutant protein still has sugar-modifying enzyme activity.

[0041] In another preferred embodiment, the amino acid sequence of the sucrose synthase mutant has a homology of at least 80% with the sequence shown in SEQ ID NO. 2, preferably at least 85% or 90%, more preferably at least 95%, and most preferably at least 98%.

[0042] In a third aspect of the present invention, there is provided a cascade catalytic system comprising:

[0043] (a) the glycosyltransferase mutant according to the first aspect of the present invention; and

[0044] (b) The sucrose synthase SUS variant according to the second aspect of the present invention.

[0045] In another preferred embodiment, the molar ratio of component (a) to component (b) is 1-10:1, preferably, 1-6:1, and more preferably, 1-4:1.

[0046] In another preferred embodiment, the catalytic system catalyzes the reaction of tripterygium wilfordii to generate tripterygium wilfordii glycoside derivatives.

[0047] In another preferred embodiment, the structure of the tripterygium wilfordii is as shown in Formula I:

[0048]

[0049] In another preferred embodiment, the catalytic system transfers the glycosyl of the glycosyl donor to the C-3 and / or C-29 hydroxyl groups of celastrol, thereby synthesizing celastrol glycoside derivatives.

[0050] In another preferred embodiment, the glycosyl donor is selected from: UDP-Xylose, UDP-Rhamnose, UDP-Arabinose, UDP-Glucuronic acid, or a combination thereof.

[0051] In another preferred embodiment, the structure of the tripterygium wilfordii glycoside derivative is as shown in Formula II or III:

[0052]

[0053] Wherein, R1 and R2 are each independently a monosaccharide group or a polysaccharide group.

[0054] In another preferred embodiment, the monosaccharides include glucose (Glc), rhamnose (Rha), acetylglucose (Glc(6)Ac), arabinofuranose (Araf), arabinopyranose (Arap), and xylose (Xyl).

[0055] In another preferred embodiment, the polysaccharide includes 2-4 monosaccharides such as Glc(2-1)Glc, Glc(6-1)Glc, Glc(6)Ac, Glc(2-1)Rha, Glc(6-1)Arap, Glc(6-1)Xyl, Glc(6-1)Araf, Glc(3-1)Glc(3-1), Glc(2-1)Glu(6)Ac, or Glc(6-1)Arap(3-1)Xyl.

[0056] In another preferred embodiment, R1 and R2 are each independently a monosaccharide group, and the monosaccharide is glucose (Glc).

[0057] In another preferred embodiment, the catalytic system catalyzes the following reaction:

[0058]

[0059] Wherein, R1 and R2 are each independently a monosaccharide group or a polysaccharide group.

[0060] In another preferred embodiment, the reaction has one or more characteristics selected from the following group:

[0061] (i) the pH of the reaction system is 5.0-9.0, preferably 7.0-8.0, more preferably 7.4-7.6;

[0062] (ii) The reaction temperature is 20-60°C, preferably 25-60°C, more preferably 30-55°C.

[0063] In the fourth aspect of the present invention, a polynucleotide is provided, wherein the polynucleotide encodes the glycosyltransferase mutant as described in the first aspect of the present invention or the sucrose synthase SUS mutant as described in the second aspect of the present invention.

[0064] In another preferred embodiment, the polynucleotide further contains auxiliary elements selected from the following group on the flank of the mutant's ORF: a signal peptide, a secretory peptide, a tag sequence (such as 6His), or a combination thereof.

[0065] In another preferred embodiment, the polynucleotide is selected from the following group: a DNA sequence, an RNA sequence, or a combination thereof.

[0066] In the fifth aspect of the present invention, a vector is provided, wherein the vector contains the polynucleotide described in the fourth aspect of the present invention.

[0067] In another preferred embodiment, the vector includes an expression vector and an integration vector.

[0068] In the sixth aspect of the present invention, a host cell is provided, wherein the host cell contains the vector described in the fifth aspect of the present invention, or the polynucleotide described in the fourth aspect of the present invention is integrated into its genome.

[0069] In another preferred embodiment, the host cell is a eukaryotic cell, such as a yeast cell or a plant cell.

[0070] In another preferred embodiment, the host cell is a prokaryotic cell, such as Escherichia coli.

[0071] In the seventh aspect of the present invention, a method for producing the glycosyltransferase mutant of the first aspect of the present invention or the sucrose synthase SUS mutant of the second aspect of the present invention is provided, comprising the steps of:

[0072] Under conditions suitable for expression, the host cell described in the sixth aspect of the present invention is cultured to express the glycosyltransferase mutant or the sucrose synthase SUS mutant; and the glycosyltransferase mutant or the sucrose synthase SUS mutant is isolated.

