CN114426958A - Glycosyltransferase-bimetal organic framework composite catalytic material, preparation method thereof and application thereof in synthesis of disaccharide and polysaccharide - Google Patents
Glycosyltransferase-bimetal organic framework composite catalytic material, preparation method thereof and application thereof in synthesis of disaccharide and polysaccharide Download PDFInfo
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- CN114426958A CN114426958A CN202111644754.7A CN202111644754A CN114426958A CN 114426958 A CN114426958 A CN 114426958A CN 202111644754 A CN202111644754 A CN 202111644754A CN 114426958 A CN114426958 A CN 114426958A
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- glycosyltransferase
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- organic framework
- catalytic material
- composite catalytic
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/12—Disaccharides
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a glycosyltransferase-bimetal organic framework composite catalytic material, a preparation method thereof and application thereof in synthesis of disaccharide and polysaccharide. The composite catalytic material of the invention takes a metal organic framework doped with bimetallic ions as a novel immobilized enzyme material, and glycosyltransferase is wrapped in the immobilized enzyme material, so that the glycosyltransferase-bimetallic organic framework composite catalytic material is prepared, and is added into a glycosyl donor and a glycosyl acceptor to carry out catalytic glycosylation reaction to obtain heparin disaccharide and polysaccharide. 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 maintain higher catalytic activity.
Description
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 disaccharide and polysaccharide.
Background
Heparan Sulfate (HS) and Heparin (Heparin) are linear sulfated acidic polysaccharides having polydispersity composed of disaccharide repeating units linked by hexuronic acid (HexA) and Glucosamine (glcne) through 1-4 glycosidic linkages. Modifications such as N-sulfation, N-acetylation, and O-sulfation can occur on the disaccharide building blocks, making heparin a complex variable sequence. HS is widely distributed on the surface of animal cells and extracellular matrix, and has physiological and pharmacological effects in aspects of anticoagulation, embryonic development, inflammatory reaction, bacterial/viral infection and the like. Heparin, as a clinically applied drug widely used for anticoagulation and treatment of thrombotic diseases, is classified into: common heparin (unfractionated heparin, UFH, MWavg 16,000Da), low molecular weight heparin (low molecular weight heparins, LMWH, MWavg 3500-6000 Da) and ultra-low molecular weight heparin (ultra-low molecular weight heparins, ULMWH, MW <2000Da) [1,10-12] in recent years, new functions and new applications of heparin compounds and analogues thereof have been discovered, for example, low molecular weight heparin can protect vascular endothelial cells and prevent pulmonary microvascular embolism, so that after the prevention of severe pneumonia of novel coronavirus is improved, good effects can be achieved in the treatment scheme of severe patients with COVID-19. meanwhile, the simultaneous use of drug atomized heparin and N-acetylcysteine can also improve the pulmonary function of severe patients with new coronavirus, the wide application range of heparin drugs and the application of low molecular weight heparin derivatives greatly increase the market demands on heparin raw materials, currently, the worldwide usage of heparin has increased to approximately 100 tons per year, and it is therefore of great importance to ensure stable availability and high safety of heparin compounds. The low molecular weight heparin widely used at present is mainly obtained by separating and extracting organs such as pig intestines, cattle lungs and the like and degrading the low molecular weight heparin by a chemical method or an enzymatic method. However, since heparin derived from animals is a mixture having a heterogeneous structure, clinical applications have many problems, such as low bioavailability, unpredictable drug efficacy, and many side effects. "sodium heparin" can cause death in humans, exposing animal-derived heparin to safety and reliability problems. Meanwhile, heparin has a complex and diverse structure, and products with different sources and batches often have fine structure differences, so that the research on the interaction between the heparin and protein and the new biological activity by directly using common heparin and low molecular weight heparin also faces huge challenges.
Currently, there are two main methods for artificially synthesizing heparin, i.e., chemical synthesis and enzymatic synthesis. The chemical synthesis method has the advantages of low cost and accurate and controllable product structure. But has the disadvantages of multiple reaction steps, complex reaction and extremely low yield. Compared with a chemical method, the enzymatic method has high synthesis selectivity, strong specificity, no need of protecting group operation and high synthesis yield, thereby greatly reducing the investment of time and money. In addition, the method has mild reaction conditions, and reduces the potential safety hazard and the harm to the environment pollution. However, the free enzyme is used for catalysis, and the natural enzyme has high price, poor stability and difficult recycling, so the catalysis cost is high. Therefore, a novel composite catalyst is urgently needed to maintain the activity of the enzyme and realize recycling, and a Metal Organic Framework (MOF) is taken as a high-crystallinity porous material with a controllable structure, can be taken as a metal armor of the enzyme by embedding the enzyme, and protects the stability of the enzyme to the maximum extent and realizes recycling.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems 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 size, good thermal stability, good chemical stability and the like. The enzyme can be protected while the enzyme catalysis efficiency is improved, so that the enzyme can tolerate certain denaturation conditions such as temperature, pH and organic solvent; the activity of the enzyme is maintained, the repeated utilization of the enzyme is realized for a plurality of times, the problems that the enzyme is easily influenced by the environment, the activity is difficult to be repeatedly utilized and the catalytic cost is high in catalytic reaction are solved, and the glycosyltransferase with unstable immobilization property and high price of the multi-metal composite catalytic material is realized for the first time.
The invention also provides a preparation method and application of the glycosyltransferase-bimetallic organic framework composite catalytic material, and the glycosyltransferase-bimetallic organic framework composite catalytic material is used as a catalyst to solve the problem that the activity of glycosyltransferase is easily influenced by the environment and is difficult to be repeatedly utilized in the process of catalytically synthesizing heparin oligosaccharide by glycosyltransferase.
