CN114426958B - Glycosyltransferase-bimetallic organic framework composite catalytic material, preparation method thereof and application thereof in disaccharide and polysaccharide synthesis - Google Patents

Glycosyltransferase-bimetallic organic framework composite catalytic material, preparation method thereof and application thereof in disaccharide and polysaccharide synthesis Download PDF

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CN114426958B
CN114426958B CN202111644754.7A CN202111644754A CN114426958B CN 114426958 B CN114426958 B CN 114426958B CN 202111644754 A CN202111644754 A CN 202111644754A CN 114426958 B CN114426958 B CN 114426958B
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heparin
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CN114426958A (en
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张幸
乔梦
纪元
李乐
沈宝星
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Nanjing Normal University
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Abstract

The invention discloses a glycosyltransferase-bimetal organic framework composite catalytic material, a preparation method thereof and application thereof in disaccharide and polysaccharide synthesis. The composite catalytic material takes a metal organic framework doped with bimetallic ions as a novel immobilized enzyme material, and glycosyltransferase is wrapped in the novel immobilized enzyme material. The composite catalytic material can efficiently catalyze the glycosylation reaction of monosaccharide, has good thermal stability and chemical stability, can improve the enzymatic reaction rate of immobilized enzyme compared with the common metal organic framework material, and can realize repeated recycling and simultaneously maintain higher catalytic activity.

Description

Glycosyltransferase-bimetallic organic framework composite catalytic material, preparation method thereof and application thereof in disaccharide and polysaccharide synthesis
Technical Field
The invention relates to the technical field of catalysts, in particular to a glycosyltransferase-bimetal organic framework composite catalytic material, a preparation method thereof and application thereof in synthesis of disaccharides and polysaccharides.
Background
Heparan Sulfate (HS) and Heparin (Heparin) are linear sulfated acidic polysaccharides with polydispersity consisting of disaccharide repeating units of hexuronic acid (HexA) and Glucosamine (GlcN) linked by 1-4 glycosidic linkages. Modifications such as N-sulfation, N-acetylation, and O-sulfation can occur in disaccharide constituent units, making heparin a complex variable sequence. HS is widely distributed on the surfaces of animal cells and extracellular matrixes, and has physiological and pharmacological actions in aspects of anticoagulation, embryo development, inflammatory reaction, bacterial/viral infection and the like. Heparin is widely used as a clinically applied drug for anticoagulation and treatment of thrombotic diseases, and can be classified into: the novel functions and novel applications of heparin compounds and analogues thereof are also discovered in recent years, such as the novel functions and novel applications of the heparin compounds and analogues thereof, such as the novel heparin compounds can protect vascular endothelial cells and prevent pulmonary microvascular embolism, thereby improving the prognosis of severe pneumonia of novel coronaviruses, the novel heparin compound can achieve good effects in the treatment scheme of patients with severe coronary viruses, simultaneously, the medicament atomized heparin and N-acetylcysteine are simultaneously used, the pulmonary function of the novel heparin patients with severe coronary viruses can be improved, the use range of a breathing machine is reduced, the application range of heparin derivatives is greatly increased, the current worldwide heparin application amount is increased to about 100 tons, and therefore, the stable acquisition and high-safety of heparin compounds are ensured, the chemical structures of the heparin compounds are also widely separated from the main organs and the animal structures of the novel heparin compound, the heparin derivatives are different from the prior art, the heparin derivatives are also different in clinical structures, such as the differential structures and the toxicity of the heparin derivatives are not easily obtained, and the heparin derivatives are widely separated from the animal structures, and the heparin derivatives are widely used in various animal structures are simultaneously, the clinical structures are different from the animal structures, and the heparin derivatives are easily obtained, direct studies of their interactions with proteins and new biological activities with plain heparin and low molecular weight heparin also present significant challenges.
Currently, there are two main methods for artificially synthesizing heparin, namely chemical synthesis and enzymatic synthesis. The chemical synthesis method has the advantages of low cost and accurate and controllable product structure. But has the defects of multiple reaction steps, complex reaction and extremely low yield. Compared with chemical method, the enzyme method has high synthesis selectivity, strong specificity, no need of protecting group operation and high synthesis yield, thus greatly reducing the investment of time and money. In addition, the method has mild reaction conditions, and reduces the hidden danger of unsafe and the harm to environmental pollution. However, the free enzyme is used for catalysis, and the cost of catalysis is high due to the high price, poor stability and difficult recycling of the natural enzyme. Therefore, a novel composite catalyst is urgently needed to maintain the activity of enzyme and realize recycling, and a Metal Organic Framework (MOF) is used as a high-crystallinity porous material with controllable structure, and can be used as the metal armor of the enzyme by embedding the enzyme, so that the stability of the enzyme is protected to the greatest extent and the recycling is realized.
Disclosure of Invention
The invention aims to: aiming at the problems existing in the prior art, the invention provides a glycosyltransferase-bimetal organic framework composite catalytic material. The invention introduces a second metal ion into the existing single metal organic framework material, and screens out the bimetallic immobilization material suitable for glycosyltransferase through experiments. The synthesized glycosyltransferase-bimetal organic framework composite catalytic material has the advantages of high porosity, large specific surface area, adjustable pore diameter, good thermal stability, chemical stability and the like. The enzyme can be protected while the enzyme catalysis efficiency is improved, so that the enzyme can withstand certain denaturation conditions such as temperature, pH, organic solvents and the like; the method maintains the activity of the enzyme, realizes repeated use of the enzyme for many times, solves the problems that the activity of the enzyme is easily influenced by the environment, the enzyme is difficult to recycle and the catalysis cost is high in the catalytic reaction, and realizes the glycosyltransferase with unstable immobilization property and high price of the multi-metal composite catalytic material for the first time.
The invention also provides a preparation method and application of the glycosyltransferase-bimetal organic framework composite catalytic material, and the glycosyltransferase-bimetal organic framework composite catalytic material is used as a catalyst to solve the problem that the glycosyltransferase activity is easily influenced by the environment and is difficult to repeatedly use for many times in the process of synthesizing heparin oligosaccharide by using glycosyltransferase as a catalyst.
The technical scheme is as follows: in order to achieve the above purpose, the glycosyltransferase-bimetallic organic framework composite catalytic material is formed by taking a Zn-based MOFs material as a main body, doping non-Zn metal ions and wrapping glycosyltransferase in the composite catalytic material.
