Efficient enzymatic synthesis method of holothurian glycosaminoglycans
By employing a one-pot enzymatic synthesis strategy, using α1,3-fucosyltransferase and its mutants, along with FKP enzyme, a structurally homogeneous sea cucumber glycosaminoglycan was successfully prepared. This solved the synthesis problem in existing technologies, achieving efficient and simple synthesis of sea cucumber glycosaminoglycans. It exhibits significant anticoagulant activity and is suitable for development into a novel anticoagulant drug.
Patent Information
- Application Number
- CN202411132030.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-16
- Publication Date
- 2025-10-31
AI Technical Summary
Existing technologies make it difficult to efficiently synthesize high molecular weight sea cucumber glycosaminoglycans with uniform structures. Furthermore, chemical synthesis methods are cumbersome and have low yields. Sea cucumber glycosaminoglycans obtained by natural extraction methods have uneven structures, which affects the development of novel anticoagulant drugs.
A one-pot enzymatic synthesis strategy was adopted, using α1,3-fucosyltransferase and its mutants and FKP enzyme, with sulfated fucose and chondroitin sulfate as substrates, to prepare sea cucumber glycosaminoglycans with anticoagulant activity in the presence of ATP and GTP.
The structure of sea cucumber glycosaminoglycans was made uniform and the synthesis was efficient. The operation was simple and the yield was high. The synthesized product has significant endogenous anticoagulant activity and is suitable for development into a safe and efficient new anticoagulant drug.
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Figure CN120866448A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biochemistry technology, specifically relating to a highly efficient enzymatic synthesis method for sea cucumber glycosaminoglycans. Background Technology
[0002] Sea cucumber glycosaminoglycans are high-molecular-weight mucopolysaccharides composed of chondroitin sulfate as the main chain and sulfated fucose as the side chain. They possess significant anticoagulant, antioxidant, anti-inflammatory, and antitumor activities, with their anticoagulant activity being the most notable, surpassing that of clinical heparin-like drugs. Furthermore, sea cucumber glycosaminoglycans act on the endogenous anticoagulant pathway, exhibiting extremely low bleeding risk and other side effects. Therefore, they are promising candidates for developing novel, safe, and highly effective anticoagulant drugs. However, currently, sea cucumber glycosaminoglycans are mainly extracted from natural sea cucumbers, requiring multiple extraction and purification steps. Moreover, naturally obtained sea cucumber glycosaminoglycans are mixtures of various sulfated configurations, resulting in heterogeneous structures that cannot be obtained using traditional separation methods to achieve a single-structure sea cucumber glycosaminoglycan. Additionally, the structures and compositions of glycosaminoglycans from different sea cucumber sources vary considerably, posing challenges to the development of novel anticoagulant drugs. Artificial synthesis is an effective method for obtaining structurally homogeneous sea cucumber glycosaminoglycans. Currently, a chemical synthesis pathway for low molecular weight sea cucumber glycosaminoglycans (nonaglycones) has been established through 12 chemical reactions. However, this requires multiple steps of adding and removing protecting groups, making the process cumbersome and yielding only 3%. Moreover, there is currently no chemical synthesis method for high molecular weight sea cucumber glycosaminoglycans. Summary of the Invention
[0003] The purpose of this invention is to solve the problems in the prior art and provide an efficient enzymatic synthesis route for sea cucumber glycosaminoglycans. Using sulfated fucose and chondroitin sulfate as substrates, and utilizing α1,3-fucosyltransferase and its mutants and FKP enzyme, a one-pot synthesis strategy is adopted to efficiently prepare sea cucumber glycosaminoglycans with anticoagulant activity, providing important technical support for the development of novel anticoagulant drugs.
[0004] To achieve the above objectives, the technical solution adopted by this invention is as follows: A highly efficient enzymatic synthesis method for sea cucumber glycosaminoglycans, using sulfated fucose and chondroitin sulfate as substrates, in the presence of ATP and GTP, utilizing an α1,3-fucosyltransferase (α1,3FucT, GenBank: AAD07447.1) mutant and L-fucokinase / GDP-fucosylationase (FKP, GenBank: AAX45030.1), employs a one-pot multi-enzyme synthesis strategy to prepare sea cucumber glycosaminoglycans with anticoagulant activity; wherein, the molar ratio of chondroitin sulfate to sulfated fucose... The ratio is 1:100–1:1200; the molar ratio of chondroitin sulfate to the α1,3-fucosyltransferase mutant is 1:1–3:1; the molar ratio of sulfated fucose to FKP enzyme is 25:1–75:1; the molar ratio of sulfated fucose to ATP and GTP is 1:1–1:3; the α1,3-fucosyltransferase mutant deletes 52 amino acid residues from the C-terminus of the protease, and mutates serine at position 46 to phenylalanine, glycine at position 128 to asparagine, histidine at position 129 to glutamate, and tyrosine at position 132 to isoleucine.