The technical scheme is as follows: in order to achieve the purpose, the composite catalytic material of the glycosyltransferase-bimetal organic framework is formed by taking Zn-based MOFs materials as a main body, doping non-Zn metal ions and wrapping the glycosyltransferase in the composite catalytic material.
Wherein the Zn-based MOFs material is ZIF-90, and the doped non-Zn metal ions comprise Mn2+,Fe2+,Co2+,Mg2+,Ca2+,Cu2+,Ni2+One or more of.
Wherein the glycosyltransferase is heparin synthase 2 (HS 2), including pmHS2(Pasteurella multocida HS2) or syntactical construct HS 2.
The preparation method of the glycosyltransferase-bimetallic 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 a zinc nitrate solution and another non-Zn metal ion solution;
(2) and (2) carrying out centrifugal washing and air drying on the reaction liquid obtained in the step (1) to obtain the glycosyltransferase-bimetal organic framework composite catalyst.
Wherein the stirring reaction in the step (1) is a stirring reaction at 35-40 ℃ for 15-25 h to obtain a reaction solution containing the composite catalyst; the centrifugation condition of 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 the zinc ions to the 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 Mg2+The ratio of the zinc ions to the organic ligands is 1:1, and the ratio of the zinc ions to the organic ligands is 1: 4.
Preferably, the adding and stirring reaction in the step (1) is carried out at 37 ℃ and 500rpm for 24h to obtain a reaction solution containing the composite catalyst.
The glycosyltransferase-bimetallic organic framework composite catalytic material disclosed by the invention is applied to preparation of disaccharide and polysaccharide synthesis.
Wherein the preparation of the disaccharide and the polysaccharide comprises the following steps:
(1) mixing a glycosyl acceptor, a glycosyl donor and a divalent metal ion;
(2) adding glycosyltransferase-bimetal organic framework composite catalytic material, and oscillating for reaction;
(3) and (3) centrifuging the reaction solution oscillated in the step (2), and taking the supernatant to perform TLC separation to obtain heparin disaccharide or heparin polysaccharide.
Wherein, the glycosyl donor in the step (1) is uridine diphosphate-N-acetylglucosamine or a derivative thereof; the glycosyl receptor 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, the uridine diphosphate-N-acetylglucosamine includes 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, N-uridine diphosphate, N-acetylglucosamine, N-uridine diphosphate, N-acetylglucosamine, N-acetylglucosamine, N-acetylglucosamine, N-N, N, 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, and 6-mercapto-uridine diphosphate-N-acetylglucosamine.
Wherein, the adding amount of the glycosyltransferase-bimetal organic framework composite catalytic material in the step (2) is 5 to 10 percent of the mass of the reaction liquid in the step (1); the oscillation reaction is a water bath oscillation reaction 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-acetylglucosamine and derivatives thereof.
In particular, 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)2·6H22mL glycosyltransferase (0.1-0.5 mg `) was added to O (40mM) solutionmL), 2-imidazole-carbonate (HICA,160mM) was added and the volume was increased with ultrapure water. Reacting for 15-25 h at 37 ℃ and 500 rpm. After the reaction is finished, centrifuging at 8,000-12,000 rpm for 4-6 min, and recovering the precipitate. The glycosyltransferases that were not encapsulated by the MOFs were then removed by washing with ultrapure water, sonication, and centrifugation three times.
A second part: preparation of glycosyltransferase-bimetal organic framework composite catalytic material
HS2@ZIF-90(Mg)
In Zn (NO)3)2·6H2Adding 2mL glycosyltransferase (0.1-0.5 mg/mL) into O (40mM) solution, stirring, and adding MgCl2·4H2O (40mM), then 2-imidazole-carbonate (HICA,160mM) was added and the volume was increased with ultrapure water. Reacting for 15-25 h at 37 ℃ and 500 rpm. After the reaction is finished, centrifuging at 8,000-12,000 rpm for 4-6 min, and recovering the precipitate. Then washed with ultrapure water, sonicated and centrifuged three times to remove glycosyltransferases not encapsulated by MOFs
And a third part: glycosyltransferase-bimetallic organic framework composite catalytic material catalyzes glycosylation reaction.
Mixing glycosyl acceptor and glycosyl donor (monosaccharide molecule) with the molar ratio of 1: 1-6: 1, and adding Mg with the final concentration of 8-12mM2+Or a ferrous ion salt solution and a manganese ion salt solution are placed in a water bath oscillator at the temperature of 35-45 ℃, fully mixed for 15-25 h at a certain rotating speed (100-300 rpm), after reactants are dissolved, 5% -10% of glycosyltransferase-bimetal organic framework composite catalytic material is added, and the reaction is finished after fully reacting for 10-15 h at a certain temperature (20-50 ℃) and a rotating speed (100-300 rpm). The reaction solution was centrifuged, and the supernatant was collected for TLC separation.
By adopting the combined process, 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 compared with that of a single-metal organic framework material. The composite catalyst provided by the invention can be used for efficiently catalyzing glycosylation reaction, has good thermal stability and chemical stability, can improve the enzymatic reaction rate, and can realize repeated recycling and maintain higher catalytic activity.