Wherein the Zn-based MOFs material is ZIF-90, and the doped non-Zn metal ions comprise Mn 2+ ，Fe 2+ ，Co 2+ ，Mg 2+ ，Ca 2+ ，Cu 2+ ，Ni 2+ One or more of the following.
Wherein the glycosyltransferase is heparin synthase 2 (heparosan synthase B, HS 2), including pmHS2 (Pasteurella multocida HS 2) or syntheticconstruct HS2.
The preparation method of the glycosyltransferase-bimetal organic framework composite catalytic material comprises the following steps:
(1) Uniformly mixing and stirring glycosyltransferase, a plurality of metal ion solutions and a 2-formaldehyde imidazole ligand solution, and reacting to obtain a reaction solution containing a composite catalytic material, wherein the plurality of metal ion solutions are formed by zinc nitrate solution and another non-Zn metal ion solution;
(2) And (3) centrifugally washing and airing the reaction liquid in the step (1) to obtain the glycosyltransferase-bimetal organic framework composite catalyst.
Wherein, in the step (1), stirring reaction is carried out for 15-25 hours at 35-40 ℃ to obtain a reaction solution containing the composite catalyst; the centrifugation condition in the step (2) is 8,000-12,000 rpm for 4-6 min.
Wherein, the molar ratio of zinc ions to non-Zn metal ions in the step (1) is 1:1-3:1, and the molar ratio of zinc ions to 2-formaldehyde imidazole organic ligand is 1:4-5; the preferred molar ratio is 1:4.
Preferably, the non-Zn metal ion in the step (1) is Mg 2+ The ratio of zinc ions to organic ligand is 1:1, and the ratio of zinc ions to organic ligand is 1:4.
Preferably, the adding and stirring reaction in the step (1) is carried out at 37 ℃ and 500rpm for 24 hours to obtain a reaction solution containing the composite catalyst.
The glycosyltransferase-bimetallic organic framework composite catalytic material is applied to the preparation of disaccharide and polysaccharide synthesis.
Wherein the preparation of disaccharides and polysaccharides comprises the steps of:
(1) Mixing a glycosyl acceptor, a glycosyl donor and a divalent metal ion;
(2) Adding glycosyltransferase-bimetal organic framework composite catalytic material, and carrying out oscillation reaction;
(3) Centrifuging the reaction liquid after the oscillation in the step (2), and taking the supernatant for TLC separation to obtain heparin disaccharide or heparin polysaccharide.
Wherein the glycosyl donor in the step (1) is uridine diphosphate-N-acetylglucosamine or derivatives thereof; the glycosyl acceptor is p-nitrophenyl-beta-D-glucuronic acid and other glucuronic acid derivatives; the molar ratio of the glycosyl acceptor to the glycosyl donor is 1:1-6:1.
Preferably, the glycosyl donor is uridine diphosphate-N-acetylglucosamine or a derivative thereof. Specifically comprises uridine diphosphate-N-acetamido glucose, including 4-azido-uridine diphosphate-N-acetylglucosamine, 4-amino-uridine diphosphate-N-acetylglucosamine, 4-fluoro-uridine diphosphate-N-acetylglucosamine, 4-mercapto-uridine diphosphate-N-acetylglucosamine, 3-azido-uridine diphosphate-N-acetylglucosamine, 3-amino-uridine diphosphate-N-acetylglucosamine, 3-fluoro-uridine diphosphate-N-acetylglucosamine, 3-mercapto-uridine diphosphate-N-acetylglucosamine, 2-azido-uridine diphosphate-N-acetylglucosamine, 2-amino-uridine diphosphate-N-acetylglucosamine, 2-fluoro-uridine diphosphate-N-acetylglucosamine, 2-mercapto-uridine diphosphate-N-acetylglucosamine, 6-azido-uridine diphosphate-N-acetylglucosamine, 6-amino-uridine diphosphate-N-acetylglucosamine, 6-fluoro-uridine diphosphate-N-acetylglucosamine, or a few-mercapto-uridine diphosphate-N-acetylglucosamine.
Wherein, the adding amount of the glycosyltransferase-bimetallic organic framework composite catalytic material in the step (2) is 5-10% of the mass of the reaction liquid in the step (1); the oscillating reaction is carried out in a water bath at 20-50 ℃, the rotating speed is 100-300 rpm, and the reaction time is 10-15 h.
Wherein, the catalytic reaction route of the enzyme composite catalyst in the steps (1) and (2) is as follows:
the glycosyl donor in the invention is uridine diphosphate-N-acetamido glucose and derivatives thereof.
Specifically, the preparation of the present invention preferably comprises three parts:
a first part: preparation of glycosyltransferase-metal organic framework composite catalytic material
HS2@ZIF-90
In Zn (NO) 3 ) 2 ·6H 2 To the O (40 mM) solution, 2mL of glycosyltransferase (0.1-0.5 mg/mL) was added, and after stirring well, 2-imidozole-carboxaldyde (HICA, 160 mM) was added, and ultrapure water was added to fix the volume. Reacting for 15-25 h at 37 ℃ and 500 rpm. After the reaction, the mixture is centrifuged at 8,000 to 12,000rpm for 4 to 6 minutes, and the precipitate is recovered. The glycosyltransferases not encapsulated by MOFs material were then removed by washing with ultrapure water, sonicating and centrifuging three times.
A second part: preparation of glycosyltransferase-bimetallic organic framework composite catalytic material
HS2@ZIF-90(Mg)
In Zn (NO) 3 ) 2 ·6H 2 Adding 2mL glycosyltransferase (0.1-0.5 mg/mL) into O (40 mM) solution, stirring, adding MgCl 2 ·4H 2 O (40 mM), then 2-imidozole-carboxaldyde (HICA, 160 mM) was added, and ultrapure water was added to fix the volume. Reacting for 15-25 h at 37 ℃ and 500 rpm. After the reaction, the mixture is centrifuged at 8,000 to 12,000rpm for 4 to 6 minutes, and the precipitate is recovered. Then washed with ultrapure water, sonicated and centrifuged three times to remove glycosyltransferases not encapsulated by MOFs material
Third section: glycosyltransferase-bimetallic organic framework composite catalytic material catalyzes glycosylation reactions.
