An immobilized multi-enzyme material, its preparation method and application

Abstract

The present invention discloses an immobilized multi-enzyme material, its preparation method and application. The immobilized multi-enzyme material includes a first enzyme, a second enzyme and a third enzyme. The first enzyme and the second enzyme are immobilized inside ZIF-90, and the third enzyme is immobilized on the surface of ZIF-90. The first enzyme is N-acetylhexosamine 1-kinase, the second enzyme is uridine transferase, and the third enzyme includes polyphosphate kinase, heparin synthase, hyaluronic acid synthase, chondroitin polymerase or N-acetylglucosamine transferase. The immobilized multi-enzyme material of the present invention can shorten the transmission distance and reaction time of the substrate, reduce the accumulation and inhibition of intermediates, thereby significantly improving the catalytic efficiency of the entire multi-enzyme cascade reaction. It can be applied to the synthesis of various sugars, and the immobilized multi-enzyme system has good stability, can realize the recycling of enzymes, and can be used repeatedly for many times, greatly reducing the amount of enzymes required for the catalytic reaction and reducing production costs.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme catalysis and enzyme immobilization, and in particular relates to an immobilized multi-enzyme material and a preparation method and application thereof. Background Art

[0002] Carbohydrate compounds are an important class of information molecules, which are widely involved in life processes such as pathogen infection, immune response, cell signal transduction, cell division and differentiation, inflammatory response, sperm-egg recognition and tumor metastasis. On the other hand, carbohydrates are also important medicinal molecules, such as anticoagulant heparin, aminoglycoside antibiotics and bacterial polysaccharide conjugate vaccines, which are widely used in disease treatment and prevention. In addition, carbohydrates are often used as "chiral pool" starting materials to synthesize natural products or pharmaceutical intermediates with important biological activity and application value. At present, the most commonly used methods for synthesizing carbohydrates at home and abroad are chemical synthesis, fermentation and enzymatic synthesis. In carbohydrate synthesis, chemical catalysis usually involves multiple steps of activation, protection, glycosylation and deprotection. The process is cumbersome and the yield is low. There are also problems such as expensive reagents and environmental pollution. Whole-cell fermentation can achieve large-scale production, but due to the complex synthetic generation pathway, the coordination of multi-step metabolism and the regulation of metabolic flow are difficult. In contrast, enzyme-catalyzed reactions have the advantages of high efficiency and simplicity, strong stereospecificity, good regioselectivity, mild reaction conditions and environmental friendliness. In practical applications, enzymes in free solution state have the disadvantages of difficulty in subsequent separation, recovery and repeated use, and poor stability. At the same time, carbohydrates have a variety of structures, and their material conversion and energy metabolism require the participation of multiple enzymes to complete synergistically. The compatibility of reaction conditions between key enzymes limits the overall catalytic efficiency. In addition, the in vitro multi-enzyme catalysis of carbohydrates is limited by substrate concentration and is difficult to adapt to the high-concentration substrate environment in industrial applications. In recent years, with the rapid development of simulated biology and industrial biocatalysis, constructing an ideal in vitro multi-enzyme cascade reaction system to deeply explore the complex functions of organisms and develop new green and efficient catalytic methods has become one of the major challenges faced by scientific researchers. Therefore, it is urgent to develop an immobilization strategy for multi-enzyme cascades to enhance the stability of enzyme molecules, give full play to the performance of synergistic catalysis between different enzymes, and have the advantages of stability, easy storage, reusability and continuous operation. On the other hand, due to the diversity of carbohydrates and the complexity of multi-enzyme catalytic systems in practical applications, this strategy should be universal and universal, and can be widely used in the effective fixation of different multi-enzyme cascade catalytic systems, and can solve some problems of in vitro enzyme catalytic synthesis of carbohydrates in industrial applications. Summary of the invention

[0003] Object of the Invention: The present invention provides an immobilized multi-enzyme material, which can significantly improve the catalytic efficiency of multi-enzyme cascade reactions, enhance the stability of multi-enzymes, achieve the recyclability of enzymes, and reduce production costs. At the same time, this immobilized material can be applied to catalytic reactions with high-concentration substrates and has great potential for industrial applications. In particular, the immobilization strategy using this immobilized multi-enzyme material is universal for different types of multi-enzyme catalytic systems.

[0004] The present invention also provides a preparation method and application of the above-mentioned immobilized multi-enzyme material.

[0005] Technical Solution: To achieve the above object, an immobilized multi-enzyme material of the present invention includes a first enzyme, a second enzyme, and a third enzyme. The first enzyme and the second enzyme are immobilized inside ZIF-90, and the third enzyme is immobilized on the surface of ZIF-90. The first enzyme is N-acetylhexosamine 1-kinase, the second enzyme is uridine transferase, and the third enzyme includes any one or several of polyphosphate kinase, heparin synthase, hyaluronic acid synthase, chondroitin polymerase, and N-acetylglucosamine transferase.

[0006] The preparation method of the immobilized multi-enzyme material of the present invention includes the following steps:

[0007] (1) Pour the mixed enzyme solution containing the first enzyme and the second enzyme into an imidazole-2-carboxaldehyde solution and mix evenly; quickly add a zinc nitrate solution, stir and react, wash with deionized water, and perform centrifugal separation to obtain dual-enzymes@ZIF-90.

[0008] (2) Uniformly mix the solution containing the amino ligand with dual-enzymes@ZIF-90, heat in a water bath, wash the solid with water until the supernatant is clear, and perform centrifugation to obtain dual-enzymes@ZIF-90-NH2.

[0009] (3) Mix dual-enzymes@ZIF-90-NH2 with NHS-PEG n -NHS and the third enzyme for reaction, after solid-liquid separation, collect the solid product triple-enzymes@ZIF-90, which is the immobilized multi-enzyme material.

[0010] Among them, in step (1), the concentrations of the zinc nitrate solution and the imidazole-2-carboxaldehyde mother liquor are 80-160 mM and 160-320 mM respectively, and the final concentration ratio of zinc nitrate to imidazole-2-carboxaldehyde is 1:2 to 1:6.

[0011] Preferably, the final molar ratio of zinc nitrate to imidazole-2-carboxaldehyde in the system is 1:4.

[0012] Among them, the solution containing the amino ligand in step (2) is a Tris-HCl solution containing the amino ligand; the amino ligand is any one of 3-amino-1,2,4-triazole, 2-aminobenzimidazole, 6-aminopurine, and 2'-amino-[1,1':4',1''-terphenyl]-4,4''-dicarboxylate, and the concentration of the prepared stock solution is 20-80 mM.

[0013] Among them, the amino ligand solution is prepared by dissolving the amino ligand in a Tris-HCl buffer solution with a pH of 7-8 and a concentration of 50-100 mM.

[0014] Among them, the water bath heating in step (2) is water bath heating and stirring at 40-50 °C for 0.5-1 h, and the rotation speed is 100-200 rpm.

[0015] Among them, the NHS-PEG n -NHS in which n = 3-200, and the concentration of NHS-PEG n -NHS is 20-80 mM.

[0016] Among them, the concentration ratio of the first enzyme, the second enzyme, and the third enzyme is 1:1:1 to 1:1:4, and the final average concentration in the system is 0.5-2 mg / mL.