The invention uses metal organic framework Materials (MOFs), which are two-dimensional or three-dimensional crystal structures formed by self-assembly between metal ions and organic ligands by using metal ions as connecting points and using the organic ligands as supports. However, in the process of forming a composite material by MOFs-coated enzyme, most MOFs synthesis conditions are harsh, and are not suitable for enzyme coating, and meanwhile, an organic ligand required in the synthesis of MOFs may affect the activity of the enzyme, so that difficulties such as screening the optimal MOFs, adjusting the optimal ratio of metal ions and organic ligands to the enzyme, and the like exist in the process of coating the enzyme by MOFs. In the invention, the ZIF-90 with good biocompatibility is utilized, and various metal ions are screened, so that the molar ratio of the bimetallic ions, the organic ligands and the enzymes which can keep the catalytic activity of the glycosyltransferase to the maximum extent is found. Compared with free enzyme, the enzyme-bimetal organic framework material composite obtained by the invention has the following advantages in enzyme catalytic reaction: the metal organic framework material limits the glycosyltransferase in a certain spatial range, and avoids the flocculation inactivation of enzyme molecules caused by disordered movement among enzymes; the second metal ions are introduced, so that the stability of the enzyme after immobilization 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 a substrate, and the reaction rate is improved; the method can be effectively separated from the product, reduce feedback inhibition and realize reutilization; enhances the mechanical property of the enzyme and improves the operation stability.
The innovation point of the invention is that 1) magnesium ions capable of improving the catalytic activity of glycosyl transferase are screened from a plurality of metal ions; 2) compared with the more stable and easily obtained lipase, the glycosyltransferase is extremely unstable, the reaction enzyme is easy to flocculate and inactivate at room temperature in a common reaction, and the supplementary material is needed to ensure the continuous reaction, and the stability of the glycosyltransferase can be obviously improved by using MOF (metal-organic framework) coating; 3) at present, the research on MOF (metal organic framework) coated enzymes focuses on cheap and easily available enzymes such as lipase, peroxidase, catalase and glucose oxidase, and the catalytic reactions are simple reactions such as hydrogen peroxide hydrolysis.
According to the invention, 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 enzyme after immobilization can be influenced, and the bimetallic organic framework material capable of improving the enzyme catalytic efficiency is screened out. By using the glycosyltransferase-bimetallic organic framework material, the stability of the glycosyltransferase can be improved, the catalytic efficiency of the enzyme can be improved, and the glycosylation cost can be further reduced.
In addition, because the properties of glycosyltransferase are extremely unstable, flocculation inactivation is possible after the overnight reaction of free enzyme, and in order to improve the stability of glycosyltransferase, the invention finds that the loss of glycosyltransferase activity in the material synthesis process can be avoided only under the condition of specific metal ion and organic ligand ratio.
The invention introduces double metal MOF, introduces multiple divalent metal ions to simulate a biological catalysis microenvironment, introduces second metal ions to assist electron transfer in glycosylation reaction, 2) stabilizes the structure of glycosyltransferase, 3) activates the catalytic activity of glycosyltransferase, and 4) compared with electronegative free enzyme, the electropositive composite material can attract glycosyl donor and acceptor with negative charges, thereby achieving the effect of substrate enrichment and accelerating the reaction rate.
According to the invention, the divalent metal solvent is added during heparin synthesis, so that the requirement of free enzyme catalysis can be met, but in the process of immobilized enzyme, as the MOF forms a compact frame around the enzyme, the divalent metal solvent added during heparin synthesis cannot be uniformly dispersed around the enzyme, and further through the synthesized bimetallic composite material, the distance between the divalent metal ion activator and the enzyme can be reduced to the greatest extent, the microenvironment of biocatalysis is imitated to the greatest extent, and a slow release and continuous function is achieved.
Has the advantages 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 a single metal organic framework material, the composite catalytic material formed by coating glycosyltransferase can greatly maintain the efficient, mild and specific enzyme catalytic activity of zymogen. Meanwhile, the method overcomes the defects of free enzyme, improves the storage stability of the enzyme, is easy to recover, improves the repeated utilization rate, and reduces the reaction cost.
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 efficiency of enzymatic reaction is accelerated. The composite catalyst of the invention can efficiently carry out glycosylation reaction of monosaccharide, has good thermal stability and chemical stability, can improve the enzymatic reaction rate, and can realize repeated recycling and maintain higher catalytic activity.
The invention has simple preparation and convenient use, can be used for preparing the heparin oligosaccharide with uniform carbon chain length, and can be widely applied to the industries of medicine, food and the like.
When heparin is synthesized by traditional enzyme catalysis, enzyme is usually removed by extreme operation such as adding organic solvent, and enzyme impurities can be removed only by centrifugation without adding any biologically incompatible reagent.
Drawings
FIG. 1 is a graph showing real-time monitoring of the catalytic disaccharide synthesis ability of glycosyltransferase Free enzyme, glycosyltransferase-metal organic framework composite catalytic material and glycosyltransferase-double metal organic framework composite catalytic material (three curves are PmHS2@ Mg-ZIF-90, Free PmHS2, PmHS2@ ZIF-90 from top to bottom);
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 a graph showing the acid stability of glycosyltransferase free enzyme, glycosyltransferase-metal organic framework composite catalytic material, and glycosyltransferase-bimetallic organic framework composite catalytic material;
FIG. 4 is an LC-MS spectrum of glycosyltransferase-bimetallic organic framework composite catalytic material for synthesizing trisaccharide;
FIG. 5 shows the synthesis of polysaccharide by glycosyltransferase-bimetallic organic framework composite catalytic material1H NMR spectrum.
Detailed Description
The invention will be better understood from the following examples. It is easily understood by those skilled in the art that the descriptions of the embodiments are only for illustrating the present invention and should not be construed as limiting the present invention as detailed in the claims. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The experimental procedures, in which specific conditions are not indicated in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturer.