[0073] In the eighth aspect of the present invention, an enzyme preparation is provided, wherein the enzyme preparation comprises the glycosyltransferase mutant according to the first aspect of the present invention and the sucrose synthase SUS mutant according to the second aspect of the present invention.

[0074] In another preferred embodiment, in the enzyme preparation, the molar ratio of the glycosyltransferase mutant to the sucrose synthase mutant SUSm is 1-10:1, preferably, 1-6:1, and more preferably, 1-4:1.

[0075] In another preferred embodiment, the enzyme preparation includes an injection and / or a lyophilized preparation.

[0076] In the ninth aspect of the present invention, there is provided a method for preparing a tripterygium wilfordii glycoside derivative, comprising the steps of:

[0077] (i) contacting the catalytic system described in the third aspect of the present invention or the enzyme preparation described in the eighth aspect of the present invention with a reaction substrate to carry out a catalytic reaction, thereby obtaining the tripterygium wilfordii glycoside derivative; and

[0078] (ii) optionally, isolating and purifying the tripterygium wilfordii glycoside derivative.

[0079] In another preferred embodiment, the reaction substrate is tripterygium wilfordii.

[0080] In another preferred embodiment, in step (i), the temperature of the catalytic reaction is 20-60°C, preferably 25-60°C, more preferably 30-55°C.

[0081] In another preferred embodiment, in step (i), the pH of the catalytic reaction is 5.0-9.0, preferably, 7.0-8.0, more preferably, 7.4-7.6.

[0082] In the tenth aspect of the present invention, there is provided a use of the catalytic system described in the third aspect of the present invention or the enzyme preparation described in the eighth aspect of the present invention, which is used to catalyze celastrol to produce celastrol glycoside derivatives, or is used to prepare a catalytic preparation for catalyzing celastrol to produce celastrol glycoside derivatives.

[0083] In the eleventh aspect of the present invention, there is provided a use of the mutant described in the first aspect of the present invention, the catalytic system described in the third aspect of the present invention, the host cell described in the sixth aspect of the present invention, or the enzyme preparation described in the eighth aspect of the present invention for preparing tripterygium wilfordii glycoside derivatives.

[0084] In the twelfth aspect of the present invention, there is provided a use of the catalytic system according to the third aspect of the present invention or the enzyme preparation according to the eighth aspect of the present invention, selected from the following group:

[0085] 1) Used to catalyze the synthesis of tripterygium wilfordii glycoside derivatives;

[0086] 2) Used for self-circulation to produce UDP-glucose (UDPG) as a sugar donor.

[0087] In the thirteenth aspect of the present invention, a method for synthesizing a tripterygium wilfordii glycoside derivative is provided, comprising the steps of:

[0088] 1) reacting sucrose and UDP in the presence of the catalytic system described in the third aspect of the present invention or the enzyme preparation described in the eighth aspect of the present invention to obtain UDPG;

[0089] 2) directly using the product obtained in step 1) as a raw material, reacting it with tripterygium wilfordii to obtain uridine diphosphate (UDP) and tripterygium wilfordii glycoside derivatives.

[0090] In another preferred embodiment, the reaction efficiency of the method is 50-100% (preferably 50-95%, more preferably 50-90%).

[0091] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 Agarose gel electrophoresis of the glycosyltransferase UGT109A3m gene (A) and 12% SDS-PAGE electrophoresis of the recombinantly expressed UGT109A3m protein after purification (B).

[0093] Figure 2 The figure shows the agarose gel electrophoresis of the sucrose synthase mutant SUSm gene and the 12% SDS-PAGE electrophoresis of the recombinantly expressed SUSm protein after purification.

[0094] Figure 3 This is the HPLC analysis chart of tripterygium wilfordii and its glycoside products, in which the peak of tripterygium wilfordii appears at 11.491 min, and the peak of tripterygium wilfordii glycoside is at 6.552 min.

[0095] Figure 4 LC-ESI-MS spectra of the substrate tripterygium wilfordii and the product tripterygium wilfordii glycoside (tripterygium wilfordii [M+H]+ion peak at m / z:473.32; tripterygium wilfordii monosaccharide [M+H]+ion peak at m / z:635.41; tripterygium wilfordii disaccharide [M+H]+ion peak at m / z:797.48).