[0017] Preferably, the concentration ratio of the first enzyme, the second enzyme, and the third enzyme is 1:1:1

[0018] Preferably, the preparation of the immobilized triple-enzyme cascade system includes the following steps:

[0019] (1) Pour the mixed enzyme solution containing 0.5-2 mg / mL of the first enzyme and 0.5-2 mg / mL of the second enzyme into a 160-320 mM imidazole-2-carbaldehyde solution and mix evenly; quickly add an equal volume of 80-160 mM zinc nitrate solution, stir and react for 0.5-1 h, with a rotation speed of 100-200 rpm, wash with deionized water, and centrifuge at 8,000-12,000 rpm for 5-10 min to obtain double-enzyme@ZIF-90.

[0020] (2) Prepare a Tris-HCl solution of an amino ligand with a concentration of 20-80 mM, uniformly mix the amino ligand solution with the double-enzyme@ZIF-90 obtained above, heat and stir in a water bath at 40-50 °C for 0.5-1 h, with a rotation speed of 100-200 rpm, wash the solid with deionized water until the supernatant is clear, and centrifuge at 8,000-12,000 rpm for 5-10 min to obtain double-enzyme@ZIF-90-NH2.

[0021] (3) Mix the double-enzyme@ZIF-90-NH2 obtained above with 20-80 mM of NHS-PEGn The - NHS solution and the third enzyme at a concentration of 0.5 - 2 mg / mL were uniformly mixed, crosslinked for 0.5 - 1 h, washed three times with deionized water, centrifuged at 6,000 - 8,000 rpm for 5 min, and freeze-dried under vacuum to obtain the immobilized three-enzyme cascade catalytic material, three-enzyme@ZIF-90.

[0022] Use of the immobilized multi-enzyme material described in the present invention in the preparation of glycosyl donors, heparin, hyaluronic acid, chondroitin, and human milk oligosaccharides.

[0023] Among them, the specific preparation process of the said use is as follows:

[0024] Synthesis of glycosyl donor: In a 2 mL centrifuge tube, add ATP, UTP, monosaccharide substrate, MgCl2, three-enzyme@ZIF-90, and 100 mM Tris-HCl buffer solution with a pH of 7.5, oscillate the reaction, and detect the glycosyl donor by high performance liquid chromatography.

[0025] Among them, the final concentration of ATP is 5 - 15 mM, the final concentration of UTP is 5 - 15 mM, the final concentration of the monosaccharide substrate is 5 - 15 mM, the final concentration of MgCl2 is 5 - 10 mM, and the final concentration of three-enzyme@ZIF-90 is 0.5 - 1 mg / mL.

[0026] Among them, the oscillating reaction is a metal bath oscillating reaction at 35 - 40 °C, the rotation speed is 700 - 1,000 rpm, and the reaction time is 36 - 48 h.

[0027] (2) Disaccharide synthesis: In a 2 mL centrifuge tube, add ATP, UTP, monosaccharide substrate, GlcA-pNP, MgCl2, three-enzyme@ZIF-90, and 100 mM Tris-HCl buffer solution with a pH of 7.5, the immobilized multi-enzyme material provided by each example, oscillate the reaction, and detect the disaccharide by high performance liquid chromatography.

[0028] Among them, the final concentration of ATP is 5 - 15 mM, the final concentration of UTP is 5 - 15 mM, the final concentration of GlcA-pNP is 5 - 10 mM, the final concentration of the monosaccharide substrate is 5 - 15 mM, the final concentration of MgCl2 is 5 - 10 mM, and the final concentration of three-enzyme@ZIF-90 is 0.5 - 1 mg / mL.

[0029] Among them, the oscillating reaction is a metal bath oscillating reaction at 35 - 40 °C, the rotation speed is 700 - 1000 rpm, and the reaction time is 36 - 48 h.

[0030] (3) Glycan synthesis: Add ATP, UTP, monosaccharide substrate, GlcA-pNP, UDP-GlcA, MgCl2, triple enzyme@ZIF-90 and 100 mM Tris-HCl buffer with pH 7.5 into 2 mL centrifuge tubes. For the immobilized multi-enzymes provided in each of the above examples, conduct an oscillating reaction and detect the glycan by polyacrylamide gel electrophoresis.

[0031] Among them, the final concentration of ATP is 5 - 15 mM, the final concentration of UTP is 5 - 15 mM, the final concentration of GlcA-pNP is 5 - 10 mM, the final concentration of UDP-GlcA is 5 - 10 mM, the final concentration of monosaccharide substrate is 5 - 15 mM, the final concentration of MgCl2 is 5 - 10 mM, and the final concentration of triple enzyme@ZIF-90 is 0.5 - 1 mg / mL.

[0032] Among them, the oscillating reaction is a metal bath oscillating reaction at 35 - 40 °C, the rotation speed is 700 - 1000 rpm, and the reaction time is 36 - 48 h.

[0033] Preferably, the application of the immobilized triple enzyme cascade system is as follows:

[0034] (1) Glycosyl donor synthesis

[0035] Take ATP (5 - 15 mM), UTP (5 - 15 mM), monosaccharide substrate (5 - 15 mM), MgCl2 (5 - 10 mM), immobilized multi-enzyme material (0.5 - 1 mg / mL) and 100 mM Tris-HCl buffer with pH 7.5 respectively, conduct the reaction at 37 °C for 48 h, and detect the glycosyl donor by high performance liquid chromatography.

[0036] (2) Disaccharide synthesis

[0037] Take ATP (5 - 15 mM), UTP (5 - 15 mM), monosaccharide substrate (5 - 15 mM), GlcA-pNP (5 - 10 mM), MgCl2 (5 - 10 mM), immobilized multi-enzyme material (0.5 - 1 mg / mL) and 100 mM Tris-HCl buffer with pH 7.5 respectively, conduct the reaction at 37 °C for 48 h, and detect the glycosyl donor by high performance liquid chromatography.

[0038] (3) Glycan synthesis

[0039] ATP (5 - 15 mM), UTP (5 - 15 mM), monosaccharide substrate (5 - 15 mM), GlcA-pNP (5 - 15 mM), UDP-GlcA (5 - 10 mM), MgCl2 (5 - 10 mM), immobilized multi-enzyme material (0.5 - 1 mg / mL), and 100 mM Tris-HCl buffer at pH 7.5 were taken respectively and reacted at 37 °C for 48 h, and the sugar chains were detected by polyacrylamide gel electrophoresis.

[0040] Advantages: Compared with the prior art, the present invention has the following advantages:

[0041] 1. In the present invention, N-acetylhexosamine 1-kinase and uridyltransferase related to glycosyl donor synthesis are immobilized inside ZIF-90 by in-situ coating method, and enzymes for cofactor regeneration or glycosidic bond synthesis are immobilized outside the material by chemical cross-linking method. This compartmentalized immobilization strategy can reduce the interference between different enzymes.

[0042] 2. The immobilized multi-enzyme catalytic material prepared in the present invention combines three different enzymes with ZIF-90 in an orderly manner by in-situ coating method and cross-linking method. Compared with free enzymes, it shortens the transmission distance between substrates and the reaction time, reduces the inhibition of intermediates, and can obtain higher catalytic efficiency.

[0043] 3. The present invention proposes a method for ligand exchange applied to post-synthetic modification of ZIF-90. On the one hand, the amino ligand can endow ZIF-90 with amino groups through ligand exchange, which is very important for the connection of the third enzyme. On the other hand, ligand exchange can improve the structure and porosity of ZIF-90 and promote substrate transfer.

