Preparation method of chondroitin

Abstract

The invention belongs to the field of biological medicine, particularly relates to a preparation method of chondroitin with controllable molecular weight, and particularly discloses chondroitin which takes an initiator as an initial polymerization substrate, n comonomers are sequentially bonded to the non-reducing tail end of the initiator, the comonomer is formed by acetylated hexosamine and uronic acid through a glucosidic bond; the comonomer and an initiator are polymerized under the catalysis of polymerase to form polysaccharide with a certain molecular weight, namely the chondroitin disclosed by the invention. The chondroitin preparation technology disclosed by the invention can be used for preparing chondroitin with linear change of molecular weight, wide controllable molecular weight range and narrow molecular weight distribution, and the molecular weight range can be 1-5 million; the chondroitin prepared by the technology has the advantages of convenience in operation, high efficiency, reliability and the like, and the chondroitin prepared by the technology can be used for preparing the chondroitin. The chondroitin can be used for preparing the chondroitin from the chondroitin prepared by the technology by the aid of the preparation technology of the chondroitin, and the chondroitin can be used for preparing the chondroitin with the preparation technology.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and particularly relates to a method for preparing chondroitin with controllable molecular weight. Background Art

[0002] Glycosaminoglycans are linear acidic polysaccharides, also known as mucopolysaccharides, primarily found in the connective tissues of higher animals. They perform a variety of functions in living organisms, including structural support, lubrication and protection, transport, tissue repair and regeneration, and signal transduction and regulation. Glycosaminoglycans can be classified into different types, including hyaluronic acid, heparin and sulfated heparin, chondroitin and sulfated chondroitin, sulfated dermatan, and sulfated keratin, based on structural characteristics such as monosaccharide composition, sulfation modification sites, and degree of modification. They are primarily composed of repeating disaccharide units, including hexosamine (N-acetyl-D-glucosamine or N-acetyl-D-galactosamine) and uronic acid (D-glucuronic acid or L-iduronic acid). Chondroitin, primarily composed of repeating disaccharide units of N-acetyl-D-galactosamine and D-glucuronic acid, is primarily found in cartilage tissue and plays an important role in maintaining normal joint function, primarily repairing, protecting, and lubricating articular cartilage. It has broad application prospects in biotechnology and medicine. However, like other natural products, chondroitin is difficult to obtain, and it is impossible to obtain a clear and single chondroitin structure. This greatly limits the application scenarios of chondroitin and people's thorough research on the biological functions of chondroitin, and it is impossible to apply chondroitin more comprehensively in the field of life.

[0003] In recent years, researchers have developed a variety of chondroitin polysaccharide preparation technologies. Based on the principles of these technologies, they can be divided into three categories: natural chondroitin extraction, microbial fermentation preparation, and enzyme-catalyzed synthesis.

[0004] Specifically, natural extraction is mainly through using natural materials containing chondroitin as extraction starting materials. Common sources mainly include marine organisms (such as shrimp, crab, fish), cartilage tissue (such as bovine trachea, bovine cartilage), etc., and then through steps such as crushing, solvent extraction, filtration, concentration and purification, finally obtaining chondroitin. This process is very time-consuming, and the process conditions involved need to be adjusted according to different starting materials. Moreover, due to seasonal changes, environmental factors, raw material sources and the inherent differences between animal species, the differences of natural chondroitin are increased, and a series of chondroitins with different sulfated modification degrees are obtained. This will result in batch differences, and thus the results cannot be reproduced well, and the problem of unspecific function will occur, and further structure-activity relationship research is limited. Therefore, it is very important to prepare homogeneous chondroitin with higher purity.