[0096] Figure 5 The structural diagram of tripterygium wilfordii aglycone and glycoside products. A. Tripterygium wilfordii molecule; B. Tripterygium wilfordii 3-monosaccharide glycoside; C. Tripterygium wilfordii 3,29-disaccharide glycoside.

[0097] Figure 6 The effect of different pH values ​​and temperatures on the conversion rate of the synthesis of triptolide glycosides by the UGT109A3m-SUSm multi-enzyme catalytic cascade system. The data are the mean ± standard deviation of three measurements. DETAILED DESCRIPTION

[0098] After extensive and in-depth research, the inventors have discovered for the first time a glycosyltransferase mutant, which has sugar-modifying enzyme activity, and the mutant has mutations in the following core amino acids associated with enzyme catalytic activity corresponding to SEQ ID NO.1 of the wild-type glycosyltransferase: alanine (A) at position 45; lysine (K) at position 73; cysteine ​​(C) at position 127; and threonine (T) at position 229; and the present invention also discovered a sucrose synthase mutant SUSm, which has improved stability, and the variant has mutations in the following amino acids associated with enzyme stability corresponding to SEQ ID NO.2 of the wild-type sucrose synthase: glycine (G) at position 154; glutamate (E) at position 358; and threonine (K) at position 792.

[0099] The present invention also found that the combination of the glycosyltransferase mutant UGT109A3m and the sucrose synthase mutant SUSm in a certain ratio can achieve synergistic cascade catalysis to obtain high-yield tripterygium wilfordii glycoside derivatives. On this basis, the present invention was completed.

[0100] the term

[0101] As used herein, the term "UDP" refers to uridine diphosphate, which has the structural formula

[0102]

[0103] As used herein, the term "UDPG" refers to uridine diphosphate glucose, the structural formula of which is

[0104]

[0105] Mutants of the present invention and nucleic acids encoding them

[0106] As used herein, the terms "glycosyltransferase mutant", "mutant", "mutant protein", and "mutant protein of the present invention" are used interchangeably and refer to non-naturally occurring glycosyltransferase mutants, and the mutant protein is an artificially modified protein based on the protein shown in SEQ ID No. 1, wherein the mutant protein contains core amino acids related to the enzyme catalytic activity, and at least one of the core amino acids is artificially modified; and the mutant protein of the present invention has sugar-modifying enzyme activity.

[0107] The term "core amino acids" refers to a sequence based on SEQ ID No. 1, and having at least 80% homology to SEQ ID NO. 1, such as 84%, 85%, 90%, 92%, 95%, 98%, wherein the corresponding positions are specific amino acids described herein, such as based on the sequence shown in SEQ ID No. 1, the core amino acids are:

[0108] Alanine (A) at position 45;

[0109] Lysine (K) at position 73;

[0110] Cysteine ​​(C) at position 127; and

[0111] The 229th position of the threonine (T), and the mutant obtained by mutating the above core amino acids has the activity of catalyzing the reaction of tripterygium wilfordii to form tripterygium wilfordii glycoside derivatives.

[0112] Preferably, in the present invention, the core amino acids of the present invention are mutated as follows:

[0113] Alanine (A) at position 45 was mutated to leucine (L);

[0114] Lysine (K) at position 73 was mutated to alanine (A);

[0115] The cysteine ​​(C) at position 127 was mutated to tyrosine (Y);

[0116] The threonine (T) at position 229 was mutated to isoleucine (I).

[0117] As used herein, the terms "sucrose synthase SUS mutant", "mutant", "mutant protein", and "mutant protein of the present invention" are used interchangeably and all refer to non-naturally occurring sucrose synthase SUS mutants, and the mutant protein is an artificially modified protein based on the protein shown in SEQID No.2, wherein the mutant protein contains core amino acids related to the catalytic activity of the enzyme, and at least one of the core amino acids is artificially modified; and the mutant protein of the present invention has sucrose synthase activity.

[0118] The term "core amino acids" refers to a sequence based on SEQ ID No. 2, and having at least 80% homology to SEQ ID NO. 2, such as 84%, 85%, 90%, 92%, 95%, 98%, wherein the corresponding positions are specific amino acids described herein, such as based on the sequence shown in SEQ ID No. 2, the core amino acids are:

[0119] Glycine (G) at position 154;

[0120] Glutamic acid (E) at position 358; and

[0121] Threonine (K) at position 792.