[0044] 4. The present invention proposes a molecule NHS-PEG n -NHS with adjustable length for the connection between inorganic materials and enzyme molecules. The succinimides at both ends of NHS-PEG n -NHS undergo amidation reactions with the amino groups on the surface of enzymes and ZIF-90 to achieve the immobilization of the third enzyme. This bridging molecule can achieve the immobilization of enzymes under mild conditions, reducing the loss of enzyme activity. NHS-PEG n -NHS has good biocompatibility and can form a microenvironment conducive to enzyme catalysis; NHS-PEG n -NHS has an adjustable length and can be applicable to enzymes with different characteristics.

[0045] 5. The immobilized multi-enzyme catalytic material prepared in the present invention maintains the original high-efficiency, mild and specific enzyme catalytic activity of the enzyme, while overcoming the deficiencies of free enzymes, improving the storage stability of the enzyme, increasing the reuse rate, and reducing the reaction cost.

[0046] 6. The immobilized multi-enzyme catalytic material prepared by the present invention exhibits great potential for industrial application. In the sugar nucleotide synthesis pathway, it still maintains good activity in a catalytic environment with a substrate concentration as high as 400 mM. In the synthesis of disaccharides and sugar chains, it still maintains good activity in a catalytic environment with a substrate concentration as high as 300 mM.

[0047] 7. The preparation method of the immobilized multi-enzyme catalytic material of the present invention has good generality and wide universality, and is applicable to multi-enzyme cascade systems for synthesizing different sugars (including heparin, chondroitin, hyaluronic acid, and oligosaccharide enzymatic synthesis systems for human milk oligosaccharides), greatly reducing the workload and cost of immobilization research on different multi-enzyme cascade systems. Description of the Drawings

[0048] Figure 1 It is the potentiometric test chart of the immobilized multi-enzyme provided in Example 1.

[0049] Figure 2 It is the scanning electron microscope image of the immobilized multi-enzyme provided in Example 1.

[0050] Figure 3 It is the mass spectrum of the glycosyl donor UDP-GlcNAc prepared in Example 1.

[0051] Figure 4 It is the nuclear magnetic spectrum of the glycosyl donor UDP-GlcNAc prepared in Example 1.

[0052] Figure 5 It is the mass spectrum of the glycosyl donor UDP-GlcNTFA prepared in Example 1.

[0053] Figure 6 It is the nuclear magnetic spectrum of the glycosyl donor UDP-GlcNTFA prepared in Example 1.

[0054] Figure 7 It is the comparison chart of product conversion rates in Example 2 and Comparative Example 1.

[0055] Figure 8 It is the mass spectrum of the heparin disaccharide prepared in Example 3.

[0056] Figure 9 It is the nuclear magnetic spectrum of the heparin disaccharide prepared in Example 3.

[0057] Figure 10 It is the mass spectrum of the hyaluronic acid disaccharide prepared in Example 4.

[0058] Figure 11 It is the nuclear magnetic spectrum of the hyaluronic acid disaccharide prepared in Example 4.

[0059] Figure 12It is the mass spectrum of chondroitin disaccharide prepared in Example 5.

[0060] Figure 13 It is the NMR spectrum of chondroitin disaccharide prepared in Example 5.

[0061] Figure 14 It is the mass spectrum of neo-N-lactotriose prepared in Example 6.

[0062] Figure 15 It is the NMR spectrum of neo-N-lactotriose prepared in Example 6.

[0063] Figure 16 It is a schematic diagram of the preparation of hyaluronic acid and chondroitin sugar chains using the immobilized multi-enzyme system provided in Example 7 and Example 8.

[0064] Figure 17 It is a schematic diagram of the enzyme activity of the immobilized multi-enzyme system provided in Example 9 under different environments.

[0065] Figure 18 It is a bar chart of the multiple recycling of hyaluronic acid disaccharide prepared using the immobilized multi-enzyme system provided in Example 10.

[0066] Figure 19 It is the storage stability of the immobilized multi-enzyme system provided in Example 1. Detailed implementation manners

[0067] According to the following examples, the present invention can be better understood. Those skilled in the art can easily understand that the content described in the examples is only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims.

[0068] The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. The experimental methods without specific conditions noted in the examples are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Among them, N-acetylglucosamine 1-kinase (ANC68241.1), uridine transferase (ACO75977.1), polyphosphate kinase (CAD6006995.1), heparin synthase (AAQ55110.1), hyaluronic acid synthase (AAC38318.2), chondroitin polymerase (CAD5992240.1), N-acetylglucosamine transferase (AAL12840.1) are artificially synthesized according to the sequences on NCBI or obtained commercially. ATP, UTP, NHS-PEG n-NHS (such as NHS-PEG5-NHS, CAS 756526-03-1), 3-amino-1,2,4-triazole, 2-aminobenzimidazole, 6-aminopurine, and 2'-amino-[1,1':4',1''-terphenyl]-4,4''-dicarboxylate were purchased from Macklin Reagent Co., Ltd. Lactose, GlcNAc, and GalNAc were purchased from Aladdin Reagent Co., Ltd. MgCl2 and zinc nitrate were purchased from Sinopharm Chemical Reagent Co., Ltd. UDP-GlcA and GlcA-pNP were purchased from Shanghai Baosen Reagent Co., Ltd. The monosaccharide such as GlcNAc in the present invention can also be replaced with the monosaccharide derivative GlcNTFA, and its structure can be synthesized according to the literature (Li Xiaoyan, Qi Chen, Wei Peilian, Huang Lei, Cai Jin, Xu Zhinan. Efficient chemoenzymatic synthesis of uridine 5'-diphosphate N-acetylglucosamine and uridine 5′-diphosphate N-trifluoacetyl glucosamine with three recombinant enzymes. Preparative Biochemistry and Biotechnology, 2017, 852-859)

[0069] The monosaccharide such as GlcNAc in the present invention can also be replaced with the monosaccharide derivatives GlcNAz, GlcNBu, GlcNPr, GlcNBz for reaction, and their structures are Compounds 2, 3, 4, 5 in the following literature respectively, and they are synthesized according to the literature ((Li Cai, Wanyi Guan, Motomitsu Kitaoka, Jie Shen, Chengfeng Xia, Wenlan Chena and Peng George Wang. A chemoenzymatic route to N-acetylglucosamine-1-phosphate analogues. Chem. Commun., 2009, 2944-2946)

[0070] As used herein, the term "NahK" refers to N-acetylhexosamine 1-kinase.

[0071] As used herein, the term "GlmU" refers to uridine transferase.

[0072] As used herein, the term "PPK" refers to polyphosphate kinase.

[0073] As used herein, the term "PmHS2" refers to heparin synthase.

[0074] As used herein, the term "PmHAs" refers to hyaluronic acid synthase.

[0075] As used herein, the term "KfoC" refers to chondroitin polymerase.

[0076] As used herein, the term "LgtA" refers to N-acetylglucosamine transferase.

[0077] As used herein, the term "ATP" refers to adenosine triphosphate.

[0078] As used herein, the term "UTP" refers to uridine triphosphate.

[0079] As used herein, the term "NHS-PEG n -NHS" refers to succinimidyl ester-polyethylene glycol n-succinimidyl ester.