Wherein, glycosyltransferase PmHS2 (purchased from Qingdao sugar science biotechnology, Ltd., HS2 for short); uridine diphosphate-N-acetylglucosamine and its derivatives (both available from Aladdin reagents, Inc.), uridine diphosphate-glucuronic acid, p-nitrophenyl- β -D-glucuronic acid (available from national drug group chemical reagents, Inc.); 2-Imidazole-carboxaldehydee (2-carboxaldehyde Imidazole, available from Aladdin reagents, Inc.). Among them, 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 No.: 10344-94-2.
Example 1
1. Glycosyltransferase-metal organic framework composite catalytic material HS2@ ZIF-90
In 2mL of Zn (NO)3)2·6H22mL glycosyltransferase (0.33mg/mL) was added to O (40mM) solution, stirred well, and 2mL2-imidazole-carboxaldehyde (HICA,160mM) was added) And adding ultrapure water to the solution until the volume is 10 mL. The reaction was carried out at 37 ℃ and 500rpm for 24 h. After the reaction was completed, centrifugation was carried out at 11,000rpm for 4min, and the precipitate was recovered and washed three times with ultrapure water to remove glycosyltransferase not coated with the MOFs material, and air-dried.
2. Preparation of glycosyltransferase-bimetal organic framework composite catalytic material HS2@ ZIF-90(Mg)
In 2mL of Zn (NO)3)2·6H22mL of glycosyltransferase (0.33mg/mL) was added to the O (40mM) solution, and after stirring well, 2mL of MgCl was added2·4H2O (40mM), then 2mL2-imidazole-carboxaldehyde (HICA,160mM) was added and the volume was made 10mL with ultra pure water. The reaction was carried out at 37 ℃ and 500rpm for 24 h. After completion of the reaction, the reaction mixture was centrifuged at 11,000rpm for 4min to recover the precipitate. The glycosyltransferases that were not encapsulated by the MOFs were then removed by washing with ultrapure water, sonication and centrifugation three times, and air dried.
Example 2
Glycosyltransferase-metal organic framework composite catalytic material and application of glycosyltransferase-bimetallic organic framework composite catalytic material in disaccharide synthesis
1. HS2@ ZIF-90 catalyzes glycosylation of monosaccharide to generate heparin disaccharide
Uridine diphosphate-N-acetylglucosamine (final concentration: 10mM), p-nitrophenyl- β -D-glucuronic acid (final concentration: 10mM), and magnesium chloride (final concentration: 10mM) were added to a Tris-HCl buffer (50mM, pH 7.4) and mixed by shaking in a water bath at 37 ℃ for 5 minutes at 150rpm, and after mixing, the HS2@ ZIF-90 composite catalyst prepared in example 1, the mass of the reaction mixture being 5% was added thereto, followed by shaking in a water bath at 37 ℃ at 150rpm, and the reaction was completed after a sufficient reaction time of 12 hours. And centrifuging the reaction solution, separating the supernatant, performing TLC separation, and collecting the separated composite catalyst.
Quantitative analysis of heparin-disaccharide by high performance liquid chromatography column: YMC-Pack Polyamine II, mobile phase A: 0.7M NaCl, mobile phase B: h2O, time program: 0-40min 100% mobile phase a, column temperature: at 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 1-trans)The ratio of the residual glycosyl donor after reaction to the glycosyl donor before reaction) the conversion rate can reach 75%, and then the HS2@ ZIF-90 composite catalyst is recycled by using the same glycosylation method and the recycling rate is calculated, wherein the conversion rate is 63% when the catalyst is recycled for 6 times.
2. HS2@ ZIF-90(Mg) catalyzes glycosylation of monosaccharide to generate heparin disaccharide
Uridine diphosphate-N-acetylglucosamine (final concentration: 10mM), p-nitrophenyl- β -D-glucuronic acid (final concentration: 10mM), and magnesium chloride (final concentration: 10mM) were added to a Tris-HCl buffer (50mM, pH 7.4) and mixed by shaking in a water bath at 37 ℃ for 5min at 150rpm, and after mixing, the HS2@ ZIF-90(Mg) complex catalyst, which is 5% of the total mass of the reaction mixture and prepared in example 1, was added thereto, and the mixture was stirred in a water bath at 37 ℃ at 150rpm, and the reaction was sufficiently reacted for 12 hours, thereby completing the reaction. And centrifuging the reaction solution, separating the supernatant, performing TLC separation, and collecting the separated composite catalyst.
The heparin disaccharide is subjected to quantitative analysis by the high performance liquid HS2 (the liquid phase is the same as the above), the maximum absorption wavelength of the heparin disaccharide 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 can reach 100%, then the HS2@ ZIF-90(Mg) composite catalyst is subjected to repeated utilization by the same glycosylation method, the repeated utilization rate is calculated, and the conversion rate can still reach 90.0% after 6 times of repeated utilization.
3. HS2@ ZIF-90 catalyzes monosaccharide glycosylation to generate heparin disaccharide under the condition of high-concentration magnesium ions
Uridine diphosphate-N-acetylglucosamine (final concentration: 10mM), p-nitrophenyl- β -D-glucuronic acid (final concentration: 10mM), and magnesium chloride (final concentration: 50mM) were added to a Tris-HCl buffer (50mM, pH 7.4) and mixed by shaking in a water bath at 37 ℃ for 5 minutes at 150rpm, and after mixing, HS2@ ZIF-90 composite catalyst prepared in example 1, which is 5% by mass of the reaction solution, was added to the mixture and then mixed by shaking in a water bath at 37 ℃ at 150rpm, and the reaction was completed after a sufficient reaction time of 12 hours. And centrifuging the reaction solution, separating the supernatant, performing TLC separation, and collecting the separated composite catalyst.