[0122] Preferably, in the present invention, the core amino acids of the present invention are mutated as follows:

[0123] The glycine (G) at position 154 was mutated to alanine (A);

[0124] Mutation of glutamic acid (E) at position 358 to isoleucine (I); and

[0125] The threonine (K) at position 792 was mutated to phenylalanine (F).

[0126] It should be understood that the amino acid numbering in the mutants of the present invention is based on SEQ ID No. 1 or 2. When a specific mutant protein has a homology of 80% or more with the sequence shown in SEQ ID No. 1 or 2, the amino acid numbering of the mutant may be misplaced relative to the amino acid numbering of SEQ ID No. 1 or 2, such as misplacement of 1-5 positions to the N-terminus or C-terminus of the amino acid. Using conventional sequence alignment techniques in the art, those skilled in the art can generally understand that such misplacement is within a reasonable range, and mutants with a homology of 80% (such as 90%, 95%, 98%) and the same or similar sugar-modifying enzyme activity should not be excluded from the mutants of the present invention due to the misplacement of amino acid numbering.

[0127] Mutants of the present invention are synthetic proteins or recombinant proteins, i.e., can be the product of chemical synthesis, or can be produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants) using recombinant technology. Depending on the host used in the recombinant production scheme, the mutants of the present invention can be glycosylated, or can be non-glycosylated. Mutants of the present invention may also include or not include the initial methionine residue.

[0128] The present invention also includes fragments, derivatives and analogs of the mutants. As used herein, the terms "fragment", "derivative" and "analog" refer to proteins that substantially retain the same biological function or activity as the mutants.

[0129] The mutant fragments, derivatives or analogs of the present invention may be (i) mutants with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) mutants with substitution groups in one or more amino acid residues, or (iii) mutants formed by fusion of a mature mutant with another compound (such as a compound that prolongs the half-life of the mutant protein, such as polyethylene glycol), or (iv) mutants formed by fusion of an additional amino acid sequence to the mutant sequence (such as a leader sequence or secretory sequence or a sequence or protein sequence used to purify the mutant, or a fusion protein formed with an antigen IgG fragment). According to the teachings of this article, these fragments, derivatives and analogs belong to the scope known to those skilled in the art. In the present invention, the conservatively substituted amino acids are preferably produced by amino acid substitution according to Table 1.

[0130] Table 1

[0131]

[0132]

[0133] Preferably, the mutant is as shown in SEQ ID No. 3 or 4. It should be understood that the mutant protein of the present invention generally has a higher homology (identity) than the sequence shown in SEQ ID No. 1 or 2. Preferably, the homology of the mutant protein to the sequence shown in SEQ ID No. 3 or 4 is at least 80%, preferably at least 85%-90%, more preferably at least 95%, and most preferably at least 98%.

[0134] In addition, the mutants of the present invention may also be modified. Modifications (usually without changing the primary structure) include: chemical derivatives of the mutants in vivo or in vitro, such as acetylation, carboxylation or glycosylation. Also included are mutants that have been modified to improve their anti-proteolytic properties or optimize their solubility properties.

[0135] The term "polynucleotide encoding a mutant" may be a polynucleotide encoding a mutant of the present invention, or may be a polynucleotide further comprising additional coding and / or non-coding sequences.

[0136] The present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides or mutant proteins having the same amino acid sequence as the present invention. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the mutant encoded by it.

[0137] The present invention also relates to polynucleotides that hybridize with the above sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis.

[0138] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0139] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0140] At present, the DNA sequence encoding the mutant of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. Then the DNA sequence can be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the mutant sequence of the present invention by chemical synthesis.

[0141] Wild-type glycosyltransferase

[0142] As used herein, "wild-type glycosyltransferase" refers to a naturally occurring, unmodified glycosyltransferase, whose nucleotide sequence can be obtained by genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR), etc., and whose amino acid sequence can be derived from the nucleotide sequence. The amino acid sequence of the wild-type glycosyltransferase is shown in SEQ ID No.: 1.

[0143] UGT109A3 is a new enzyme isolated from Bacillus subtilis. The glycosyltransferase UGT109A3 with strong substrate promiscuity has great potential in the heterologous synthesis of plant triterpenoid saponins. Studies have shown that the enzyme can catalyze the glycosylation of tetracyclic triterpenoid compounds protoginsenosides PPD and PPT at multiple sites to produce non-natural ginsenosides with higher activity.