[0080] Example 1

[0081] The immobilized multi-enzyme system of this example was prepared by the following method:

[0082] (1) Pour the mixed enzyme solution containing 2 mg / mL of N-acetylhexosamine 1-kinase and 2 mg / mL of uridine transferase into 2 volumes of 320 mM imidazole-2-carboxaldehyde solution and mix evenly; quickly add 160 mM zinc nitrate solution with the same volume as the enzyme solution, stir and react for 0.5 h at a rotation speed of 150 rpm, wash 3 times with deionized water, and resuspend the obtained solid NahK-GlmU@ZIF-90 in deionized water (50 mg / mL).

[0083] (2) Prepare a Tris-HCl solution of 3-amino-1,2,4-triazole with a concentration of 20 mM, and the Tris-HCl buffer is a Tris-HCl buffer with a pH of 7.5 and a concentration of 100 mM. Mix the 3-amino-1,2,4-triazole solution and the NahK-GlmU@ZIF-90 solution obtained above evenly according to a volume ratio of 1:1, heat and stir in a water bath at 40 °C for 1 h at a rotation speed of 150 rpm, wash 3 times with deionized water, and resuspend the obtained solid NahK-GlmU@ZIF-90-NH2 in deionized water (50 mg / mL).

[0084] (3) Resuspend the obtained NahK-GlmU@ZIF-90-NH2 in a solution of 20 mM NHS-PEG5-NHS and 2 mg / mL polyphosphate kinase at a volume ratio of 5:1:3, mix and stir at room temperature for 0.5 h, wash three times with deionized water, centrifuge at 6,000 rpm for 5 min, and freeze-dry the precipitate under vacuum to obtain the immobilized three-enzyme cascade catalytic material NahK-GlmU@ZIF-90@PPK.

[0085] (4) Take ATP (15 mM), UTP (15 mM), monosaccharide substrate GlcNAc (15 mM), MgCl2 (5 mM), and NahK-GlmU@ZIF-90@PPK (0.5 mg / mL) according to the final concentrations in the system. The solvent is 100 mM Tris-HCl buffer with a pH of 7.5. React at 37 °C for 48 h, and detect the glycosyl donor by high-performance liquid chromatography and mass spectrometry. High-performance liquid chromatography conditions: Inertsil ODS-3 chromatographic column (150 mm × 4.6 mm, 5 μm). Mobile phase A is a phosphate buffer composed of 20 mmol / L sodium dihydrogen phosphate and disodium hydrogen phosphate (pH adjusted to 6.2 with phosphoric acid), and mobile phase B is pure methanol. The sample tray temperature is 25 °C, the chromatographic temperature is 25 °C. The detection wavelength is 254 nm, and the injection volume is 20 μL. The mass spectrometry detects sugar nucleotides using the negative ion mode.

[0086] The cascade catalytic reaction involved is as follows:

[0087]

[0088] Use an Amerigo high-resolution zeta potential analyzer to measure the potential of different samples. As Figure 1 shown, the potential of ZIF-90 (synthesis method see step 3 of Comparative Example 3) is 18.5 mV, and the potential of NahK-GlmU@ZIF-90 is -2.7 mV, indicating that the enzyme was successfully encapsulated into the material. The potential of NahK-GlmU@ZIF-90-NH2 is 19.2 mV, indicating successful amination of NahK-GlmU@ZIF-90. The potential of NahK-GlmU@ZIF-90@PPK decreased to -6.8 mV, indicating that the third enzyme was successfully immobilized on the surface of ZIF-90. The morphology of the immobilized material is as Figure 2 shown, and the encapsulation and amination modification of the enzyme do not affect the crystal shape of ZIF-90.

[0089] In the immobilized triple-enzyme cascade system of N-acetylhexosamine 1-kinase, uridine transferase, and polyphosphate kinase, the reaction catalyzed by NahK uses the monosaccharide GlcNAc (Ⅰ) and ATP as substrates to generate GlcNAc-1-P (Ⅱ) and ADP. The reaction catalyzed by GlmU uses GlcNAc-1-P (Ⅱ) and UTP as substrates to generate the sugar nucleotide UDP-GlcNAc (Ⅲ) and UDP. The polyphosphate kinase catalyzes the regeneration of ADP into ATP. The mass spectrometry results are as Figure 3 shown: The theoretical molecular weight of UDP-GlcNAc is 607.08. In the negative ion mode, [M-H] - = 606.2, which is consistent with the theoretical value, indicating that the immobilized multi-enzyme cascade system we established successfully synthesized UDP-GlcNAc. The results of monitoring the generated UDP-GlcNAc using high-performance liquid chromatography showed that the conversion rate of UDP-GlcNAc was 92%; the nuclear magnetic resonance hydrogen spectrum is as Figure 4 shown: 1H NMR (400 MHz, D2O) δ 7.97 (d, J = 8.1 Hz, 1H), 5.98 (dd, J = 7.8, 6.4 Hz, 2H), 5.53 (dd, J = 7.3, 3.3 Hz, 1H), 4.40 - 4.36 (m, 2H), 4.31 - 4.28 (m, 1H), 4.25 (ddd, J = 11.7, 4.6, 2.6 Hz, 1H), 4.20 (ddd, J = 11.8, 5.6, 3.1 Hz, 1H), 4.00 (dt, J = 10.5, 3.0 Hz, 1H), 3.94 (ddd, J = 10.1, 4.4, 2.3 Hz, 1H), 3.90 - 3.86 (m, 1H), 3.84 - 3.80 (m, 2H), 3.56 (dd, J = 10.0, 9.3 Hz, 1H), 2.09 (s, 3H).

[0090] Example 2

[0091] This example is different from Example 1 in that the monosaccharide GlcNAc in step (4) is changed to GlcNTFA, and the cascade catalytic reaction involved is as follows:

[0092]

[0093] In the immobilized triple-enzyme cascade system of N-acetylhexosamine 1-kinase, uridine transferase, and polyphosphate kinase, the reaction catalyzed by NahK uses the monosaccharide GlcNTFA (Ⅰ) and ATP as substrates to generate GlcNTFA-1-P (Ⅱ) and ADP. The reaction catalyzed by GlmU uses GlcNTFA-1-P (Ⅱ) and UTP as substrates to synthesize the sugar nucleotide UDP-GlcNTFA (Ⅲ). The polyphosphate kinase catalyzes the regeneration of ADP into ATP. The mass spectrometry results are as Figure 5As shown: The theoretical molecular weight of UDP-GlcNTFA is 661.05. In the negative ion mode, [M-H] - = 660.02, which is consistent with the theoretical value, indicating that the established immobilized multi-enzyme cascade system successfully synthesized UDP-GlcNTFA. The results generated by high performance liquid chromatography showed that the conversion rate of UDP-GlcNTFA was 90%; the nuclear magnetic resonance hydrogen spectrum was as Figure 6 shown: 1H NMR (400 MHz, D2O) δ 7.97 (d, J = 8.1 Hz, 1H), 5.98 (dd, J = 8.8, 6.4 Hz, 2H), 5.62 (dd, J = 7.0, 3.3 Hz, 1H), 4.37 (p, J = 5.2 Hz, 2H), 4.29 (dd, J = 5.5, 2.9 Hz, 1H), 4.24 (ddd, J = 11.7, 4.5, 2.6 Hz, 1H), 4.19 (ddd, J = 11.8, 5.6, 3.0 Hz, 1H), 4.11 (dt, J = 10.6, 2.9 Hz, 1H), 4.00 - 3.92 (m, 2H), 3.89 (dd, J = 12.5, 2.3 Hz, 1H), 3.83 (dd, J = 12.5, 4.3 Hz, 1H), 3.59 (dd, J = 9.9, 9.4 Hz, 1H).