Mixing glycosyl acceptor and glycosyl donor (monosaccharide molecule) in the molar ratio of 1:1-6:1, and adding Mg with the final concentration of 8-12mM 2+ Or ferrous ion salt solution and manganese ion salt solution, then placing the mixed solution in a water bath oscillator at 35-45 ℃ and fully mixing the mixed solution for 15-25 h at a certain rotating speed (100-300 rpm), adding 5-10% glycosyltransferase-bimetal organic framework composite catalytic material after the reactants are dissolved, and fully reacting for 10-15 h at a certain temperature (20-50 ℃) and a rotating speed (100-300 rpm) to finish the reaction. The reaction mixture was centrifuged, and the supernatant was separated by TLC.
By adopting the combination process, glycosyltransferase is wrapped in the bimetal organic framework material, so that the influence of the environment on the enzyme activity is reduced, the enzyme activity is maintained, the mechanical property of the enzyme is enhanced, the operation stability is improved, and the efficiency of the enzymatic reaction is accelerated compared with that of the single metal organic framework material. The composite catalyst disclosed by the invention can efficiently catalyze glycosylation reaction, has good thermal stability and chemical stability, can also improve enzymatic reaction rate, and can realize repeated recycling and simultaneously maintain higher catalytic activity.
The invention uses metal organic frame Materials (MOFs), which are two-dimensional or three-dimensional crystal structures formed by self-assembly between metal ions and organic ligands by taking the metal ions as connection points and organic ligands as supports. However, in the process of forming a composite material by coating MOFs with enzymes, most MOFs are difficult to synthesize, and are not suitable for coating enzymes, and meanwhile, organic ligands required in the process of synthesizing MOFs possibly affect the activity of enzymes, so that the process of coating MOFs with enzymes has the difficulties of screening optimal MOFs, adjusting the optimal proportion of metal ions and organic ligands and enzymes, and the like. The invention utilizes ZIF-90 with better biocompatibility and screens a plurality of metal ions, thereby finding out the mole ratio of the bimetallic ion, the organic ligand and the enzyme which can furthest maintain the catalytic activity of glycosyltransferase. In the enzyme-catalyzed reaction, the enzyme-bimetallic organic framework material compound obtained by the invention has the following advantages compared with free enzyme: the glycosyltransferase is limited in a certain space range by the metal organic framework material, so that flocculation inactivation of enzyme molecules caused by disordered movement among enzymes is avoided; the second metal ion is introduced, so that the stability of the immobilized enzyme is improved, and the reaction efficiency of the enzyme is improved; in the reaction process, the porous characteristic of the metal organic framework material can promote the contact of enzyme and substrate, and the reaction rate is improved; can be effectively separated from the product, reduces feedback inhibition, and realizes recycling; the mechanical property of the enzyme is enhanced, and the operation stability is improved.
The innovation point of the invention is that 1) magnesium ions which can improve the catalytic activity of glycosyltransferase are screened out from a plurality of metal ions; 2) Compared with the relatively stable and easily available lipase, the glycosyltransferase is extremely unstable, in the common reaction, the reactive enzyme is easily flocculated and deactivated at room temperature, and the feeding is needed to ensure the continuous operation of the reaction, and the MOF wrapping can obviously improve the stability of the glycosyltransferase; 3) The research on MOF coated enzyme is focused on lipase, peroxidase, catalase, glucose oxidase and other cheap and easily available enzymes, and the catalyzed reaction is also a simple reaction such as hydrogen peroxide hydrolysis.
The second metal ion is introduced into the metal organic framework material for synthesizing the single metal ion, so that the catalytic activity of the immobilized enzyme can be influenced, and the bimetallic organic framework material capable of improving the catalytic efficiency of the enzyme can be screened out. The glycosyltransferase-bimetal organic framework material can improve the stability of glycosyltransferase, improve the catalytic efficiency of the enzyme and further reduce the glycosylation cost.
In addition, since glycosyltransferases are extremely unstable, free enzymes may flocculate and deactivate after overnight reaction, and in order to improve the stability of glycosyltransferases, the present invention has found that the loss of glycosyltransferase activity during material synthesis can be avoided only at specific metal ion and organic ligand ratios.
The invention introduces bimetallic MOF, the invention introduces a plurality of divalent metal ions to simulate the biocatalytic microenvironment, the introduction of the second metal ions can 1) assist the transfer of electrons in glycosylation reaction, 2) stabilize the structure of glycosyltransferase, 3) activate the catalytic activity of glycosyltransferase, 4) compared with electronegative free enzyme, the electropositive composite material can attract negatively charged glycosyl donor and acceptor, thereby achieving the effect of substrate enrichment and accelerating the reaction rate.
The divalent metal solvent is added during heparin synthesis, so that the requirement of free enzyme catalysis can be met, however, in the enzyme immobilization process, as the MOF forms a compact framework around the enzyme, the divalent metal solvent added during heparin synthesis cannot be uniformly dispersed around the enzyme, the distance between the divalent metal ion activator and the enzyme can be reduced to the greatest extent through the synthesized bimetal composite material, the micro-environment of biocatalysis is imitated to the greatest extent, and the slow release and continuous acting functions are achieved.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
the glycosyltransferase-bimetal organic framework composite catalytic material prepared by the invention is a stable material with high porosity, high specific surface area and adjustable structure. Compared with the monometal organic framework material, after the glycosyltransferase is wrapped to form the composite catalytic material, the original high-efficiency, mild and specific enzyme catalytic activity of the enzyme can be maintained to a great extent. Meanwhile, the defect of free enzyme is overcome, the storage stability of the enzyme is improved, the recovery is easy, the recycling rate is improved, and the reaction cost is reduced.
According to the invention, glycosyltransferase is wrapped in the bimetallic organic framework material, so that the influence of the environment on the enzyme activity is reduced, the enzyme activity is maintained, the mechanical property of the enzyme is enhanced, the operation stability is improved, and the enzymatic reaction efficiency is accelerated. The composite catalyst disclosed by the invention can be used for high-efficiency monosaccharide glycosylation reaction, has good thermal stability and chemical stability, can also be used for improving the enzymatic reaction rate, and can be recycled for multiple times while maintaining higher catalytic activity.
The invention has simple preparation and convenient use, can be used for preparing heparin oligosaccharides with uniform carbon chain length, and can be widely applied to industries such as medicines, foods and the like.
In the conventional enzyme catalyzed heparin synthesis, the enzyme is often removed by an extreme operation such as adding an organic solvent, and the enzyme impurities can be removed only by centrifugation without adding any biocompatible reagent.