[0005] With the development of microbial fermentation engineering, in recent years, researchers have developed a method for preparing chondroitin by microbial fermentation, which has largely solved the problem of uncertain natural extraction sources and cumbersome process conditions. This process mainly uses microorganisms for production. First, it is necessary to develop a more suitable strain, including fungi, bacteria or yeast, etc., and it is necessary to re-edit their genomes and introduce genes that efficiently express chondroitin synthase to give the host strain the ability to prepare chondroitin. Secondly, the culture medium is placed in a fermentation vessel together with the strain, and the growth of the microorganism is achieved by controlling the fermentation conditions (temperature, pH value, oxygen concentration) etc. As the microorganism grows, a steady stream of chondroitin will gradually be secreted into the culture medium. After fermentation is completed, the chondroitin in the culture medium can be collected. Finally, chondroitin can be obtained through a series of purification methods. However, there are many problems that need to be solved in order to obtain medical grade or even research grade chondroitin, such as heat source, purity and microbial residue. With regard to the structure of the chondroitin prepared, the molecular weight dispersity of the chondroitin prepared by this method is difficult to be accurately controlled, resulting in a wider molecular weight distribution of the chondroitin finally prepared, which limits its application in some research fields. Patent CN112708571B discloses a recombinant yeast for fermentation production of controllable molecular weight chondroitin sulfate and its application. Utilizing synthetic biology technology and genetic engineering means, with Pichia pastoris GS115 as the starting strain, heterologous expression of chondroitin sulfate synthesis pathway-related proteins in the cell: KfoC and KfoA from Escherichia coli K4, chondroitin sulfotransferase C4ST or C6ST from mice, UDP-glucose dehydrogenase TuaD from Bacillus subtilis, and chondroitin sulfate lyase ABCI from Proteus vulgaris is achieved, thereby obtaining a production strain for synthesizing chondroitin sulfate A (CSA) and chondroitin sulfate C (CSC). Chondroitin sulfates A and C of specific molecular weight can be obtained by controlling the concentration of the inducer methanol and the time of induction. This has achieved the first time that chondroitin sulfates of specific configuration with controllable molecular weight are synthesized using microbial fermentation carbon sources. CN106755205A and CN111621533A disclose the successful synthesis of chondroitin sulfate with a specific configuration using a microbial enzyme method. However, this production method requires the purification of a large amount of enzyme, has complicated steps, and cannot achieve the purpose of regulating the molecular weight.

[0006] Enzymatic synthesis of chondroitin utilizes specific enzyme-catalyzed reactions to synthesize chondroitin. Currently, researchers have developed two chondroitin synthases: P. multocida chondroitin synthetase (PmCS) and E. coli K4 strain chondroitin synthetase (KfoC). These enzymes primarily produce chondroitin by simultaneously transferring UDP-GalNAc and UDP-GlcA to an initial oligosaccharide substrate, alternating and repeating the process. Unlike previously mentioned natural extraction and microbial fermentation methods, enzymatic production of chondroitin significantly simplifies the production process. Simply preparing sufficient chondroitin synthase and controlling catalytic conditions such as temperature, additives, initiators, and comonomer concentrations allows for the synthesis of chondroitin in an in vitro solution system. This significantly overcomes the time-consuming and source-dependent nature of chondroitin extraction, as well as the purity issues associated with microbial fermentation. Patent CN106755205A discloses an enzymatic method for preparing chondroitin sulfate. Chondroitin is used as a substrate, and chondroitin 4-sulfotransferase heterologously expressed in microbial cells catalyzes the formation of biologically active chondroitin sulfate A or chondroitin 6-sulfotransferase to form biologically active chondroitin sulfate C. For the first time, microbial cells are used to express animal-derived C4ST and C6ST to obtain biologically active enzymes. Chondroitin and ASST are integrated to obtain biologically active chondroitin sulfates CSA and CSC, with a conversion rate of 10-30%.

[0007] In summary, natural extraction, microbial fermentation, and enzymatic synthesis methods each have their own advantages, but none can achieve the controlled preparation of monodisperse chondroitin with a controlled molecular weight. This is fundamentally due to the fact that sugar synthesis in organisms is not like protein synthesis, which can be directly controlled by genes. Instead, it requires genetically controlled expression of enzymes involved in sugar synthesis, which in turn controls sugar synthesis. This process results in uncontrollable sugar sequence, which directly leads to a broad molecular weight distribution. Furthermore, chondroitin molecular weight significantly influences its properties and functions, and differences in molecular weight can even lead to completely opposite biological functions. For example, high-molecular-weight chondroitin is used clinically to treat inflammation, while low-molecular-weight chondroitin exhibits pro-inflammatory properties. High-molecular-weight chondroitin generally has low solubility, making it difficult to be absorbed by the digestive system and may also cause activation of coagulation factor XII and platelet aggregation. However, the degradation of high-molecular-weight chondroitin to produce low-molecular-weight chondroitin significantly overcomes these issues and is widely used in pharmaceuticals and dietary supplements, particularly for the prevention of osteoarthritis. Furthermore, high-molecular-weight chondroitin generally exhibits improved adhesion, viscosity, and lubricity, making it suitable for joint protection and lubrication. It also allows for better interactions with other biomolecules, such as cell receptor binding and signal transduction. However, this high-molecular-weight chondroitin itself presents a significant synthetic challenge.