Quantitative analysis of heparin-disaccharide by high performance liquid chromatography column: YMC-Pack Polyamine II, mobile phase A: 0.7M NaCl, mobile phase B: h2O, time program: 0-40min 100% mobile phase a, column temperature: at 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 to be 60%, which indicates that the addition of equivalent free magnesium ions can not accelerate the reaction and can inhibit the catalytic capability of HS2@ ZIF-90, and the HS2@ ZIF-90(Mg) composite catalyst obtained by covalently doping magnesium ions into the ZIF-90 has ultrahigh catalytic capability.
4. HS2 free enzyme catalyzing monosaccharide glycosylation to heparin disaccharide
Adding uridine diphosphate-N-acetylglucosamine (final concentration 10mM), p-nitrophenyl-beta-D-glucuronic acid (final concentration 10mM) and magnesium chloride (final concentration 10mM) into Tris-HCl buffer (50mM pH 7.4), shaking and mixing in a water bath at 37 ℃ for 5min at the rotation speed of 150rpm, after mixing uniformly, adding HS2 enzyme (HS 2@ ZIF-90(Mg) in step 2 is added to the reaction solution, the amount of the enzyme is about 3.3Mg/mL) at the rotation speed of 150rpm in the water bath shaking at 37 ℃ after mixing uniformly, and fully reacting for 12h to finish the reaction. The reaction solution was boiled at 100 ℃ for 5min, centrifuged, and the supernatant was fractionated by TLC.
Quantitative analysis of heparin-disaccharide by high performance liquid chromatography column: YMC-Pack Polyamine II, mobile phase A: 0.7M NaCl, mobile phase B: h2O, time program: 0-40min 100% mobile phase a, column temperature: at 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 1-the ratio of the residual glycosyl donor after the reaction to the glycosyl donor before the reaction) is calculated and is 98 percent.
5. HS2 free enzyme for catalyzing glycosylation of monosaccharide to heparin disaccharide under high-concentration magnesium ion condition
Uridine diphosphate-N-acetylglucosamine (final concentration: 10mM), p-nitrophenyl- β -D-glucuronic acid (final concentration: 10mM), and magnesium chloride (final concentration: 50mM) were added to a Tris-HCl buffer (50mM, pH 7.4) and mixed by shaking in a water bath at 37 ℃ for 5min at 150rpm, and after mixing, HS2 enzyme (final concentration: 5mg/ml) was added thereto, followed by reaction at 150rpm in a water bath shaking at 37 ℃ for 12 hours, thereby completing the reaction. And centrifuging the reaction solution, separating the supernatant, performing TLC separation, and collecting the separated composite catalyst.
Quantitative analysis of heparin-disaccharide by high performance liquid chromatography column: YMC-Pack Polyamine II, mobile phase A: 0.7M NaCl, mobile phase B: h2O, time program: 0-40min 100% mobile phase a, column temperature: at 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 1-the ratio of the residual glycosyl donor after the reaction to the glycosyl donor before the reaction) is calculated to be 99 percent.
The above experiments also show that the addition of an equal amount of free magnesium ions does not accelerate the reaction to a great extent, further demonstrating the ultra-high catalytic ability of the HS2@ ZIF-90(Mg) composite catalyst obtained by covalently incorporating magnesium ions into ZIF-90.
Example 3
Real-time monitoring of glycosyltransferase free enzyme, glycosyltransferase-metal organic framework composite catalytic material and catalytic disaccharide synthesis capacity of glycosyltransferase-double-metal organic framework composite catalytic material
1. Real-time monitoring of ability of HS2@ ZIF-90 to catalyze glycosylation of monosaccharide to heparin disaccharide
Uridine diphosphate-N-acetylglucosamine (final concentration 10mM), p-nitrophenyl- β -D-glucuronic acid (final concentration 10mM), and magnesium chloride (final concentration 10mM) were added to Tris-HCl buffer (50mM pH 7.4) and mixed in a 37 ℃ water bath with shaking at 150rpm for 5min, after mixing, HS2@ ZIF-90 composite catalyst prepared in example 1, which is 5% of the mass of the reaction solution, was added to the mixture, and then the mixture was mixed in a 37 ℃ water bath with shaking at 150rpm, and the reaction solution was centrifuged at 1 st, 2 nd, 3 th, 6 th, 9 th, 12 th, 24 th, and 36 th hours of the reaction, and a portion of the supernatant was collected and subjected to HPLC analysis.
The change rule of the HS2@ ZIF-90 catalytic conversion rate along with time is detected by high performance liquid chromatography to judge the catalytic efficiency, YMC-Pack Polyamine II, and the mobile phase A: 0.7M NaCl, mobile phase B: h2O, time ofThe procedure is as follows: 0-40min 100% mobile phase a, column temperature: at 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 1-the ratio of the residual glycosyl donor after the reaction to the glycosyl donor before the reaction) is calculated. The catalytic reaction reached substantial equilibrium at 9 hours with a final conversion of 75% (fig. 1).
2. Real-time monitoring of ability of HS2@ ZIF-90(Mg) to catalyze glycosylation of monosaccharides to heparin disaccharides
Uridine diphosphate-N-acetylglucosamine (final concentration 10mM), p-nitrophenyl- β -D-glucuronic acid (final concentration 10mM), and magnesium chloride (final concentration 10mM) were added to Tris-HCl buffer (50mM pH 7.4) and mixed by shaking in a 37 ℃ water bath at 150rpm for 5min, after mixing, HS2@ ZIF-90(Mg) composite catalyst prepared in example 1, which was 5% by mass of the reaction solution, was added, and then the mixture was mixed in a 37 ℃ water bath at 150rpm, and the reaction solutions were centrifuged at 1 st, 2 nd, 3 rd, 6 th, 9 th, 12 th, 24 th, and 36 th hours of the reaction, respectively, and a part of the supernatant was fractionated and analyzed by HPLC.