Drawings
FIG. 1 shows the real-time monitoring of the ability of the glycosyltransferase Free enzyme, the glycosyltransferase-metal organic framework composite catalyst material and the glycosyltransferase-bimetal organic framework composite catalyst material to catalyze disaccharide synthesis (the three curves are PmHS2@Mg-ZIF-90, free PmHS2, pmHS2@ZIF-90 from top to bottom in sequence);
FIG. 2 is a graph showing the temperature stability of glycosyltransferase free enzyme, glycosyltransferase-metal organic framework composite catalytic material and glycosyltransferase-bimetallic organic framework composite catalytic material;
FIG. 3 is an acid stability of glycosyltransferase free enzyme, glycosyltransferase-metal organic framework composite catalytic material and glycosyltransferase-bimetallic organic framework composite catalytic material;
FIG. 4 is a LC-MS spectrum of a glycosyltransferase-bimetallic organic framework composite catalytic material for the synthesis of trisaccharides;
FIG. 5 is a schematic illustration of the synthesis of polysaccharide from glycosyltransferase-bimetallic organic framework composite catalytic material 1 H NMR spectrum.
Detailed Description
The invention will be better understood from the following examples. It will be readily understood by those skilled in the art that the description of the embodiments is provided for illustration only and should not limit the invention as described in detail in the claims. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. The experimental methods for which specific conditions are not specified in the examples are generally conducted under conventional conditions or under conditions recommended by the manufacturer.
Wherein glycosyltransferase PmHS2 (available from peninsula science biotechnology limited, abbreviated HS 2); uridine diphosphate-N-acetylglucosamine and derivatives thereof (all purchased from Arraga Ding Shiji Co., ltd.), uridine diphosphate-glucuronic acid, p-nitrophenyl-beta-D-glucuronic acid (purchased from Guogu Chemicals Co., ltd.); 2-Imidazole-carboxaldehde (2-formylimidazole, available from Arraga Ding Shiji Co.). Wherein uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), also known as uridine diphosphate-N-acetylglucosamine, CAS:91183-98-1; p-nitrophenyl-beta-D-glucuronic acid (GlcA-pNP), also known as p-nitrophenyl-beta-D-glucuronide, 4-nitrophenyl-beta-D-glucuronic acid, CAS number: 10344-94-2.
Example 1
1. Glycosyltransferase-metal organic framework composite catalytic material HS2@ZIF-90
In 2mL Zn (NO) 3 ) 2 ·6H 2 To a solution of O (40 mM) was added 2mL of glycosyltransferase (0.33 mg/mL) and stirred well, 2mL of 2-imidozole-carboxaldyde (HICA, 160 mM) was added, and ultrapure water was added to a constant volume of 10mL. The reaction was carried out at 37℃and 500rpm for 24 hours. After the completion of the reaction, the mixture was centrifuged at 11,000rpm for 4 minutes, and the precipitate was recovered and washed three times with ultrapure water to remove glycosyltransferases not coated with MOFs material, and dried in the air.
2. Preparation of glycosyltransferase-bimetallic organic framework composite catalytic Material HS2@ZIF-90 (Mg)
In 2mL Zn (NO) 3 ) 2 ·6H 2 After adding 2mL of glycosyltransferase (0.33 mg/mL) to a solution of O (40 mM), stirring well, 2mL of MgCl was added 2 ·4H 2 O (40 mM), then 2mL of 2-imidozole-carboxaldyde (HICA, 160 mM) was added, and ultrapure water was added to fix the volume to 10mL. The reaction was carried out at 37℃and 500rpm for 24 hours. After the completion of the reaction, the mixture was centrifuged at 11,000rpm for 4 minutes, and the precipitate was recovered. Then washed with ultrapure water, sonicated and centrifuged three times to remove glycosyltransferases not encapsulated by MOFs material, and air dried.
Example 2
Glycosyltransferase-metal organic framework composite catalytic material and application of glycosyltransferase-bimetal organic framework composite catalytic material in disaccharide synthesis
1. HS2@ZIF-90 catalyzed monosaccharide glycosylation to generate heparin disaccharide
Uridine diphosphate-N-acetylglucosamine (final concentration 10 mM), p-nitrophenyl-beta-D-glucuronic acid (final concentration 10 mM) and magnesium chloride (final concentration 10 mM) are added into Tris-HCl buffer solution (50 mM pH 7.4) and mixed in a water bath at 37 ℃ under shaking at 150rpm for 5min, after the uniform mixing, the HS2@ZIF-90 composite catalyst prepared in example 1, which is 5% of the mass of the reaction solution, is added, and the reaction is completed after the full reaction for 12h at 150rpm in the water bath shaking at 37 ℃. The reaction solution was centrifuged, and the supernatant was separated by TLC, and the separated composite catalyst was collected.
Quantitative analysis of heparin disaccharide was performed by high performance liquid chromatography, chromatographic column: YMC-Pack Polyamine II, mobile phase A:0.7M NaCl, mobile phase B: h 2 O, time program: 0-40min 100% mobile phase A, column temperature: 25 ℃. The maximum absorption wavelength of the heparin disaccharide product is about 310nm, the conversion rate of the glycosylation reaction (the conversion rate is the ratio of the residual glycosyl donor after 1-reaction to the glycosyl donor before reaction) can reach 75%, then the HS2@ZIF-90 composite catalyst is repeatedly utilized by using the same glycosylation method, the recycling rate is calculated, and the conversion rate is 63% when the composite catalyst is repeatedly utilized for 6 times.
2. HS2@ZIF-90 (Mg) catalyzed monosaccharide glycosylation to form heparin disaccharide
Uridine diphosphate-N-acetylglucosamine (final concentration 10 mM), p-nitrophenyl-beta-D-glucuronic acid (final concentration 10 mM) and magnesium chloride (final concentration 10 mM) are added into Tris-HCl buffer solution (50 mM pH 7.4) and mixed in a water bath at 37 ℃ under shaking at 150rpm for 5min, after the uniform mixing, HS2@ZIF90 (Mg) composite catalyst prepared in example 1 is added in an amount of 5% of the total mass of the reaction solution, the reaction is completed after the full reaction for 12h under shaking at 150rpm in the water bath at 37 ℃. The reaction solution was centrifuged, and the supernatant was separated by TLC, and the separated composite catalyst was collected.