[0008] To address the above issues, it is necessary to develop a chondroitin with a broadly distributed and monodispersed molecular weight to meet different functional requirements. Summary of the Invention

[0009] Based on the current demand for chondroitin with different molecular weights, this application uses an initiator as a substrate, and forms a reaction package through the initiator, a matching catalytic enzyme, and a comonomer. After the reaction of this reaction package, the molecular weight of the chondroitin obtained changes linearly, the controllable molecular weight range is wide, and the molecular weight distribution is narrow. Based on this, the present invention is completed;

[0010] In a first aspect, the present invention provides a chondroitin, wherein the chondroitin uses an initiator as an initial polymerization substrate, and n comonomers are sequentially bonded to the non-reducing end of the initiator, wherein the comonomers are formed by acetylated hexosamine and uronic acid through glycosidic bonds; the comonomers and the initiator are polymerized under the catalysis of a polymerase to form a polysaccharide of a certain molecular weight, namely the chondroitin of the present invention.

[0011] Furthermore, the range of n includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000.

[0012] Furthermore, the initiator is selected from one or more of the following: GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (trisaccharide), GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (tetrasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (pentasaccharide) , GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (hexasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalN Acβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (heptasaccharide), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3 Galβ1-4GlcNAcαorβProN3 (octasaccharide), GalNAcβ1-4Galβ1-4GlcNAcαorβProN3 (GN-1), GalNAcβ1-4Galβ1-3Galβ1-4GlcN AcαorβProN3(GN-2), GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc(CH4) and GalNAcβ1-4GlcAβ1-3GalNAcαorβProN3(CH3).

[0013] Furthermore, the acetylated hexosamine is selected from N-acetylgalactosamine, UDP-GalNAc (uridine-5'-diphosphate-N-acetyl-galactosamine sodium salt), UDP-GalNAz (uridine-5'-diphosphate-N-azidoacetylgalactosamine), UDP-GalNTFA (uridine-5'-diphosphate-N-trifluoroacetylgalactosamine), UDP-GalN (uridine-5'-diphosphate-galactosamine), UDP-GalNAalk (uridine-5'-diphosphate-N-alkynylacetylgalactosamine), One or more of UDP-glucosamine, UDP-GlcNAc (uridine-5'-diphosphate-N-acetyl-glucosamine sodium salt), UDP-GlcNAz (uridine-5'-diphosphate-N-azidoacetylglucosamine), UDP-GlcNTFA (uridine-5'-diphosphate-N-trifluoroacetylglucosamine), UDP-GlcN (uridine-5'-diphosphate-glucosamine) and UDP-GlcNAalk (uridine-5'-diphosphate-N-alkynyl acetylglucosamine).

[0014] Furthermore, the uronic acid can be selected from glucuronic acid and / or UDP-GlcA (uridine-5'-diphosphoglucuronic acid trisodium salt).

[0015] Furthermore, the molecular weight range of the chondroitin includes 1-5 million; 100,000-4 million, 250,000-3 million, 350,000-2 million, 500,000-1 million, 200,000-300,000, 1,000-70,000, 70,000-300,000, 350,000-660,000 and 380,000-900,000.

[0016] Furthermore, the polymerase is selected from one or more of Pasteurella multicida heparosan synthase 2 (PmHS2), Escherichia coli K4 from Chondroitin synthase (KfoC), Pasteurella multicida HA synthase (PmHAS) and Pasteurella multocida from Chondroitin synthase (PmCS).

[0017] In a second aspect, the present invention provides a method for synthesizing chondroitin; the method comprises the following steps:

[0018] S1. Acetylated hexosamine and uronic acid bonded nucleotides form comonomers;

[0019] S2. The comonomer and the initiator generate polysaccharide, namely the chondroitin of the present invention, through polymerization reaction catalyzed by polymerase.