[0094] Comparative Example 1

[0095] This Comparative Example 1 was the same as the method of Example 2, except that step (4) was directly carried out, and free N-acetylglucosamine 1-position kinase, uridine transferase, and polyphosphate kinase were used (ensuring that the usage amounts of the three enzymes were the same as the enzyme amounts on the immobilized material). The results were as Figure 7 shown: The immobilized enzyme exhibited better catalytic activity. After reacting for 48 h, the conversion rate of UDP-GlcNTFA in the immobilized enzyme system of Example 2 could reach about 90%, while the conversion rate of UDP-GlcNTFA catalyzed by the free enzyme was only 55%.

[0096] Example 3

[0097] This example was the same as the method of Example 1, except that the polyphosphate kinase in step (3) was changed to heparin synthase, the substrate GlcA-pNP with a final concentration of 10 mM was added in step (4), and the reaction was carried out at 37 °C for 48 h. The cascade catalytic reaction involved was as follows:

[0098]

[0099] In the immobilized three-enzyme cascade system of N-acetylhexosamine 1-kinase, uridine transferase, and heparin synthase, the reaction catalyzed by NahK uses GlcNAc(I) and ATP as substrates to generate GlcNAc-1-P(II) and ADP. The reaction catalyzed by GlmU uses GlcNAc-1-P(II) and UTP as substrates to generate the sugar nucleotide UDP-GlcNAc(III) and UDP. Heparin synthase uses the generated UDP-GlcNAc(III) and GlcA-pNP as substrates to synthesize heparin disaccharide(IV). The mass spectrometry results are as Figure 8 shown: The theoretical molecular weight of heparin disaccharide is 518.14. In the negative ion mode, [M-H] - = 517.09, which is consistent with the theoretical value, indicating that the established immobilized multi-enzyme cascade system successfully synthesized heparin disaccharide. The heparin disaccharide generated by high performance liquid chromatography showed a conversion rate of 88%; the nuclear magnetic resonance hydrogen spectrum is as Figure 9 shown: 1H NMR(400MHz, D2O): δ(ppm)4.43(d, J = 7.9, 1H), 3.81-3.83(m, 2H), 3.74-3.78(m, 2H), 3.67-3.69(m, 1H), 3.66(d, J = 8.8, 1H), 3.60-3.53(m, 3H), 3.47-3.41(m, 3H), 3.28(s, 3H), 1.93(s, 1H).

[0100] Example 4

[0101] This example is the same as the method in Example 1, except that the polyphosphate kinase in step (3) is changed to hyaluronic acid synthase, and the substrate GlcA-pNP with a final concentration of 10 mM is added in step (4), and the reaction is carried out at 37 °C for 48 h. The cascade catalytic reaction involved is as follows:

[0102]

[0103] In the immobilized three-enzyme cascade system of N-acetylhexosamine 1-kinase, uridine transferase, and hyaluronic acid synthase, the reaction catalyzed by NahK uses GlcNAc(I) and ATP as substrates to generate GlcNAc-1-P(II) and ADP. The reaction catalyzed by GlmU uses GlcNAc-1-P(II) and UTP as substrates to generate the sugar nucleotide UDP-GlcNAc(III) and UDP. Hyaluronic acid synthase uses the generated UDP-GlcNAc(III) and GlcA-pNP as substrates to synthesize hyaluronic acid disaccharide(IV). The mass spectrometry results are as Figure 10 shown: The theoretical molecular weight of hyaluronic acid disaccharide is 518.14. In the negative ion mode, [M-H] -= 517.09 is consistent with the theoretical value, indicating that the established immobilized multi-enzyme cascade system successfully synthesized hyaluronic acid disaccharide. The hyaluronic acid disaccharide generated by high-performance liquid chromatography showed a conversion rate of 95% of hyaluronic acid disaccharide; the nuclear magnetic resonance hydrogen spectrum is as Figure 11 shown: 1 H NMR (500 MHz, D2O): δ (ppm) 4.85 (d, J = 3.5, 1H), 4.35 (d, J = 7.5, 1H), 3.82 (dd, J = 8.5, 10.5, 1H), 3.77 (dd, J = 2.0, 12.0, 1H), 3.73 - 3.70 (m, 1H), 3.66 (ddd, J = 2.0, 4.5, 9.5, 1H), 3.59 - 3.61 (m, 1H), 3.53 (t, J = 9.0, 1H), 3.39 - 3.33 (m, 2H), 3.28 (s, 3H), 3.20 - 3.17 (m, 2H).

[0104] Example 5

[0105] This example is the same as the method of Example 1, except that the polyphosphate kinase in step (3) is changed to chondroitin polymerase, the monosaccharide substrate in step (4) is changed from GlcNAc to GalNAc, and the substrate GlcA-pNP with a final concentration of 10 mM is added and reacted at 37 °C for 48 h. The cascade catalytic reaction involved is as follows:

[0106]

[0107] In the immobilized three-enzyme cascade system of N-acetylhexosamine 1-position kinase, uridine transferase and chondroitin synthase, the reaction catalyzed by NahK uses GalNAc (Ⅰ) and ATP as substrates to generate GalNAc-1-P (Ⅱ) and ADP. The reaction catalyzed by GlmU uses GalNAc-1-P (Ⅱ) and UTP as substrates to generate the sugar nucleotide UDP-GalNAc (Ⅲ) and UDP. Chondroitin synthase uses the generated UDP-GalNAc (Ⅲ) and GlcA-pNP as substrates to synthesize chondroitin disaccharide (Ⅳ). The mass spectrometry results are as Figure 12 shown: The theoretical molecular weight of chondroitin disaccharide is 518.14. In the negative ion mode, [M-H] - = 517.09 is consistent with the theoretical value, indicating that the established immobilized multi-enzyme cascade system successfully synthesized chondroitin disaccharide. The chondroitin disaccharide generated by high-performance liquid chromatography showed a conversion rate of 82% of chondroitin disaccharide; the nuclear magnetic resonance hydrogen spectrum is as Figure 13 shown: 11H NMR (400 MHz, D2O): δ (ppm) 4.85 (d, J = 3.2, 1H), 4.54 (d, J = 5.6, 1H), 4.05 (dd, J = 2.8, 8.0, 1H), 3.98 (dd, J = 6.3, 8.1, 1H), 3.93 (dd, J = 2.1, 9.8, 1H), 3.88 (dd, J = 3.5, 8.8, 1H), 3.82 (ddd, J = 1.7, 3.5, 7.7, 1H), 3.75 (d, J = 7.0, 1H), 3.71 (dd, J = 6.3, 7.0, 1H), 3.54 - 3.50 (m, 2H), 3.39 (s, 3H), 3.37 - 3.34 (m, 1H).