The change rule of the catalytic conversion rate of HS2@ ZIF-90(Mg) along with time is detected by high performance liquid chromatography to judge the catalytic efficiency, YMC-Pack Polyamine II, and the mobile phase A: 0.7M NaCl, mobile phase B: h2O, time program: 0-40min 100% mobile phase a, column temperature: at 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 1-the ratio of the residual glycosyl donor after the reaction to the glycosyl donor before the reaction) is calculated. The catalytic reaction reached essentially equilibrium at 6 hours with a final conversion of 100% (FIG. 1). The catalytic capability and catalytic efficiency of the HS2@ ZIF-90(Mg) composite catalyst are greatly improved.
3. Real-time monitoring of ability of HS2 free enzyme to catalyze glycosylation of monosaccharide to heparin disaccharide
Uridine diphosphate-N-acetylglucosamine (final concentration 10mM), p-nitrophenyl- β -D-glucuronic acid (final concentration 10mM), and magnesium chloride (final concentration 10mM) were added to Tris-HCl buffer (50mM pH 7.4) and mixed by shaking in a 37 ℃ water bath at 150rpm for 5min, and after mixing, HS2 free enzyme (final concentration 5mg/ml) was added and then mixed by shaking in a 37 ℃ water bath at 150rpm, and portions of the reaction solution were separated at 1, 2, 3, 6, 9, 12, 24, and 36 hours of the reaction and analyzed by HPLC, respectively.
The change rule of the HS2 catalytic conversion rate along with time is detected by high performance liquid chromatography to judge the catalytic efficiency, YMC-Pack Polyamine II, mobile phase A: 0.7M NaCl, mobile phase B: h2O, time program: 0-40min 100% mobile phase a, column temperature: at 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 1-the ratio of the residual glycosyl donor after the reaction to the glycosyl donor before the reaction) is calculated. The catalytic reaction reached substantial equilibrium at 12 hours with a final conversion of 98% (fig. 1).
In addition, real-time monitoring data of a substrate product shows that the HS2@ ZIF-90(Mg) synthesized by the method can reach the conversion rate of 97 percent in three hours, and the catalytic activity and the efficiency of the method are far higher than those of free enzyme and common immobilization, and the method is high in speed and conversion rate.
Example 4
And (3) testing the temperature stability of the glycosyltransferase free enzyme, the glycosyltransferase-metal organic framework composite catalytic material and the glycosyltransferase-bimetal organic framework composite catalytic material.
1. Temperature stability test of HS2@ ZIF-90
Uridine diphosphate-N-acetylglucosamine (final concentration 10mM), p-nitrophenyl-beta-D-glucuronic acid (final concentration 10mM) and magnesium chloride (final concentration 10mM) were added to Tris-HCl buffer (50mM, pH 7.4) and mixed by shaking at 150rpm for 5min, and after mixing, HS2@ ZIF-90 composite catalyst prepared in example 1, which is 5% by mass of the reaction solution, was added to the mixture, and the mixture was incubated in a water bath at 50, 60 and 70 ℃ for 30min, followed by reaction at 150rpm for 12h, thereby completing the reaction. The reaction solution was centrifuged, and the supernatant was fractionated by TLC and HPLC. The final conversions were 70%, 64%, 59%, respectively (fig. 2).
2. Temperature stability test of HS2@ ZIF-90(Mg)
Uridine diphosphate-N-acetylglucosamine (final concentration: 10mM), p-nitrophenyl-beta-D-glucuronic acid (final concentration: 10mM) and magnesium chloride (final concentration: 10mM) were added to a Tris-HCl buffer (50mM, pH 7.4) and mixed by shaking at 150rpm for 5min, and after mixing uniformly, HS2@ ZIF-90(Mg) composite catalyst prepared in example 1, which is 5% of the mass of the reaction solution, was added to the mixture, and the mixture was incubated in a water bath at 50, 60 and 70 ℃ for 30min, followed by reaction at 150rpm for 12h, thereby terminating the reaction. The reaction solution was centrifuged, and the supernatant was fractionated by TLC and HPLC. The final conversions were 92%, 85%, 74%, respectively (fig. 2).
3. Temperature stability test of HS2 free enzyme
Adding uridine diphosphate-N-acetylglucosamine (final concentration is 10mM), p-nitrophenyl-beta-D-glucuronic acid (final concentration is 10mM) and magnesium chloride (final concentration is 10mM) into a Tris-HCl buffer (50mM, pH 7.4), shaking and mixing at the rotating speed of 150rpm for 5min, adding HS2 free enzyme with the final concentration of 5mg/ml after uniform mixing, incubating in a water bath at 50, 60 and 70 ℃ for 30min respectively, and then reacting at the rotating speed of 150rpm for 12h to finish the reaction. The supernatant was fractionated by TLC and HPLC. The final conversions were 65%, 29%, 13%, respectively (FIG. 2).
Compared with free enzyme, the temperature stability of the metal organic framework immobilized enzyme is remarkably improved, wherein compared with the traditional ZIF-90 encapsulated enzyme, the HS2@ ZIF-90(Mg) composite catalyst embodies better temperature stability, and lays a foundation for 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 (50mM, 5mL) at pH 3, 4, 5, 6, 7 for 30min, and after completion, the buffer was removed by centrifugation at 8000 rpm.