The quantitative analysis (liquid phase is the same as that above) is carried out on the heparin disaccharide by the high-efficiency liquid phase HS2, the maximum absorption wavelength of the heparin disaccharide is about 310nm, the conversion rate (the conversion rate is the ratio of the residual glycosyl donor after 1-reaction to the glycosyl donor before reaction) of the glycosylation reaction is calculated, the conversion rate can reach 100%, then the HS2@ZIF-90 (Mg) composite catalyst is repeatedly utilized by using the same glycosylation method, the recycling rate is calculated, and the conversion rate can still reach 90.0% when the composite catalyst is repeatedly utilized for 6 times.
3. HS2@ZIF-90 catalyzes monosaccharide glycosylation under high-concentration magnesium ion condition to generate heparin disaccharide
Uridine diphosphate-N-acetylglucosamine (final concentration 10 mM), p-nitrophenyl-beta-D-glucuronic acid (final concentration 10 mM) and magnesium chloride (final concentration 50 mM) are added into Tris-HCl buffer solution (50 mM pH 7.4) and mixed in a water bath at 37 ℃ under shaking at 150rpm for 5min, after the uniform mixing, HS2@ZIF-90 composite catalyst prepared in example 1, which is 5% of the mass of the reaction solution, is added, and the reaction is completed after the full reaction for 12h at 150rpm in the water bath shaking at 37 ℃. The reaction solution was centrifuged, and the supernatant was separated by TLC, and the separated composite catalyst was collected.
Quantitative analysis of heparin disaccharide was performed by high performance liquid chromatography, chromatographic column: YMC-Pack Polyamine II, mobile phase A:0.7M NaCl, mobile phase B: h 2 O, time program: 0-40min 100% mobile phase A, column temperature: 25 ℃. The maximum absorption wavelength of the heparin disaccharide product is about 310nm, the conversion rate of the glycosylation reaction (the conversion rate is 1-the ratio of the residual glycosyl donor after the reaction to the glycosyl donor before the reaction) is calculated, and the conversion rate is 60%, which indicates that the addition of equivalent free magnesium ions cannot accelerate the reaction, but can inhibit the catalytic capacity of HS2@ZIF-90, and only the HS2@ZIF-90 (Mg) composite catalyst obtained by covalently doping magnesium ions into ZIF-90 has ultrahigh catalytic capacity.
4. Hs2 free enzyme catalyzed monosaccharide glycosylation to heparin disaccharide
Uridine diphosphate-N-acetylglucosamine (final concentration 10 mM), p-nitrophenyl-. Beta. -D-glucuronic acid (final concentration 10 mM) and magnesium chloride (final concentration 10 mM) were added to Tris-HCl buffer (50 mM pH 7.4) and mixed by shaking in a water bath at 37℃at a rotation speed of 150rpm for 5 minutes, and after the mixture was homogenized, HS2 enzyme (HS2@ZIF-90 (Mg) was added to the reaction solution in step 2 in an enzyme amount of about 3.3 Mg/mL) was added to the solution, and the reaction was completed after the completion of the reaction for 12 hours at 150rpm in a water bath shaking at 37 ℃. The reaction mixture was boiled at 100℃for 5min, centrifuged, and the supernatant was separated by TLC.
Quantitative analysis of heparin disaccharide was performed by high performance liquid chromatography, chromatographic column: YMC-Pack Polyamine II, mobile phase A:0.7M NaCl, mobile phase B: h 2 O, time program: 0-40min 100% mobile phase A, column temperature: 25 ℃. The maximum absorption wavelength of the heparin disaccharide product is about 310nm, and the conversion rate of the glycosylation reaction (the conversion rate is the ratio of the residual glycosyl donor after the reaction to the glycosyl donor before the reaction) is calculated to be 98%.
5. HS2 free enzyme catalyzes monosaccharide glycosylation to generate heparin disaccharide under high concentration magnesium ion condition
Uridine diphosphate-N-acetylglucosamine (final concentration 10 mM), p-nitrophenyl-. Beta. -D-glucuronic acid (final concentration 10 mM) and magnesium chloride (final concentration 50 mM) were added to Tris-HCl buffer (50 mM pH 7.4) and mixed by shaking in a water bath at 37℃at a rotation speed of 150rpm for 5 minutes, and after the uniform mixing, HS2 enzyme was added at a final concentration of 5mg/ml and then the mixture was stirred in a water bath at 37℃at a rotation speed of 150rpm, followed by completion of the reaction for 12 hours. The reaction solution was centrifuged, and the supernatant was separated by TLC, and the separated composite catalyst was collected.
Quantitative analysis of heparin disaccharide was performed by high performance liquid chromatography, chromatographic column: YMC-Pack Polyamine II, mobile phase A:0.7M NaCl, mobile phase B: h 2 O, time program: 0-40min 100% mobile phase A, column temperature: 25 ℃. The maximum absorption wavelength of the heparin disaccharide product is about 310nm, and the conversion rate of the glycosylation reaction (the conversion rate is the ratio of the residual glycosyl donor after the reaction to the glycosyl donor before the reaction) is calculated to be 99%.
The above experiments also demonstrate that the addition of an equivalent amount of free magnesium ions does not greatly accelerate the reaction, further demonstrating the ultra-high catalytic capacity of the hs2@zif-90 (Mg) composite catalyst obtained by covalently incorporating magnesium ions into ZIF-90.
Example 3
Glycosyltransferase free enzyme, glycosyltransferase-metal organic framework composite catalytic material and real-time monitoring of disaccharide synthesis catalytic capability of glycosyltransferase-bimetal organic framework composite catalytic material
1. Real-time monitoring of ability of HS2@ZIF-90 to catalyze glycosylation of monosaccharides to heparin disaccharides
Uridine diphosphate-N-acetylglucosamine (final concentration 10 mM), p-nitrophenyl-. Beta. -D-glucuronic acid (final concentration 10 mM) and magnesium chloride (final concentration 10 mM) were added to Tris-HCl buffer (50 mM pH 7.4) and mixed by shaking in a water bath at 37℃at a rotation speed of 150rpm for 5 minutes, and after adding 5% by mass of the reaction mixture of the HS2@ZIF-90 composite catalyst prepared in example 1 to the mixture, the mixture was centrifuged at 150rpm in a water bath at 37℃and the reaction mixture was subjected to HPLC analysis at 1,2,3,6,9, 12, 24 and 36 hours, respectively.