[0108] Example 6

[0109] This example is the same as the method of Example 1, except that the polyphosphate kinase in step (3) is changed to N - acetylglucosaminyltransferase, and a substrate lactose Lac with a final concentration of 10 mM is added in step (4). The cascade catalytic reaction involved is as follows:

[0110]

[0111] In the immobilized three - enzyme cascade system of N - acetylhexosamine 1 - kinase, uridyltransferase, and N - acetylglucosaminyltransferase, the reaction catalyzed by NahK uses monosaccharide GlcNAc (Ⅰ) and ATP as substrates to generate GlcNAc - 1 - P (Ⅱ) and ADP. The reaction catalyzed by GlmU uses GlcNAc - 1 - P (Ⅱ) and UTP as substrates to generate sugar nucleotide UDP - GlcNAc (Ⅲ). N - acetylglucosaminyltransferase uses UDP - GlcNAc (Ⅲ) and lactose as substrates to generate neo - N - lactotriose (Ⅳ). The mass spectrometry results are as Figure 14 shown: The theoretical molecular weight of neo - N - lactotriose is 545.59. In the negative ion mode, [M - H] - = 544.15, which is consistent with the theoretical value, indicating that the immobilized multi - enzyme cascade system we established successfully synthesized neo - N - lactotriose, and its conversion rate is 77%; the nuclear magnetic resonance hydrogen spectrum is as Figure 15 shown: 1 1H NMR (400 MHz, D2O): δ (ppm) 5.16 (d, J = 3.8 Hz, 0.38H), 4.63 (dd, J = 8.4, 1.9 Hz, 1H), 4.38 (d, J = 7.9 Hz, 0.63H), 3.90 - 3.63 (m, 16H), 3.56 (d, J = 33.3 Hz, 2H), 3.40 (d, J = 7.8 Hz, 2H), 3.22 (d, J = 1.2 Hz, 1H), 1.98 (d, J = 0.8 Hz, 3H).

[0112] Example 7

[0113] This example is the same as the method of Example 1, except that the polyphosphate kinase in step (3) is changed to hyaluronan synthase, and in step (4), the substrates GlcA-pNP and UDP-GlcA with a final concentration of 10 mM are added and reacted at 37 °C for 48 h. The cascade catalytic reaction involved is as follows:

[0114]

[0115] In the immobilized three-enzyme cascade system of N-acetylhexosamine 1-kinase, uridyltransferase, and hyaluronan synthase, the reaction catalyzed by NahK uses the monosaccharide GlcNAc (Ⅰ) and ATP as substrates to generate GlcNAc-1-P (Ⅱ) and ADP. The reaction catalyzed by GlmU uses GlcNAc-1-P (Ⅱ) and UTP as substrates to generate the sugar nucleotide UDP-GlcNAc (Ⅲ). The hyaluronan synthase PmHAs uses UDP-GlcNAc (Ⅲ) and GlcA-pNP as substrates to synthesize hyaluronic acid disaccharide (Ⅳ). Next, the hyaluronan synthase uses hyaluronic acid disaccharide, UDP-GlcNAc (Ⅲ), and UDP-GlcA as substrates to alternately occur through Step a and Step b to synthesize hyaluronic acid. In this example, whether a sugar chain is formed is judged based on whether the reaction solution is stained and whether there are bands on the gel. The polyacrylamide gel electrophoresis results are as Figure 16 shown, and a clear hyaluronic acid band was observed on the gel, indicating that the established immobilized multi-enzyme cascade system successfully synthesized hyaluronic acid.

[0116] Example 8

[0117] This example is the same as the method of Example 1, except that the polyphosphate kinase in step (3) is changed to chondroitin synthase, and in step (4), the substrates GlcA-pNP and UDP-GlcA with a final concentration of 10 mM are added and reacted at 37 °C for 48 h. The cascade catalytic reaction involved is shown in the following figure:

[0118]

[0119] In the immobilized three-enzyme cascade system of N-acetylhexosamine 1-kinase, uridine transferase, and chondroitin synthase, the reaction catalyzed by NahK uses monosaccharide GalNAc (Ⅰ) and ATP as substrates to generate GalNAc-1-P (Ⅱ) and ADP. The reaction catalyzed by GlmU uses GalNAc-1-P (Ⅱ) and UTP as substrates to generate sugar nucleotide UDP-GalNAc (Ⅲ). Chondroitin synthase KfoC synthesizes chondroitin disaccharide (Ⅳ) using the generated UDP-GalNAc (Ⅲ) and GlcA-pNP as substrates. Next, chondroitin synthase uses chondroitin disaccharide, UDP-GlcA, and UDP-GalNAc as substrates and alternates between Step c and Step d to synthesize chondroitin. In this example, whether a sugar chain is formed is judged based on whether the reaction solution is stained and whether there are bands on the gel. The results of polyacrylamide gel electrophoresis are as Figure 16 shown. A hyaluronic acid band was clearly observed on the gel, indicating that the established immobilized multi-enzyme cascade system successfully synthesized chondroitin.

[0120] Example 9

[0121] Test the enzyme activity of the immobilized three-enzyme cascade system in Example 4 of the present invention under different environments, and compare it with the enzyme activity of the non-immobilized three-enzyme mixture. The immobilized three-enzyme and free enzyme (the enzyme amount is the same as that of the immobilized enzyme) in Example 4 were respectively placed in solutions at different temperatures (40 °C, 50 °C, 60 °C, 70 °C, and 80 °C) and different pH values (pH 3, pH 4, pH 5, pH 6, pH 7) for 1 h. The immobilized enzyme and free enzyme obtained after treatment were reacted at pH 7.5 and 37 °C for 48 h to synthesize hyaluronic acid disaccharide. The disaccharide produced in each cycle was detected by high-performance liquid chromatography and the conversion rate of the disaccharide was calculated. The results are as Figure 17 shown. After treatment with high temperature or acid, the immobilized enzyme maintained higher activity than the free enzyme.

[0122] Example 10

[0123] Chondroitin disaccharide was prepared by the method provided in Example 5 of the present invention, and solid-liquid separation was carried out. The immobilized multi-enzyme was collected and recycled in the same way to prepare chondroitin disaccharide. The concentration of the disaccharide produced in each cycle was detected by high-performance liquid chromatography, and the conversion rate of the disaccharide was calculated (expressed as the relative disaccharide production concentration, and the concentration of chondroitin disaccharide produced in the first cycle reaction was set as 100%). The results are as Figure 18 shown. The reusability of the three-enzyme immobilized catalytic material was evaluated. When reused 9 times, its conversion rate could still reach more than 75%.

[0124] Example 11

[0125] This example is the same as the method in Example 1, except that the substrate concentration in step (4) is increased. ATP (400 mM), UTP (400 mM), monosaccharide substrate GlcNAc (400 mM), MgCl2 (15 mM), 100 mM Tris-HCl buffer at pH 7.5. The above immobilized triple-enzyme cascade catalytic material is 0.5 mg / mL of the immobilized multi-enzyme provided in each of the above examples, and the reaction is carried out at 37 °C for 48 h. Heparin disaccharide generated using high-performance liquid chromatography shows that the conversion rate of UDP-GlcNAc is 82%; in this example, due to the high-concentration substrate, the reaction solution becomes viscous, affecting mass transfer. Although the conversion rate decreases, compared with the low-concentration reaction system, more product is obtained within the same time.

[0126] Comparative Example 2

[0127] This Comparative Example 2 is the same as the method in Example 1, except that step (4) is directly carried out, and free N-acetylhexosamine 1-kinase, uridine transferase, and polyphosphate kinase are used (ensuring that the usage amounts of the three enzymes are the same as the enzyme amounts on the immobilized material). At the same time, the substrate concentration in step (4) is increased. ATP (400 mM), UTP (400 mM), monosaccharide substrate GlcNAc (400 mM), MgCl2 (15 mM), 100 mM Tris-HCl buffer at pH 7.5, and the reaction is carried out at 37 °C for 48 h. Heparin disaccharide generated using high-performance liquid chromatography shows that the conversion rate of UDP-GlcNAc is 32%; compared with Example 11, the results prove that the immobilized material has better catalytic activity in a high-concentration substrate reaction system.