[0144] In the present invention, we analyzed the structure of this enzyme and screened out the glycosyltransferase variant UGT109A3m (i.e., the mutant described in the present invention) through semi-rational design, which can effectively improve its conversion rate in synthesizing novel tripterygium wilfordii glycosides, thus facilitating the effective preparation of tripterygium wilfordii glycosides.

[0145] The present invention constructs a cascade catalytic system by combining a glycosyltransferase UGT109A3 mutant enzyme (i.e., the mutant described in the present invention) with a sucrose synthase SUS variant described in the present invention to generate a novel celastrol triterpenoid glycoside compound with single-point glycosylation of C3-OH and double-point glycosylation of -OH of C3-OH and C29 carboxyl groups, and effectively realizes the recycling of UDP-glucose in the cascade catalytic system, thereby providing a method for efficiently preparing a novel celastrol glycoside. The prepared glycoside compound improves the structural diversity and water solubility of celastrol triterpenoid.

[0146] Expression vector

[0147] The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the mutant protein coding sequence of the present invention, and a method for producing the polypeptide of the present invention by recombinant technology.

[0148] The polynucleotide sequences of the present invention can be used to express or produce recombinant mutant proteins by conventional recombinant DNA techniques. Generally, the following steps are involved:

[0149] (1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a mutant of the present invention, or a recombinant expression vector containing the polynucleotide;

[0150] (2) Host cells cultured in a suitable culture medium;

[0151] (3) Isolate and purify proteins from culture medium or cells.

[0152] In the present invention, the polynucleotide sequence encoding the mutant protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. As long as they can replicate and be stable in the host, any plasmid and vector can be used. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0153] Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence encoding the mutant protein of the present invention and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: lac or trp promoters of Escherichia coli; lambda phage PL promoter. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0154] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0155] The vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins.

[0156] The host cell can be a prokaryotic cell, such as a bacterial cell, or a lower eukaryotic cell, such as a yeast cell.

[0157] Those skilled in the art are aware of how to select appropriate vectors, promoters, enhancers and host cells. Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, and the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0158] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0159] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0160] The main advantages of the present invention are:

[0161] (1) The glycosyltransferase of the present invention can specifically and efficiently transfer the C-3 / C-29 hydroxyl group of a quinone methylated triterpenoid compound (such as tripterygium wilfordii) substrate to glucose.

[0162] (2) The glycosyltransferase of the present invention is particularly capable of converting tripterygium wilfordii into a novel tripterygium wilfordii glycoside with good water solubility and stable properties. In addition, the cascade catalytic system reaction of the present invention has the advantages of low raw material cost and mild reaction conditions, and plays an important role in improving the bioavailability, improving the solubility of the product and potentially improving the toxic and side effects of tripterygium wilfordii.

[0163] (3) The present invention can be used to enhance the water solubility, bioavailability, activity, etc. of terpenoid compounds, and has important application prospects in the synthesis of novel tripterygium wilfordii glycosides.

[0164] (4) The present invention can synthesize novel highly active tripterygium wilfordii derivatives, further improving the structural diversity of terpenoid compounds.

[0165] (5) The present invention discovers for the first time a glycosyltransferase mutant having sugar-modifying enzyme activity, and the mutant undergoes mutations in the following core amino acids associated with enzyme catalytic activity corresponding to SEQ ID No. 1 of the wild-type glycosyltransferase: alanine (A) at position 45; cysteine ​​(C) at position 127; and threonine (T) at position 229; and the present invention also finds that combining the mutant with sucrose synthase SUS in a certain ratio can achieve synergistic cascade catalysis to obtain high-yield celastrol glycoside derivatives.

[0166] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0167] Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial channels.

[0168] General description of the sources of the biological materials described in the present invention:

[0169] 1. Primer synthesis: The primers used in the present invention were synthesized by Shanghai Ruigu Biotechnology Co., Ltd.

[0170] 2. The DpnI enzyme and other enzyme preparations used in the experiment were purchased from Thermo Scientific; PrimeSTAR Max high-fidelity polymerase was purchased from TakaRa; restriction endonucleases were purchased from Fermentas; and the DNA gel recovery kit and plasmid mini-extraction kit used were purchased from Axygen.

[0171] 3. The E. coli BL21 (DE3), E. coli DH5α strain and pET-28a (+) plasmid involved in the following examples are all from this laboratory (the above strain E. coli BL21 (DE3) can be purchased and does not need to be deposited for patent procedures). The culture media involved in the following examples are as follows: LB liquid culture medium: yeast powder 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, kanamycin 100 μg / L; LB solid culture medium: yeast powder 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, agar powder 15 g / L, kanamycin 100 μg / L.