The change rule of the HS2@ZIF-90 catalytic conversion rate with time is detected by high performance liquid chromatography to judge the catalytic efficiency, YMC-Pack Polyamine II and mobile phase A:0.7M NaCl, mobile phase B: h 2 O, time program: 0-40min 100% mobile phase A, column temperature: 25 ℃. The maximum absorption wavelength of the heparin disaccharide product is about 310nm, and the conversion rate of glycosylation reaction (conversion rate is 1-ratio of residual glycosyl donor after reaction to glycosyl donor before reaction) is calculated. The catalytic reaction reached equilibrium substantially at 9 hours, with a final conversion of 75% (fig. 1).
2. Real-time monitoring of the ability of HS2@ZIF-90 (Mg) to catalyze the glycosylation of monosaccharides to heparin disaccharides
Uridine diphosphate-N-acetylglucosamine (final concentration 10 mM), p-nitrophenyl-. Beta. -D-glucuronic acid (final concentration 10 mM) and magnesium chloride (final concentration 10 mM) were added to Tris-HCl buffer (50 mM pH 7.4) and mixed by shaking in a water bath at 37℃at a rotation speed of 150rpm for 5 minutes, and after adding 5% by mass of the reaction mixture of the HS2@ZIF-90 (Mg) composite catalyst prepared in example 1 to the mixture, the mixture was stirred in a water bath at 37℃at a rotation speed of 150rpm, and the reaction mixture was centrifuged at 1,2,3,6,9, 12, 24 and 36 hours, respectively, to obtain a fraction of the supernatant, which was subjected to HPLC analysis.
Detection by high performance liquid chromatographyThe change rule of the catalytic conversion rate of HS2@ZIF-90 (Mg) with time is measured to judge the catalytic efficiency, YMC-Pack Polyamine II, mobile phase A:0.7M NaCl, mobile phase B: h 2 O, time program: 0-40min 100% mobile phase A, column temperature: 25 ℃. The maximum absorption wavelength of the heparin disaccharide product is about 310nm, and the conversion rate of glycosylation reaction (conversion rate is 1-ratio of residual glycosyl donor after reaction to glycosyl donor before reaction) is calculated. The catalytic reaction reached equilibrium substantially at 6 hours, with a final conversion of 100% (fig. 1). Proved by the experiment, the catalytic capability and catalytic efficiency of the HS2@ZIF-90 (Mg) composite catalyst are greatly improved.
3. Real-time monitoring of the ability of HS2 free enzyme to catalyze the glycosylation of monosaccharides to heparin disaccharides
Uridine diphosphate-N-acetylglucosamine (final concentration 10 mM), p-nitrophenyl-. Beta. -D-glucuronic acid (final concentration 10 mM) and magnesium chloride (final concentration 10 mM) were added to Tris-HCl buffer (50 mM pH 7.4) and mixed by shaking in a water bath at 37℃at a rotation speed of 150rpm for 5 minutes, and after the uniform mixing, HS2 free enzyme (final concentration 5 mg/ml) was added thereto and then the mixture was stirred in a water bath at 37℃at a rotation speed of 150rpm, and a part of the reaction mixture was separated and analyzed by HPLC at 1,2,3,6,9, 12, 24 and 36 hours, respectively.
And detecting the change rule of the HS2 catalytic conversion rate along with time by high performance liquid chromatography to judge the catalytic efficiency, YMC-Pack Polyamine II and mobile phase A:0.7M NaCl, mobile phase B: h 2 O, time program: 0-40min 100% mobile phase A, column temperature: 25 ℃. The maximum absorption wavelength of the heparin disaccharide product is about 310nm, and the conversion rate of glycosylation reaction (conversion rate is 1-ratio of residual glycosyl donor after reaction to glycosyl donor before reaction) is calculated. The catalytic reaction reached equilibrium substantially at 12 hours, with a final conversion of 98% (fig. 1).
In addition, the real-time monitoring data of the substrate product show that the conversion rate of HS2@ZIF-90 (Mg) synthesized by the method can reach 97% in three hours, and the catalytic activity and the efficiency of the method are far higher than those of free enzyme and common immobilization, so that the method is high in speed and conversion rate.
Example 4
Temperature stability test of glycosyltransferase free enzyme, glycosyltransferase-metal organic framework composite catalytic material and glycosyltransferase-bimetallic organic framework composite catalytic material.
1. Temperature stability test of HS2@ZIF-90
Uridine diphosphate-N-acetylglucosamine (final concentration 10 mM), p-nitrophenyl-beta-D-glucuronic acid (final concentration 10 mM) and magnesium chloride (final concentration 10 mM) are added into Tris-HCl buffer solution (50 mM, pH 7.4) and mixed by shaking at a rotation speed of 150rpm for 5min, after the uniform mixing, HS2@ZIF90 composite catalyst prepared in example 1, which is 5% of the mass of the reaction solution, are added, and the mixture is incubated in water baths at 50, 60 and 70 ℃ for 30min, and then reacted at a rotation speed of 150rpm for 12h, thereby ending the reaction. The reaction solution was centrifuged, and the supernatant was separated for TLC separation and HPLC detection. The final conversion was 70%,64%,59%, respectively (fig. 2).
2. Temperature stability test of HS2@ZIF-90 (Mg)
Uridine diphosphate-N-acetylglucosamine (final concentration 10 mM), p-nitrophenyl-beta-D-glucuronic acid (final concentration 10 mM) and magnesium chloride (final concentration 10 mM) are added into Tris-HCl buffer solution (50 mM, pH 7.4) and mixed by shaking, the rotating speed is 150rpm, the time is 5min, after the uniform mixing, the HS2@ZIF-90 (Mg) composite catalyst prepared in example 1, which is 5% of the mass of the reaction solution, are added, and the mixture is incubated in water baths at 50, 60 and 70 ℃ for 30min respectively, and then the reaction is finished after the reaction is carried out for 12h at a rotating speed of 150 rpm. The reaction solution was centrifuged, and the supernatant was separated for TLC separation and HPLC detection. The final conversion was 92%,85%,74%, respectively (fig. 2).
3. Temperature stability test of HS2 free enzyme
Uridine diphosphate-N-acetylglucosamine (final concentration 10 mM), p-nitrophenyl-. Beta. -D-glucuronic acid (final concentration 10 mM) and magnesium chloride (final concentration 10 mM) were added to Tris-HCl buffer (50 mM, pH 7.4) and mixed by shaking at 150rpm for 5min, and after mixing uniformly, HS2 free enzyme was added at 5mg/ml, incubated in water baths at 50, 60 and 70℃for 30min, followed by reaction at 150rpm for 12h, followed by completion of the reaction. The supernatant was separated by TLC and HPLC. The final conversion was 65%,29%,13%, respectively (fig. 2).