Adding uridine diphosphate-N-acetylglucosamine (with the final concentration of 10mM), p-nitrophenyl-beta-D-glucuronic acid (with the final concentration of 10mM) and magnesium chloride (with the final concentration of 10mM) into a Tris-HCl buffer solution (with the concentration of 50mM and the pH of 7.4), shaking and mixing at the rotating speed of 150rpm for 5min, respectively adding the treated HS2@ ZIF-90 composite catalyst with the mass being 5% of the mass of a reaction solution after uniform mixing, carrying out water bath shaking reaction at the temperature of 37 ℃ and the rpm of 150 ℃ for 12h, then finishing the reaction, centrifuging the reaction solution, and separating and carrying out TLC separation and HPLC detection on a supernatant. The final conversions were 53%, 62%, 37%, 56%, 75%, respectively (FIG. 3).
2. Acid stability test of HS2@ ZIF-90(Mg)
HS2@ ZIF-90(Mg) hybrid catalyst prepared in example 1 was incubated in Tris-HCl buffer (50mM, 5mL) at pH 3, 4, 5, 6, 7 for 30min, and after completion, the buffer was removed by centrifugation at 8000 rpm.
Adding uridine diphosphate-N-acetylglucosamine (with the final concentration of 10mM), p-nitrophenyl-beta-D-glucuronic acid (with the final concentration of 10mM) and magnesium chloride (with the final concentration of 10mM) into a Tris-HCl buffer solution (with the concentration of 50mM and the pH of 7.4), shaking and mixing at the rotating speed of 150rpm for 5min, adding the treated HS2@ ZIF-90(Mg) composite catalyst accounting for 5% of the mass of the reaction solution after uniform mixing, carrying out water bath shaking reaction at the temperature of 37 ℃ and the rpm of 150 ℃ for 12h, then finishing the reaction, centrifuging the reaction solution, and separating and carrying out TLC separation and HPLC detection on the supernatant. The final conversion rates are respectively 90%, 95%, 98%, 100% and 100% (fig. 3), which proves that the HS2@ ZIF-90(Mg) composite catalyst has better acid stability compared with the traditional ZIF-90 material.
3. Acid stability test of HS2 free enzyme
HS2 was incubated for 30min in Tris-HCl buffer (50mM, 5mL) at pH 3, 4, 5, 6, 7, respectively.
Adding uridine diphosphate-N-acetylglucosamine (final concentration 10mM), p-nitrophenyl-beta-D-glucuronic acid (final concentration 10mM) and magnesium chloride (final concentration 10mM) into Tris-HCl buffer (50mM, pH 7.4), shaking and mixing at the rotation speed of 150rpm for 5min, after uniform mixing, respectively adding the above buffer solution with the final concentration of 5mg/ml HS2 free enzyme, shaking and reacting in a water bath at 37 ℃ and 150rpm for 12h, then finishing the reaction, and collecting the supernatant for TLC separation and HPLC detection. The final conversions were 68%, 70%, 68%, 96%, respectively (FIG. 3).
Example 6
HS2@ ZIF-90(Mg) catalyzed heparan trisaccharide generation
Uridine diphosphate-glucuronic acid (final concentration: 10mM), acetylglucosamine-glucuronic acid-p-nitrophenyl (final concentration: 10mM), and magnesium chloride (final concentration: 10mM) were added to a Tris-HCl buffer (50mM, pH 7.4) and mixed by shaking in a water bath at 37 ℃ for 5min at a rotation speed of 150rpm, and after mixing, the HS2@ ZIF-90(Mg) composite catalyst prepared in example 1, in an amount of 10% by mass of the reaction solution, was added thereto and then mixed by shaking in a water bath at 37 ℃ at a rotation speed of 150rpm, and the reaction was completed after a sufficient reaction for 12 hours. The reaction solution was centrifuged, the supernatant was fractionated to obtain heparin trisaccharide, and qualitative analysis was performed on heparin trisaccharide by liquid chromatography-mass spectrometry (fig. 4), with a conversion rate of 100%, indicating that the present invention can be applied not only to disaccharide synthesis but also to trisaccharide synthesis.
Example 7
HS2@ ZIF-90(Mg) catalyzed heparin polysaccharide production
Uridine diphosphate-N-acetylglucosamine (final concentration 10mM), uridine diphosphate-glucuronic acid (final concentration 10mM), p-nitrophenyl-glucuronic acid (final concentration 1mM), and magnesium chloride (final concentration 10mM) were added to Tris-HCl buffer (50mM, pH 7.4) and mixed by shaking in a 37 ℃ water bath at 150rpm for 5min, and after mixing, HS2@ ZIF-90(Mg) complex catalyst prepared in example 1, which is 10% by mass of the reaction mixture, was added to the mixture and then mixed by shaking in a 37 ℃ water bath at 150rpm, and the reaction was completed after 12 hours of complete reaction. The reaction solution was centrifuged, the supernatant was collected to obtain heparin polysaccharide, and qualitative analysis was performed on heparin polysaccharide by means of a nuclear magnetic resonance spectrometer (fig. 5), which shows that the present invention can be applied not only to the synthesis of disaccharides and trisaccharides, but also to the synthesis of polysaccharides.
Example 8
The enzyme activities of different glycosyltransferases-metal organic framework composite catalytic materials in example 1 of the present invention in different environments were tested, and compared with the enzyme activities of free glycosyltransferases not coated by the MOFs material.