[0172] Example 1: Construction of glycosyltransferase variant enzyme UGT109A3m

[0173] The specific steps are as follows:

[0174] Based on the resolved structure of glycosyltransferase UGT109A3 (SEQ ID No.1) (PDB code: 7VLB) and molecular dynamics simulation, 64 single-point mutations were designed. Through activity screening and combined mutation, we obtained the optimal mutant UGT109A3m (A45L / K73A / C127Y / T229I), encoding the gene of glycosyltransferase UGT109A3 variant enzyme with amino acid sequence as shown in SEQ ID NO.3; the mutant gene obtained by PCR amplification (see Figure 1 The product was spread on LB solid medium and cultured at 37°C for 12 h. The transformants were picked on LB solid medium and cultured in LB liquid medium. After culturing at 37°C for 12 h, the plasmid was extracted and sequenced to obtain the correctly sequenced recombinant plasmid pET28a-UGT109A3m. The correctly sequenced recombinant plasmid pET28a-UGT109Am was transformed into E.coli BL21 (DE3) to obtain the recombinant E. coli pET28a-UGT109A3m / E.coli BL21.

[0175] Example 2: Construction of sucrose synthase SUS expression vector

[0176] The gene encoding the sucrose synthase SUS with an amino acid sequence as shown in SEQ ID NO.2 was chemically synthesized; the obtained gene was subcloned into the pET-28a(+) plasmid, transformed into E.coli DH5α, the transformation product was spread on LB solid medium, cultured at 37°C for 8h, the transformant was picked on the LB solid medium, inoculated into LB liquid medium for culture, and the plasmid was extracted after culturing at 37°C for 10h, and the plasmid was sequenced to obtain the recombinant plasmid pET28a-SUS with correct sequencing. In order to enhance the expression efficiency and structural stability of SUS protein, based on SUS structural analysis and molecular dynamics simulation, the multi-subunit interface region was selected, 26 single-point mutations were designed, and the optimal mutant SUSm (G154A / E358I / K792F) was obtained through activity screening and combined mutations. The recombinant plasmid pET28a-SUSm with correct sequencing was transformed into E.coli BL21(DE3), that is, the recombinant E. coli pET28a-SUSm / E.coli BL21 was obtained.

[0177] Example 3: Heterologous expression and purification of glycosyltransferase UGT109A3m and sucrose synthase SUSm

[0178] The specific steps are as follows:

[0179] The corresponding clones with correct sequencing were transferred to LB liquid medium containing kanamycin (100 μg / L) and cultured overnight at 37°C 220rpm. The overnight culture was inoculated into an autoinduction medium containing kanamycin (purchased from Shanghai Ruigu Biotechnology Co., Ltd.) at a ratio of 1%, and cultured at 37°C 220rpm until OD600 was about 0.6. Finally, isopropyl 1-β-D-thiogalactopyranoside (IPTG, concentration of 0.1mM) was added to the culture medium and incubated at 20°C for 20 hours.

[0180] The cultured bacterial solution was centrifuged at 4°C and 5000rpm for 15min to collect the bacterial cells, and 10mL binding buffer (pH 8.0, 50mM Tris-HCl buffer, 500mM NaCl, 20mM imidazole) was added to 1g of wet bacteria, and the cells were resuspended and broken in a high-pressure homogenizer. The broken liquid was centrifuged at 4°C and 12000rpm for 30min, and the supernatant was taken. After filtering the supernatant through a 0.45μm cellulose filter, the UGT109A3 protein with 6xHis-tag at the C-terminus was purified by Ni-NTA affinity chromatography (GE Healthcare). The purification process is as follows: 15mL binding buffer equilibrated the nickel column; the supernatant was added to the nickel column tube and slowly flowed through; 15mL binding buffer was used to wash the nickel column to remove unbound proteins; 5mL elution buffer (pH8.0, 50mM Tris-HCl buffer, 500mM NaCl, 200mM imidazole) was used to elute the target protein. The eluate containing the target protein was run through a desalting column to remove imidazole. The enzyme purity was assessed by SDS-PAGE (see Figure 1 Middle B, Figure 2 ), and the protein concentration was determined using NanoDrop 2000 (Thermo Scientific).