Compared with free enzyme, the temperature stability of the metal organic framework immobilized enzyme is obviously improved, wherein compared with the traditional ZIF-90 encapsulated enzyme, the HS2@ZIF-90 (Mg) composite catalyst has better temperature stability, and lays a foundation for the large-scale and commercial application of the enzyme preparation.
Example 5
Acid stability test of glycosyltransferase free enzyme, glycosyltransferase-metal organic framework composite catalytic material and glycosyltransferase-bimetal organic framework composite catalytic material
1. Acid stability test of HS2@ZIF-90
The HS2@ZIF-90 composite catalyst prepared in example 1 was incubated in Tris-HCl buffer (50 mM,5 mL) at pH 3,4,5,6,7, respectively, for 30min, and after completion the buffer was removed by centrifugation at 8000 rpm.
Uridine diphosphate-N-acetylglucosamine (final concentration 10 mM), p-nitrophenyl-beta-D-glucuronic acid (final concentration 10 mM) and magnesium chloride (final concentration 10 mM) are added into Tris-HCl buffer solution (50 mM, pH 7.4) and mixed by shaking, the rotation speed is 150rpm, the time is 5min, after the uniform mixing, the HS2@ZIF-90 composite catalyst which is 5% of the mass of the reaction solution and treated is added, the reaction is finished after the water bath shaking reaction for 12h at 37 ℃, the reaction solution is centrifuged, and the supernatant is separated by TLC and HPLC detection. The final conversion was 53%,62%,37%,56%,75%, respectively (fig. 3).
2. Acid stability test of HS2@ZIF-90 (Mg)
The HS2@ZIF-90 (Mg) composite catalyst prepared in example 1 was incubated in Tris-HCl buffer (50 mM,5 mL) at pH 3,4,5,6,7, respectively, for 30min, and after completion the buffer was removed by centrifugation at 8000 rpm.
Adding uridine diphosphate-N-acetamido glucose (final concentration 10 mM), p-nitrophenyl-beta-D-glucuronic acid (final concentration 10 mM) and magnesium chloride (final concentration 10 mM) into Tris-HCl buffer solution (50 mM, pH 7.4), mixing under shaking at 150rpm for 5min, adding 5% of the mass of the reaction solution of the treated HS2@ZIF-90 (Mg) composite catalyst, performing water bath shaking at 37 ℃ for 12h, ending the reaction, centrifuging the reaction solution, separating supernatant, and performing TLC separation and HPLC detection. The final conversion rates are 90%,95%,98%,100%,100% (FIG. 3), respectively, which demonstrates the superior acid stability of the HS2@ZIF-90 (Mg) composite catalyst compared to the conventional ZIF-90 material.
3. Acid stability test of HS2 free enzyme
HS2 was incubated in Tris-HCl buffer (50 mM,5 mL) at pH 3,4,5,6,7, respectively, for 30min.
Uridine diphosphate-N-acetylglucosamine (final concentration 10 mM), p-nitrophenyl-. Beta. -D-glucuronic acid (final concentration 10 mM) and magnesium chloride (final concentration 10 mM) were added to Tris-HCl buffer (50 mM, pH 7.4) and mixed by shaking at a rotation speed of 150rpm for 5 minutes, and after the uniform mixing, the above-mentioned buffer having a final concentration of 5mg/ml HS2 free enzyme was added to each of the above-mentioned treated solutions, the reaction was terminated after shaking in a water bath at 37℃for 12 hours, and the supernatant was separated by TLC and HPLC. The final conversion was 68%,70%,68%,96%, respectively (fig. 3).
Example 6
Catalytic formation of heparin trisaccharide by HS2@ZIF-90 (Mg)
Uridine diphosphate-glucuronic acid (final concentration 10 mM) and acetamido glucose-glucuronic acid-p-nitrophenyl (final concentration 10 mM) are added into Tris-HCl buffer (50 mM, pH 7.4), magnesium chloride (final concentration 10 mM) is mixed by shaking in a water bath at 37 ℃ at a speed of 150rpm for 5min, after the uniform mixing, HS2@ZIF-90 (Mg) composite catalyst prepared in example 1, which is 10% of the mass of the reaction solution, is added, and the reaction is completed after the full reaction for 12h at 150rpm in the water bath shaking at 37 ℃. The reaction solution is centrifuged, the supernatant is separated to obtain heparin trisaccharide, and the heparin trisaccharide is qualitatively analyzed by liquid chromatography-mass spectrometry (figure 4) with the conversion rate of 100%, so that the invention can be applied to disaccharide synthesis as well as trisaccharide synthesis.
Example 7
Catalytic formation of heparin polysaccharide by HS2@ZIF-90 (Mg)
Uridine diphosphate-N-acetylglucosamine (final concentration 10 mM), uridine diphosphate-glucuronic acid (final concentration 10 mM) and p-nitrophenyl-glucuronic acid (final concentration 1 mM) are added into Tris-HCl buffer (50 mM, pH 7.4), and mixed by shaking in a water bath at 37 ℃ for 5 minutes at a speed of 150rpm, and after the mixture is uniformly mixed, the reaction is completed after adding the HS2@ZIF-90 (Mg) composite catalyst prepared in example 1 in an amount of 10% by mass of the reaction solution, and then stirring in a water bath at 37 ℃ at a speed of 150rpm, and the reaction is completed after the complete reaction for 12 hours. The reaction solution is centrifuged, the supernatant is separated to obtain heparin polysaccharide, and the heparin polysaccharide is qualitatively analyzed by a nuclear magnetic resonance spectrometer (figure 5), which shows that the invention can be applied to the synthesis of disaccharide and trisaccharide as well as polysaccharide.
Example 8
The enzyme activities of the different glycosyltransferase-metal organic framework composite catalytic materials in example 1 of the present invention were tested under different environments and compared to the enzyme activities of free glycosyltransferases not encapsulated by MOFs material.