[0128] Example 12

[0129] This example is different from Example 1 in that 3-amino-1,2,4-triazole in step (2) is changed to 2-aminobenzimidazole.

[0130] Example 13

[0131] This example is different from Example 2 in that NHS-PEG5-NHS in step (3) is changed to NHS-PEG 10 -NHS.

[0132] Example 14

[0133] Test the storage stability of the immobilized enzyme cascade system in Example 1 of the present invention, and compare it with the storage stability of the non-immobilized three-enzyme mixture. Store the immobilized three-enzyme cascade system and the free enzyme in Example 1 at 4°C respectively, and test the activities of the immobilized three-enzyme cascade system and the free enzyme on the 1st, 3rd, 5th, 7th, 9th, 12th, and 15th days respectively. Detect the glycosyl donor generated during each cycle by high performance liquid chromatography according to the method of Example 1 and calculate the conversion rate. The results are as Figure 19 shown that the immobilized enzyme still maintained more than 80% of its activity after 15 days of storage, while the activity of the free enzyme remained only about 10%.

[0134] Example 15

[0135] The method of Example 15 is the same as that of Example 1, except that: in step (1), the concentrations of the zinc nitrate solution and the imidazole-2-carboxaldehyde mother liquor are 80 mM and 160 mM respectively, and the final molar ratio of zinc nitrate to imidazole-2-carboxaldehyde in the system is 1:6. In step (2), the amino ligand is 2-amino-[1,1':4',1”-terphenyl]-4,4”-dicarboxylate, and the concentration is 80 mM. The amino ligand solution is prepared by dissolving the amino ligand in a Tris-HCl buffer solution with pH 7 and a concentration of 50 mM. The water bath heating in step (2) is a 50°C water bath heating and stirring for 0.5 h at a rotation speed of 100 rpm. In step (3), the concentration ratio of the first enzyme, the second enzyme, and the third enzyme is 1:1:4, and the concentration is 2 mg / mL.

[0136] Example 16

[0137] The method of Example 16 is the same as that of Example 1, except that: in step (1), the concentrations of the zinc nitrate solution and the imidazole-2-carboxaldehyde mother liquor are 120 mM and 240 mM respectively, and the final molar ratio of zinc nitrate to imidazole-2-carboxaldehyde in the system is 1:4. In step (2), the amino ligand is 3-amino-1,2,4-triazole, and the concentration is 50 mM. The amino ligand solution is prepared by dissolving the amino ligand in a Tris-HCl buffer solution with pH 7.5 and a concentration of 75 mM. The water bath heating in step (2) is a 45°C water bath heating and stirring for 1 h at a rotation speed of 150 rpm. In step (3), n = 5 in NHS-PEGn-NHS, and the concentration of NHS-PEG5-NHS is 50 mM. The concentration ratio of the first enzyme, the second enzyme, and the third enzyme is 1:1:1, and the concentration is 1 mg / mL.

[0138] Comparative Example 3

[0139] The immobilized enzyme in this example was prepared by the following method:

[0140] (1) Pour 2 mg / mL of N-acetylglucosamine 1-kinase (NahK) into 2 volumes of 320 mM imidazole-2-carboxaldehyde solution and mix well; quickly add an equal volume of 160 mM zinc nitrate solution to the enzyme solution, stir and react for 0.5 h at a rotation speed of 150 rpm, wash 3 times with deionized water, and centrifuge at 8,000 rpm for 5 min to obtain solid NahK@ZIF-90.

[0141] (2) Pour 2 mg / mL of uridine transferase (GlmU) into 2 volumes of 320 mM imidazole-2-carboxaldehyde solution and mix well; quickly add an equal volume of 160 mM zinc nitrate solution to the enzyme solution, stir and react for 0.5 h at a rotation speed of 150 rpm, wash 3 times with deionized water, and centrifuge at 8,000 rpm for 5 min to obtain solid GlmU@ZIF-90.

[0142] (3) Stir and react 320 mM imidazole-2-carboxaldehyde solution with 160 mM zinc nitrate solution for 0.5 h (4:1 / v:v) at a rotation speed of 150 rpm, wash 3 times with deionized water, and centrifuge at 8,000 rpm for 5 min to obtain solid ZIF-90, which is resuspended in deionized water (50 mg / mL). Prepare a Tris-HCl solution of 3-amino-1,2,4-triazole with a concentration of 20 mM. The Tris-HCl buffer is a Tris-HCl buffer with a pH of 7.5 and a concentration of 100 mM. Mix the 3-amino-1,2,4-triazole solution and the above-obtained ZIF-90 solution evenly at a volume ratio of 1:1, heat and stir in a water bath at 40 °C for 1 h at a rotation speed of 150 rpm, wash 3 times with deionized water, centrifuge at 8,000 rpm for 5 min, and freeze-dry under vacuum to obtain ZIF-90-NH2, which is resuspended in deionized water (50 mg / mL).

[0143] (4) Mix the above-obtained ZIF-90-NH2 suspension with 20 mM NHS-PEG5-NHS solution and 2 mg / mL of polyphosphate kinase at a volume ratio of 5:1:3, mix and stir at room temperature for 0.5 h, wash 3 times with ionized water, centrifuge at a rotation speed of 8,000 rpm for 5 min, and freeze-dry under vacuum to obtain ZIF-90@PPK.

[0144] (5) According to the final concentration of the system, take ATP (15 mM), UTP (15 mM), monosaccharide substrate GlcNAc (15 mM), and MgCl2 (5 mM) respectively. By adding a mixture of NahK@ZIF-90, GlmU@ZIF-90, and ZIF-90@PPK at 0.5 mg / mL (1:1:1 / w:w:w), with the solvent being 100 mM Tris-HCl buffer at pH 7.5, react at 37 °C for 48 h, and detect UDP-GlcNAc by high-performance liquid chromatography. The result shows that the conversion rate of UDP-GlcNAc is 36%; compared with Example 1, the catalytic efficiency of the three-enzyme co-immobilization system is 2.5 times higher than that of the catalytic system directly mixed in this comparative example.

[0145] Comparative Example 4

[0146] The immobilized enzyme in this example was prepared by the following method:

[0147] (1) Pour N-acetylhexosamine 1-kinase (NahK) containing 2 mg / mL into 2 volumes of 320 mM imidazole-2-carboxaldehyde solution and mix evenly; quickly add 160 mM zinc nitrate solution with the same volume as the enzyme solution and stir for 0.5 h at a rotation speed of 150 rpm. Wash with deionized water 3 times, centrifuge at 8,000 rpm for 5 min, and resuspend the obtained solid NahK@ZIF-90 in an aqueous solution (50 mg / mL).

[0148] (2) Pour uridine transferase (GlmU) containing 2 mg / mL into 2 volumes of 320 mM imidazole-2-carboxaldehyde solution and mix evenly; quickly add 160 mM zinc nitrate solution with the same volume as the enzyme solution and stir for 0.5 h at a rotation speed of 150 rpm. Wash with deionized water 3 times, and resuspend the obtained solid GlmU@ZIF-90 in an aqueous solution (50 mg / mL).