[0181] Example 4: Activity analysis of glycosyltransferase UGT109A3m in synthesizing tripterygium wilfordii glycoside and construction of cascade catalytic system

[0182] The specific steps are as follows:

[0183] The glycosylation reaction was carried out in 500 μL reaction buffer (50 mM Tris-HCl buffer pH 8.0) containing 0.5 mg / ml UGT109A3m enzyme, 5 mM UDP-glucose, and 0.25 mM tripterine. After incubation at 37°C for 8 hours, 1 ml of methanol was added to terminate the reaction. The results were analyzed by HPLC and LC-MS. Agilent's HPLC 1260 was used for HPLC detection, and the analytical column was Agilent's C18 column (model: ZOBAX SB-C18, 4.6×150 mm, 5 μm). Mobile phase A was ultrapure water, mobile phase B was acetonitrile, the flow rate was 1.2 mL / min, the injection volume was 10 μL, the temperature was 40°C, and the detection wavelength was 220 nm. The elution procedure was as follows:

[0184]

[0185] LC-MS analysis was performed using an HPLC 1290-MS 6230 (Agilent). Compounds were separated using an Agilent C18 column (model: ZOBAX SB-C18, 4.6×150 mm, 5 μm), mobile phase A was ultrapure water, mobile phase B was acetonitrile, flow rate was 0.8 mL / min, injection volume was 10 μL, temperature was 40°C, and detection wavelength was 220 nm. The elution procedure was as follows:

[0186]

[0187] Note: Mass spectrometry analysis uses positive ionization mode and the ionization method is electrospray ionization (ESI)

[0188] The results of HPLC analysis of the enzymatic reaction showed that an obvious product peak appeared at 6.5 min ( Figure 3 ); The collected products were analyzed by mass spectrometry, indicating the presence of tripterygium wilfordii glycoside products, which were monosaccharide products with a molecular weight of 473.32 and disaccharide products with a molecular weight of 797.48 ( Figure 4 ), the corresponding tripterygium wilfordii aglycone and the inferred corresponding monosaccharide and disaccharide glycoside product molecular structures are as follows Figure 5 shown.

[0189] In the UGT109A3m-SUSm cascade reaction of the present invention, the sugar donor UDP-glucose can be generated by sucrose synthase SUSm catalyzed by relatively low-priced UDP and sucrose, and at the same time, glycosyltransferase UGT109A3m can effectively convert tripterygium wilfordii into tripterygium wilfordii glucoside to generate UDP, forming a cascade cycle catalysis. The enzyme ratio between UGT109A3m and SUSm is one of the key factors for achieving synergistic cascade catalysis to obtain high-yield tripterygium wilfordii-glucoside. The dual enzyme ratio was explored, and the catalytic results are shown in Table 2.

[0190] Table 2: Effect of enzyme ratio on the conversion rate of tripterygium wilfordii glycoside

[0191]

[0192] The results show that the optimal ratio of UGT109A3m and SUS can be set to 2:1.

[0193] In the UGT109A3m-SUSm cascade reaction, expensive UDPG can be regenerated from UDP while efficiently converting celastrol to celastrol glucoside. In the cascade reaction, when the UGT109A3m concentration was 30 μg / mL, increasing the SUSm concentration from 20 μg / mL to 80 μg / mL only increased the conversion rate by 5.56%. However, when 60 μg / mL UGT109A3m and 20 μg / mL SUS were added to the reaction system, the optimal conversion rate could be increased by about 35.78% relative to the 1:1 ratio, indicating that the UGT109A3m catalysis is the rate-limiting step of the cascade reaction. Therefore, in further experiments, the optimal ratio of UGT109A3m and SUSm can be set to 2:1.

[0194] The test system was the same as the above reaction system, except that the buffer was changed to different pH values. The specific buffers were: pH 6.5, pH 7.0, pH 7.5, pH 8.0, pH 8.5 Tris-HCl buffer. The results of the conversion analysis of the catalytic system at different pH values ​​showed that the optimal conversion rate was obtained at pH 7.5 (see Figure 6 A). The effect of different temperatures on the activity of the UGT109A3m-SUSm cascade catalytic system was tested in the same way as the above reaction system, except for the different temperatures. The specific temperatures were: 35°C, 40°C, 45°C, 50°C, and 55°C. The results of the conversion analysis of the catalytic system at different temperatures showed that the optimal conversion rate was obtained at 40°C (see Figure 6 Therefore, 40°C, pH 7.5, 0.4 mM UDP, 500 mM sucrose and 1.0 mM tripterygium wilfordii were used to develop the UGT109A3-SUSm cascade glycosylation.