The two glycosyltransferase-metal organic framework composite catalytic materials and the free enzyme in example 1 are respectively treated in organic solvents at different temperatures for 2 hours. The two treated composite catalytic materials and the free enzyme are used for the glycosylation reaction of monosaccharide (according to the method of the embodiment 2), then heparin disaccharide is quantitatively analyzed through high performance liquid, the conversion rate of the glycosylation reaction (the conversion rate is the ratio of the residual glycosyl acceptor and glycosyl donor after the reaction) is calculated, and the enzyme activity ratio (relative activity%) is the ratio of the conversion rate of the glycosylation reaction after the treatment to the conversion rate catalyzed by the original free enzyme. According to some experimental analysis, under the condition of the same enzyme amount, free glycosyltransferase HS2 in organic solvents such as DMSO and THF is easy to inactivate and almost has no activity, and glycosyltransferases wrapped by HS2@ ZIF-90 and HS2@ ZIF-90(Mg) have good protection effect on the enzyme compared with the free enzyme, and HS2@ ZIF-90(Mg) has the best protection effect on the enzyme, and the conversion rate can reach more than 90 percent and is obviously better than HS2@ ZIF-90 (about 70 percent).
From the experiments, the glycosyltransferase-bimetallic organic framework composite catalytic material can obviously improve the conversion rate, can reach more than 10-20% under certain specific extreme conditions, has a very prominent effect (the improvement of a few percent in the conversion rate of the glycosylation reaction can be considered as the obvious improvement), and can be effectively applied to industrial processing in certain specific environments.
Example 9
Example 9 was prepared identically to example 1, except that: the non-Zn metal ion being Fe2+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 ligands is 1:5, the concentration of glycosyltransferase is 0.1mg/ml, and after uniform stirring, the reaction is carried out for 25 hours at 35 ℃. After the reaction, the mixture was centrifuged at 8000rpm for 6min to recover the precipitate.
Example 10
Example 10 was prepared in the same manner as example 1, except that: the non-Zn metal ion being Mn2+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 ligands is 1:4, the concentration of glycosyltransferase is 0.5mg/ml, and after uniform stirring, the reaction is carried out for 15 hours at 40 ℃. After completion of the reaction, the mixture was centrifuged at 12000rpm for 4min to recover the precipitate.
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-acetylglucosamine; adding 10 mass percent of HS2@ ZIF-90(Mg) composite catalyst prepared in example 1 into the reaction solution, oscillating the mixture in a water bath at 20 ℃ at the rotating speed of 300rpm, and fully reacting for 15 hours to finish the reaction. And centrifuging the reaction solution, separating the supernatant, performing TLC separation, and collecting the separated composite catalyst.
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, the HS2@ ZIF-90(Mg) composite catalyst which is prepared in the example 1 and has the mass of 8 percent of the reaction liquid is added, the rotation speed of 100rpm is carried out in water bath oscillation at the temperature of 50 ℃, and the reaction is finished after the reaction is fully carried out for 10 hours. And centrifuging the reaction solution, separating the supernatant, performing TLC separation, and collecting the separated composite catalyst.
Claims (10)
1. The glycosyltransferase-bimetal organic framework composite catalytic material is characterized in that a Zn-based MOFs material is used as a main body, non-Zn metal ions are doped, and glycosyltransferase is wrapped in the composite catalytic material to form the composite catalytic material.
2. The magnetic nanoparticle-glycosyltransferase-amorphous metal-organic framework composite catalytic material of claim 1, wherein the doped non-Zn metal ion preferably comprises Mn2+,Fe2+,Co2+,Mg2+,Ca2+,Cu2+,Ni2+One or more of.
3. The glycosyltransferase-bimetallic organic framework composite catalytic material of claim 1, wherein the glycosyltransferase is heparin synthase 2 (HS 2), including pmHS2(Pasteurella multocida HS2) or syntactical construct HS 2.
4. A method for preparing the glycosyltransferase-bimetallic organic framework composite catalytic material of claim 1, comprising the steps of:
(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 a zinc nitrate solution and another non-Zn metal ion solution;
(2) and (2) carrying out centrifugal washing and air drying on the reaction liquid obtained in the step (1) to obtain the glycosyltransferase-bimetal organic framework composite catalyst.
5. The preparation method according to claim 4, wherein the stirring reaction in the step (1) is a stirring reaction at 35-40 ℃ for 15-25 h to obtain a reaction solution containing the composite catalyst; the centrifugation condition of the step (2) is 8,000-12,000 rpm for 4-6 min.
6. The preparation method according to claim 4, wherein the molar ratio of the zinc ions to the non-Zn metal ions in the step (1) is 1:1 to 3:1, and the molar ratio of the zinc ions to the 2-formaldehyde imidazole organic ligand is 1:4 to 5.
7. Use of the glycosyltransferase-bimetallic organic framework composite catalytic material of claim 1 in the preparation of a disaccharide and polysaccharide synthesis.
8. Use according to claim 7, characterized in that it comprises the following steps:
(1) mixing a glycosyl acceptor, a glycosyl donor and a divalent metal ion;
(2) adding glycosyltransferase-bimetal organic framework composite catalytic material, and oscillating for reaction;
(3) and (3) centrifuging the reaction solution oscillated in the step (2), and taking the supernatant to perform TLC separation to obtain heparin disaccharide or heparin polysaccharide.
9. The use according to claim 7, wherein the glycosyl donor of step (1) is uridine diphosphate-N-acetylglucosamine or a derivative thereof; the glycosyl receptor 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.
10. The use of claim 7, wherein the glycosyltransferase-bimetallic organic framework composite catalytic material of step (2) is added in an amount of 5-10% by mass of the reaction solution; the oscillation reaction is a water bath oscillation reaction at 20-50 ℃, the rotating speed is 100-300 rpm, and the reaction time is 10-15 h.
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