[0020] Furthermore, the initiator is selected from one or more of the following: GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (trisaccharide), GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (tetrasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (pentasaccharide) , GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (hexasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalN Acβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (heptasaccharide), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3 Galβ1-4GlcNAcαorβProN3 (octasaccharide), GalNAcβ1-4Galβ1-4GlcNAcαorβProN3 (GN-1), GalNAcβ1-4Galβ1-3Galβ1-4GlcN AcαorβProN3(GN-2), GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc(CH4) and GalNAcβ1-4GlcAβ1-3GalNAcαorβProN3(CH3).

[0021] Furthermore, the feeding ratio of the comonomer and the initiator includes 25-5000:1; 50-4000:1; 100-3000:1; 200-2000:1; 25-400:1; 50-1600:1 and 1000-7000:1.

[0022] In one embodiment, the comonomer of the present invention is composed of acetylated hexosamine and uronic acid linked by a β-1,4 glycosidic bond.

[0023] In another embodiment, the comonomers of the present invention are linked by uronic acid and acetylated hexosamine via a β-1,3 glycosidic bond.

[0024] Furthermore, the acetylated hexosamine is selected from N-acetylgalactosamine, UDP-GalNAc (uridine-5'-diphosphate-N-acetyl-galactosamine sodium salt), UDP-GalNAz (uridine-5'-diphosphate-N-azidoacetylgalactosamine), UDP-GalNTFA (uridine-5'-diphosphate-N-trifluoroacetylgalactosamine), UDP-GalN (uridine-5'-diphosphate-galactosamine), UDP-GalNAalk (uridine-5'-diphosphate-N-alkynylacetylgalactosamine), lactosamine), N-acetylglucosamine, UDP-GlcNAc (uridine-5'-diphosphate-N-acetyl-glucosamine sodium salt), UDP-GlcNAz (uridine-5'-diphosphate-N-azidoacetylglucosamine), UDP-GlcNTFA (uridine-5'-diphosphate-N-trifluoroacetylglucosamine), UDP-GlcN (uridine-5'-diphosphate-glucosamine) and UDP-GlcNAalk (uridine-5'-diphosphate-N-alkynyl acetylglucosamine).

[0025] Furthermore, the uronic acid can be selected from glucuronic acid and / or UDP-GlcA (uridine-5'-diphosphoglucuronic acid trisodium salt).

[0026] Furthermore, the polymerase is selected from one or more of Pasteurella multicida heparosan synthase 2 (PmHS2), Escherichia coli K4 from Chondroitin synthase (KfoC), Pasteurella multicida HA synthase (PmHAS) and Pasteurella multocida or chondroitin synthase (PmCS).

[0027] In a third aspect, the present invention provides an initiator for synthesizing chondroitin, wherein the binding energy of the initiator is ±30 kcal·mol -1, the initiator is selected from one or more of the following: GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (trisaccharide), GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (tetrasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (pentasaccharide) , GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (hexasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalN Acβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (heptasaccharide), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3 Galβ1-4GlcNAcαorβProN3 (octasaccharide), GalNAcβ1-4Galβ1-4GlcNAcαorβProN3 (GN-1), GalNAcβ1-4Galβ1-3Galβ1-4GlcN AcαorβProN3(GN-2), GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc(CH4) and GalNAcβ1-4GlcAβ1-3GalNAcαorβProN3(CH3).

[0028] In a fourth aspect, the present invention provides a method for preparing an initiator, the method comprising the following steps:

[0029] S01. Synthesizing intermediate M1 by reacting a monosaccharide with a compound containing a halogen functional group;

[0030] S02. Adding an azide compound to the synthesized intermediate M1 to generate intermediate M2;

[0031] S03. Using M2 as a substrate, a glycosyltransferase, a monosaccharide or its derivative is added to synthesize a disaccharide initiator;

[0032] S04. Using disaccharide as substrate, glycosyltransferase and uronic acid are added to synthesize trisaccharide initiator;

[0033] S05. Using trisaccharide as substrate, glycosyltransferase and acetylated-hexosamine were added to synthesize tetrasaccharide initiator;

[0034] S06. Using tetrasaccharide as substrate, glycosyltransferase and uronic acid were added to synthesize pentasaccharide initiator;

[0035] S07. Using pentasaccharide as substrate, glycosyltransferase and acetylated hexosamine were added to synthesize hexasaccharide initiator;

[0036] S08. Using hexasaccharide as substrate, glycosyltransferase and uronic acid were added to synthesize heptasaccharide initiator;

[0037] S09. Using heptasaccharide as substrate, add glycosyltransferase and acetylated hexosamine to synthesize octasaccharide initiator.