[0149] (3) Prepare a Tris-HCl solution of 3-amino-1,2,4-triazole with a concentration of 20 mM. The Tris-HCl buffer is 100 mM Tris-HCl buffer at pH 7.5. Mix the 3-amino-1,2,4-triazole solution with the above-obtained NahK@ZIF-90 solution and GlmU@ZIF-90 solution evenly according to a volume ratio of 1:1, heat and stir in a water bath at 40 °C for 1 h at a rotation speed of 150 rpm, wash the solid with water until the supernatant is clear, centrifuge and separate at 8,000 rpm, and resuspend the obtained NahK@ZIF-90-NH2 and GlmU@ZIF-90-NH2 in an aqueous solution (50 mg / mL) respectively.

[0150] (4) Mix the obtained NahK@ZIF-90-NH2 and GlmU@ZIF-90-NH2 with 20 mM NHS-PEG5-NHS solution and 1 mg / mL polyphosphate kinase at a volume ratio of 5:1:3, stir at room temperature for 0.5 h, wash with deionized water three times, centrifuge at 6000 rpm for 5 min, and freeze-dry the precipitate to obtain NahK@ZIF-90@PPK and GlmU@ZIF-90@PPK.

[0151] (5) Take ATP (15 mM), UTP (15 mM), monosaccharide substrate GlcNAc (15 mM), MgCl2 (5 mM), and a mixture of NahK@ZIF-90@PPK and GlmU@ZIF-90@PPK (ensuring that the amount of each enzyme is the same as in Example 1) according to the final concentration of the system. The solvent is 100 mM Tris-HCl buffer with a pH of 7.5, and react at 37 °C for 48 h. Detect UDP-GlcNAc by high-performance liquid chromatography. The result shows that the conversion rate of UDP-GlcNAc is 45%; compared with Example 1, the catalytic efficiency of the three-enzyme co-immobilization system is 2 times higher than that of the catalytic system in this Comparative Example 4.

[0152] Comparative Example 5

[0153] (1) Pour the mixed enzyme solution containing 2 mg / mL N-acetylhexosamine 1-kinase and 2 mg / mL uridyltransferase into 2 volumes of 320 mM imidazole-2-carboxaldehyde solution and mix evenly; quickly add an equal volume of 160 mM zinc nitrate solution and stir for 0.5 h at a rotation speed of 150 rpm, wash with deionized water three times, and freeze-dry to obtain NahK-GlmU@ZIF-90.

[0154] (2) Take ATP (15 mM), UTP (15 mM), monosaccharide substrate GlcNAc (15 mM), MgCl2 (5 mM) according to the final concentration of the system. By adding a mixture of NahK-GlmU@ZIF-90 and free PPK (ensuring that the amount of each enzyme is the same as in Example 1), the solvent is 100 mM Tris-HCl buffer with a pH of 7.5, and react at 37 °C for 48 h. Detect UDP-GlcNAc by high-performance liquid chromatography. The result shows that the conversion rate of UDP-GlcNAc is 51%; compared with Example 1, the catalytic efficiency of the three-enzyme co-immobilization system is 1.8 times higher than that of the catalytic system in this comparative example.

[0155] In summary, the immobilized enzyme system prepared by the present invention can tolerate high substrate concentrations (Example 11), which is very important for industrial applications. In general glycosidase reactions, the substrate concentration is generally 5-25 mM. High substrate concentrations will inhibit the enzyme activity. Currently, the high-concentration reaction of the existing technology can reach 200 mM, and the enzyme catalytic activity will decrease sharply at higher concentrations (Comparative Example 2). However, the immobilized catalytic system of the present invention can be applied to the substrate catalysis at 400 mM. At the same time, the immobilized enzyme system of the present invention can be stored for a long time, greatly reducing the enzyme separation and purification process and lowering the cost. The characteristics of kinases, glycosynthases, and glycosyltransferases are different. This immobilization strategy of the present invention can be used in different multi-enzyme cascade systems and can maintain their good activities.

Claims

1. An immobilized multi-enzyme material, characterized in that, It includes a first enzyme, a second enzyme and a third enzyme. The first enzyme and the second enzyme are immobilized inside ZIF-90, and the third enzyme is immobilized on the surface of ZIF-90; the first enzyme is N N-acetylhexosamine 1-kinase, the second enzyme is uridine transferase, and the third enzyme is any one of polyphosphate kinase, heparin synthase, hyaluronic acid synthase, chondroitin polymerase, N N-acetylglucosamine transferase.

2. The preparation method of the immobilized multi-enzyme material according to claim 1, characterized in that, It includes the following steps: (1) Pour the mixed enzyme solution containing the first enzyme and the second enzyme into the imidazole-2-carboxaldehyde solution and mix evenly; quickly add the zinc nitrate solution, stir and react, wash with deionized water, and centrifuge to obtain dual-enzyme@ZIF-90; (2) Uniformly mix the solution containing the amino ligand with dual-enzyme@ZIF-90, heat in a water bath, wash the solid with water until the supernatant is clear, and centrifuge to obtain dual-enzyme@ZIF-90-NH2; (3) React dual-enzyme@ZIF-90-NH2 with NHS-PEG5-NHS and the third enzyme, after solid-liquid separation, collect the solid product triple-enzyme@ZIF-90, which is the immobilized multi-enzyme material.

3. The preparation method according to claim 2, wherein In step (1), the concentrations of the zinc nitrate solution and the imidazole-2-carboxaldehyde mother liquor are 80~160 mM and 160~320 mM respectively, and the final molar ratio of zinc nitrate to imidazole-2-carboxaldehyde in the system is 1:2~1:

6.

4. The preparation method according to claim 2, characterized in that, In step (2), the amino ligand is any one of 3-amino-1,2,4-triazole, 2-aminobenzimidazole, 6-aminopurine, and 2-amino-[1,1':4',1''-terphenyl]-4,4''-dicarboxylate, and the concentration is 20~80 mM.

5. The preparation method according to claim 4, wherein, The amino ligand solution is prepared by dissolving the amino ligand in a Tris-HCl buffer solution with a pH of 7~8 and a concentration of 50~100 mM.

6. The preparation method according to claim 4, characterized in that, The water bath heating in step (2) is heating and stirring in a 40~50 °C water bath for 0.5~1 h, and the rotation speed is 100~200 rpm.

7. The preparation method according to claim 2, wherein In step (3), the concentration of NHS-PEG5-NHS is 20~80 mM.

8. The preparation method according to claim 2, wherein, The concentration ratio of the first enzyme, the second enzyme, and the third enzyme is 1:1:1~1:1:4, and the concentration is 0.5~2 mg / mL.

9. Use of the immobilized multi-enzyme material according to claim 1 in the synthesis of a glycosyl donor, heparin disaccharide, hyaluronic acid, chondroitin or neo-lactotriose, wherein the glycosyl donor is UDP-GlcNAc or UDP-GlcNTFA. N -lactotriose, wherein the glycosyl donor is UDP-GlcNAc or UDP-GlcNTFA.

10. The preparation method of the immobilized multi-enzyme material according to claim 2 is applied to the multi-enzyme immobilization for synthesizing glycosyl donors, heparin disaccharide, hyaluronic acid, chondroitin, and neo- N -lactotriose, and the glycosyl donor is UDP-GlcNAc or UDP-GlcNTFA.

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