[0005] Preferably, the chondroitin sulfate is chondroitin sulfate A.
[0006] Preferably, the concentration of the sulfated fucose is 1-5 mM.
[0007] Preferably, the reaction time is 12-96 hours.
[0008] Preferably, the reaction pH is 7.0–8.0.
[0009] Preferably, the reaction temperature is 25-35℃.
[0010] Preferably, 10-30 mM MgCl2 is added to the reaction.
[0011] Compared with the prior art, the present invention has the following advantages:
[0012] (1) The glycosaminoglycan synthesized by the enzymatic method of the present invention has a uniform structure and is completely identical to that of natural sea cucumber glycosaminoglycan;
[0013] (2) This invention requires only one reaction step, is simple to operate, efficient, and has a high yield;
[0014] (3) The sea cucumber glycosaminoglycan synthesized in this invention has strong endogenous anticoagulant activity and is expected to be developed into a safe and efficient new anticoagulant drug. Attached Figure Description
[0015] Figure 1This is a schematic diagram illustrating the principle of the efficient enzymatic synthesis method for sea cucumber glycosaminoglycans according to the present invention.
[0016] Figure 2 This is a high-performance liquid chromatography (HPLC) molecular weight determination chromatogram of sea cucumber glycosaminoglycans in Example 3 of the present invention;
[0017] Figure 3 The 1H NMR spectrum of sea cucumber glycosaminoglycans in Example 3 of this invention (…) 1 H-NMR spectrum;
[0018] Figure 4 The carbon NMR spectrum of sea cucumber glycosaminoglycans in Example 3 of this invention (…) 13 C-NMR spectrum;
[0019] Figure 5 The above is the nuclear magnetic resonance (NMR) Overhauser effect (NOE) spectrum of sea cucumber glycosaminoglycans in Example 3 of this invention;
[0020] Figure 6 This is a graph showing the endogenous anticoagulant APTT activity of sea cucumber glycosaminoglycans in Example 3 of the present invention. Detailed Implementation
[0021] To facilitate understanding of the present invention, a more detailed description is provided below with reference to the accompanying drawings and specific embodiments. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.
[0022] The α1,3-fucosyltransferase mutant used in this embodiment of the invention is a mutant obtained by deleting 52 amino acid residues from the C-terminus of the protease, and mutating serine at position 46 to phenylalanine, glycine at position 128 to asparagine, histidine at position 129 to glutamate, and tyrosine at position 132 to isoleucine (α1,3FucT-S46F / A128N / H129E / Y132I / ΔC52), see Yun Hee Choi et al. Solubilization and terative Saturation Mutagenesis of α1,3-Fucosyltransferase From Helicobacter pylori to Enhance ltsCatalytic Efficiency[J]. Biotechnology and Bioengineering, vol.9999, No.xxx, 2016.
[0023] Example 1: The enzymatic synthesis reaction of sea cucumber glycosaminoglycans provided in this example is based on the following principle: Figure 1 As shown, the specific steps are as follows:
[0024] 1 mM 4-O-sulfated fucose, 1 mM ATP, 1 mM GTP, 10 μM chondroitin sulfate A (molecular weight 1 kDa), and 10 mM MgCl2 were added to 10 mM, pH 7.0 Tris-HCl. 3.3 mM α1,3-fucosyltransferase mutant and 14 μM M KP enzyme were added, and the mixture was reacted at 25 °C with shaking for 12 h. After the reaction, the enzymes were inactivated by boiling, and the supernatant was collected by centrifugation. The supernatant was dialyzed in a 500 Da dialysis bag, and the dialysate was concentrated and lyophilized to obtain the product, sea cucumber glycosaminoglycan.
[0025] Example 2 The enzymatic synthesis reaction of sea cucumber glycosaminoglycans provided in this example is based on the following principle: Figure 1 As shown, the specific steps are as follows:
[0026] 2.5 mM 4-O-sulfated fucose, 5 mM ATP, 5 mM GTP, 3 μM chondroitin A sulfate (molecular weight 30 kDa), and 20 mM MgCl2 were added to 10 mM Tris-HCl at pH 7.5. 1.5 μM α1,3-fucosyltransferase mutant and 50 μM M KP enzyme were then added, and the mixture was reacted at 30 °C with shaking for 48 h. After the reaction, the enzymes were inactivated by boiling, and the supernatant was collected by centrifugation. The supernatant was dialyzed in an 8000-14000 Da dialysis bag, and the dialysate was concentrated and lyophilized to obtain the product, sea cucumber glycosaminoglycan.