The two glycosyltransferase-metal organic framework composite catalytic materials in example 1 and the free enzyme are respectively placed in organic solvents at different temperatures for 2 hours. The two treated composite catalytic materials and free enzyme are used for the glycosylation reaction of monosaccharide (the method of example 2), and then quantitative analysis is carried out on heparin disaccharide through high-efficiency liquid phase, so as to calculate the conversion rate of the glycosylation reaction (the conversion rate is the ratio of residual glycosyl acceptor to glycosyl donor after the reaction), and the enzyme activity ratio (relative activity%) is the ratio of the conversion rate of the saccharification reaction after the treatment to the conversion rate catalyzed by the original free enzyme. It is found from some experimental analysis that, under the condition of equal enzyme amount, free glycosyltransferase HS2 in organic solvents such as (DMSO and THF) is easy to inactivate, and has little activity, and the glycosyltransferases wrapped by HS2@ZIF-90 and HS2@ZIF-90 (Mg) have good protection effect on the enzyme compared with the free enzyme, and the conversion rate can reach more than 90% compared with the protection effect of the enzyme, and is obviously better than that of the HS2@ZIF-90 (about 70%).
From the experiment, the glycosyltransferase-bimetallic organic framework composite catalytic material can remarkably improve the conversion rate, can reach more than 10-20% under certain extreme conditions, has very remarkable effect (a few percent of improvement in the conversion rate of glycosylation reaction can be regarded as remarkable improvement), and can be effectively applied to industrial processing in certain specific environments.
Example 9
Example 9 was prepared in the same manner as example 1, except that: the non-Zn metal ion is Fe 2+ The molar ratio of zinc ions to non-Zn metal ions is 3:1, the molar ratio of zinc ions to 2-formaldehyde imidazole organic ligand is 1:5, the glycosyltransferase concentration is 0.1mg/ml, and after uniform stirring, the reaction is carried out for 25h at 35 ℃. After the reaction, the mixture was centrifuged at 8000rpm for 6 minutes to collect a precipitate.
Example 10
Example 10 was prepared in the same manner as example 1, except that: the non-Zn metal ion is Mn 2+ The molar ratio of zinc ions to non-Zn metal ions is 2:1, the molar ratio of zinc ions to 2-formaldehyde imidazole organic ligand is 1:4, the glycosyltransferase concentration is 0.5mg/ml, and after uniform stirring, the reaction is carried out for 15h at 40 ℃. After the completion of the reaction, the mixture was centrifuged at 12000rpm for 4 minutes, and the precipitate was recovered.
Example 11
Example 11 is the same as example 2 except that: the molar ratio of the glycosyl acceptor to the glycosyl donor is 6:1, and the glycosyl donor is 4-F-uridine diphosphate-N-acetamido glucose; after adding 10% of the mass of the reaction solution of the HS2@ZIF-90 (Mg) composite catalyst prepared in example 1, the reaction is completed after the reaction is completed for 15 hours at a rotating speed of 300rpm in water bath oscillation at 20 ℃. The reaction solution was centrifuged, and the supernatant was separated by TLC, and the separated composite catalyst was collected.
Example 12
Example 12 is the same as example 2 except that: the molar ratio of the glycosyl acceptor to the glycosyl donor is 2:1, after the HS2@ZIF-90 (Mg) composite catalyst prepared in the example 1 accounting for 8% of the mass of the reaction solution is added, the reaction is completed after the reaction is fully performed for 10 hours at the rotating speed of 100rpm in water bath oscillation at 50 ℃. The reaction solution was centrifuged, and the supernatant was separated by TLC, and the separated composite catalyst was collected.

Claims (9)

1. A glycosyltransferase-bimetal organic framework composite catalytic material is characterized in that the composite catalytic material takes Zn-based MOFs material as a main body and is doped with Mg 2+ Wrapping glycosyltransferase inside the substrate to form the glycosyltransferase; the glycosyltransferase is heparin synthase 2 #heparosan synthase BHS 2), zinc ions and Mg 2+ The molar ratio of the zinc ions to the 2-formaldehyde imidazole organic ligand is 1:1-3:1, and the molar ratio of the zinc ions to the 2-formaldehyde imidazole organic ligand is 1:4-5.
2. The glycosyltransferase-bimetallic organic framework composite catalytic material of claim 1, wherein the glycosyltransferase is a material comprisingPasteurella multocida HS2 orsyntheticconstruct HS2。
3. A method for preparing the glycosyltransferase-bimetallic organic framework composite catalytic material of claim 1, comprising the steps of:
(1) The glycosyltransferase, a plurality of metal ion solutions and a 2-formaldehyde imidazole ligand solution are mixed and stirred uniformly, and a reaction solution containing a composite catalytic material is obtained after the reaction, wherein the plurality of metal ion solutions are prepared from
Zinc nitrate solution and Mg 2+ Forming a solution;
(2) And (3) centrifugally washing and airing the reaction liquid in the step (1) to obtain the glycosyltransferase-bimetal organic framework composite catalyst.
4. The preparation method according to claim 3, wherein the stirring reaction in the step (1) is carried out at 35-40 ℃ for 15-25 h to obtain a reaction solution containing the composite catalyst; the centrifugation condition in the step (2) is 8,000-12,000 rpm for 4-6 min.
5. The process according to claim 3, wherein in the step (1), zinc ions and Mg are mixed with each other 2+ The molar ratio of the zinc ions to the 2-formaldehyde imidazole organic ligand is 1:1-3:1, and the molar ratio of the zinc ions to the 2-formaldehyde imidazole organic ligand is 1:4-5.
6. Use of a glycosyltransferase-bimetallic organic framework composite catalytic material according to claim 1 for the preparation of a composition for heparin disaccharides and heparin polysaccharides.
7. The use according to claim 6, characterized in that it comprises the steps of:
(1) Mixing a glycosyl acceptor, a glycosyl donor and a divalent metal ion;
(2) Adding glycosyltransferase-bimetal organic framework composite catalytic material, and carrying out oscillation reaction;
(3) Centrifuging the reaction liquid after the oscillation in the step (2), and taking the supernatant for TLC separation to obtain heparin disaccharide or heparin polysaccharide.
8. The use according to claim 7, wherein the glycosyl donor of step (1) is uridine diphosphate-N-acetamido glucose or a derivative thereof; the glycosyl acceptor is p-nitrophenyl-beta-D-glucuronic acid and other glucuronic acid derivatives; the molar ratio of the glycosyl acceptor to the glycosyl donor is 1:1-6:1.
9. The use according to claim 7, wherein the glycosyltransferase-bimetallic organic framework composite catalytic material in step (2) is added in an amount of 5-10% of the mass of the reaction solution; the oscillating reaction is a water bath oscillating reaction at 20-50 ℃, the rotating speed is 100-300 rpm, and the reaction time is 10-15 h.
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