[0195] Solubility of the compound

[0196] In order to analyze the water solubility of tripterygium wilfordii and its glycosides, an excess of tripterygium wilfordii or prepared tripterygium wilfordii glucoside was added to distilled water. The suspension was mixed by vortexing at 25°C for 2 hours and centrifuged at 15,000×g for 30 minutes to remove insoluble matter. The supernatant was collected and analyzed by HPLC after methanol pretreatment. The HPLC peak at 254nm was integrated to calculate the concentration of the sample solution. The results are shown in Table 3.

[0197] Table 3 Analysis of water solubility of tripterygium wilfordii and tripterygium wilfordii glycosides

[0198]

[0199] As can be seen from Table 3, glycosylation can improve the solubility and pharmacodynamics of hydrophobic natural products. We evaluated the effect of glycosylation of tripterygium wilfordii on its water solubility. The water solubility of tripterygium wilfordii was poor, only 56 μM, while the prepared tripterygium wilfordii glycoside product was very effective in improving its water solubility, with the concentration of tripterygium wilfordii-diglucoside product reaching 1×10 5 μM, compared with the water solubility of tripterygium wilfordii, it increased by about 3.4×10 3 times.

[0200] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0201] Sequence information:

[0202] Wild-type UGT109A3 SEQ ID NO.1

[0203]

[0204] Wild-type SUS SEQ ID NO.2

[0205]

[0206] Glycosyltransferase mutant UGT109A3m SEQ ID NO.3

[0207]

[0208] Sucrose synthase SUS mutant SUSm SEQ ID NO.4

[0209]

Claims

1. A glycosyltransferase mutant, characterized in that: The mutant is a non-natural protein, and the mutant has the activity of catalyzing the reaction of tripterygium wilfordii to form tripterygium wilfordii glycoside derivatives, and the mutant has a mutation in the following core amino acids related to the enzyme catalytic activity corresponding to SEQ ID NO.1 in the amino acid sequence of the wild-type glycosyltransferase: Alanine (A) at position 45; Lysine (K) at position 73; Cysteine ​​(C) at position 127; and Threonine (T) at position 229.

2. A sucrose synthase SUS mutant, characterized in that: The mutant is a non-natural protein, and the mutant has sucrose synthase activity, and the mutant has a mutation in the following amino acid related to enzyme stability corresponding to SEQ ID NO.2 in the amino acid sequence of the wild-type sucrose synthase SUS enzyme: Glycine (G) at position 154; Glutamic acid (E) at position 358; and Threonine (K) at position 792.

3. A cascade catalytic system, characterized in that: The catalytic system comprises: (a) the glycosyltransferase mutant according to claim 1; and (b) The sucrose synthase SUS variant according to claim 2.

4. A polynucleotide, characterized in that The polynucleotide encodes the glycosyltransferase mutant according to claim 1 or the sucrose synthase SUS mutant according to claim 2.

5. A carrier, characterized in that The vector contains the polynucleotide according to claim 4.

6. A host cell, characterized in that The host cell contains the vector according to claim 5, or the polynucleotide according to claim 4 is integrated into its genome.

7. A method for producing the glycosyltransferase mutant according to claim 1 or the sucrose synthase SUS mutant according to claim 2, characterized in that: Includes steps: Cultivating the host cell according to claim 6 under conditions suitable for expression, thereby expressing the glycosyltransferase mutant or the sucrose synthase SUS mutant; and isolating the glycosyltransferase mutant or the sucrose synthase SUS mutant.

8. An enzyme preparation, characterized in that The enzyme preparation comprises the glycosyltransferase mutant according to claim 1 and the sucrose synthase SUS mutant according to claim 2.

9. A method for preparing a tripterygium wilfordii glycoside derivative, characterized in that: Includes steps: (i) contacting the catalytic system according to claim 3 or the enzyme preparation according to claim 8 with a reaction substrate to carry out a catalytic reaction, thereby obtaining the tripterygium wilfordii glycoside derivative; and (ii) optionally, isolating and purifying the tripterygium wilfordii glycoside derivative.

10. Use of the catalytic system according to claim 3 or the enzyme preparation according to claim 8, characterized in that: It is used for catalyzing tripterygium wilfordii to generate tripterygium wilfordii glycoside derivatives, or is used for preparing catalytic preparations for catalyzing tripterygium wilfordii to generate tripterygium wilfordii glycoside derivatives.

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