[0027] Example 3 The enzymatic synthesis reaction of sea cucumber glycosaminoglycans provided in this example is based on the following principle: Figure 1 As shown, the specific steps are as follows:
[0028] 5 mM 4-O-sulfated fucose, 15 mM ATP, 15 mM GTP, 4.2 μM chondroitin sulfate A (molecular weight 35 kDa), and 20 mM MgCl2 were added to 10 mM, pH 8.0 Tris-HCl. 4.2 μM α1,3-fucosyltransferase mutant and 0.2 mM FKP enzyme were added, and the mixture was reacted at 35 °C with shaking for 96 h. After the reaction, the enzymes were inactivated by boiling, and the supernatant was collected by centrifugation. The supernatant was dialyzed in an 8000-14000 Da dialysis bag. The dialysate was concentrated and lyophilized to obtain the product, sea cucumber glycosaminoglycan F. 4S CS A The yield was 63.7%. The synthesized product was subjected to high-performance liquid chromatography (HPLC) for molecular weight determination and nuclear magnetic resonance (NMR) for hydrogen spectroscopy. 1 H-NMR), carbon nuclear magnetic resonance (NMR) 13 The structure was identified and analyzed using C-NMR and Overhauser effect (NOE) nuclear magnetic resonance spectra, and the results are as follows: Figure 2 , 3 4, 5. This indicates that the sea cucumber glycosaminoglycan synthesized by the method of the present invention is completely identical to the natural sea cucumber glycosaminoglycan.
[0029] Synthetic sea cucumber glycosaminoglycan F 4S CS A The specific method for endogenous anticoagulant activity (APTT) testing is as follows: Take 10 μL of sample solutions of different concentrations, using low molecular weight heparin (LMWH) as a positive control, and chondroitin sulfate A (CS) as a positive control. A PBS isotonic buffer and other buffers were added as negative controls to a test tube preheated to 37°C. 30 μL of sheep plasma and 30 μL of APTT reagent were added, and the tube was incubated at 37°C for 5 min. Then, 30 μL of preheated 0.025 M CaCl2 solution (at 37°C) was quickly added, and the timing and clotting time were recorded simultaneously. The results are as follows: Figure 6 As shown, the sea cucumber glycosaminoglycan F synthesized by the method of the present invention 4S CS A It exhibits excellent endogenous anticoagulant activity, providing important technical support for the development of novel anticoagulant drugs.
Claims
1. A highly efficient enzymatic method for synthesizing sea cucumber glycosaminoglycans, characterized in that: Using sulfated fucose and chondroitin sulfate as substrates, and in the presence of ATP and GTP, a one-pot multi-enzyme synthesis strategy was employed to prepare sea cucumber glycosaminoglycans with anticoagulant activity, utilizing an α1,3-fucosyltransferase mutant and L-fucokinase / GDP-fucosylationase. The molar ratio of chondroitin sulfate to sulfated fucose was 1:(100–1200); the molar ratio of chondroitin sulfate to the α1,3-fucosyltransferase mutant was (1–3):1; and the sulfated fucose... The molar ratio of acidified fucose to L-fucokinase / GDP-fucopyrophosphorylase is (25-75):1; the molar ratio of sulfated fucose to ATP and GTP is 1:(1–3); the α1,3-fucosyltransferase mutant deletes 52 amino acid residues at the C-terminus of the protease, and mutates serine at position 46 to phenylalanine, glycine at position 128 to asparagine, histidine at position 129 to glutamate, and tyrosine at position 132 to isoleucine.
2. The efficient enzymatic synthesis method for sea cucumber glycosaminoglycans according to claim 1, characterized in that: The chondroitin sulfate is chondroitin sulfate A.
3. The efficient enzymatic synthesis method for sea cucumber glycosaminoglycans according to claim 1, characterized in that: The concentration of sulfated fucose is 1-5 mM.
4. The efficient enzymatic synthesis method for sea cucumber glycosaminoglycans according to claim 1, characterized in that: The reaction time is 12-96 hours.
5. The efficient enzymatic synthesis method for sea cucumber glycosaminoglycans according to claim 1, characterized in that: The reaction pH is 7.0–8.
0.
6. The efficient enzymatic synthesis method for sea cucumber glycosaminoglycans according to claim 1, characterized in that: The reaction temperature is 25-35℃.
7. The efficient enzymatic synthesis method for sea cucumber glycosaminoglycans according to claim 1, characterized in that: Add 10-30 mM MgCl2 to the reaction.