[0038] Furthermore, the monosaccharide or its derivative is selected from D-(+)-glucose, D-(+)-galactose, D-glucuronic acid, D-galacturonic acid, D-glucosamine, D-(+)-galactosamine, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine and L-(-)-fucose, D-(+)-mannose, UDP-galactose (uridine-5'-diphosphate-galactose), UDP-GalNAc (uridine-5'-diphosphate-N-acetyl-galactosamine sodium salt), UDP-GalNAz (uridine-5'-diphosphate-N-azidoacetylgalactosamine), UDP-GalNTFA (uridine-5'-diphosphate-N-trifluoroacetylgalactosamine) , UDP-GalN (uridine-5'-diphosphate-galactosamine), UDP-GalNAalk (uridine-5'-diphosphate-N-alkynyl acetylgalactosamine), N-acetylglucosamine, UDP-GlcNAc (uridine-5'-diphosphate-N-acetyl-glucosamine sodium salt), UDP-GlcNAz (uridine-5'-diphosphate-N-azidoacetylglucosamine), UDP-GlcNTFA (uridine-5'-diphosphate-N-trifluoroacetylglucosamine), UDP-GlcN (uridine-5'-diphosphate-glucosamine) and UDP-GlcNAalk (uridine-5'-diphosphate-N-alkynyl acetyl glucosamine) One or more.

[0039] Furthermore, the compounds of the halogen functional group include but are not limited to 3-chloro-1-propanol, 3-bromo-1-propanol, 2-bromoethanol, 2-chloroethanol, 1-chloro-2-propanol, 1-chloro-2-methyl-2-propanol, (S)-(+)-2-chloro-1-propanol, 2-[2-(2-chloroethoxy)ethoxy]ethanol or 2-(2-chloroethoxy)ethanol.

[0040] Furthermore, the azide compound includes but is not limited to sodium azide, potassium azide, lithium azide, lead azide, 3-azido-1-propanol, 2-azidoethanol, 2-azido-1-amine hydrobromide or 3-azido-1-propylamine.

[0041] Furthermore, the acetylated hexosamine is selected from N-acetylgalactosamine, UDP-GalNAc (uridine-5'-diphosphate-N-acetyl-galactosamine sodium salt), UDP-GalNAz (uridine-5'-diphosphate-N-azidoacetylgalactosamine), UDP-GalNTFA (uridine-5'-diphosphate-N-trifluoroacetylgalactosamine), UDP-GalN (uridine-5'-diphosphate-galactosamine), UDP-GalNAalk (uridine-5'-diphosphate-N-alkynylacetylgalactosamine), One or more of UDP-GlcNAc (uridine-5'-diphosphate-N-acetyl-glucosamine sodium salt), UDP-GlcNAz (uridine-5'-diphosphate-N-azidoacetylglucosamine), UDP-GlcNTFA (uridine-5'-diphosphate-N-trifluoroacetylglucosamine), UDP-GlcN (uridine-5'-diphosphate-glucosamine) and UDP-GlcNAalk (uridine-5'-diphosphate-N-alkynyl acetylglucosamine).

[0042] Furthermore, the uronic acid can be selected from glucuronic acid and / or UDP-GlcA (uridine-5'-diphosphoglucuronic acid trisodium salt).

[0043] In one embodiment, the intermediate M1 synthesized by the reaction of the monosaccharide and the compound containing a halogen functional group is GlcNAcProαorβBr.

[0044] In one embodiment, the intermediate M2 generated by adding an azide compound to the synthesized intermediate M1 is GlcNAcαorβProN3.

[0045] Furthermore, the glycosyltransferase is selected from: NmLgtB enzyme, NmLgtA enzyme, human α-1,3-N-galactosyltransferase (GTB), bovine α-1,3-galactosyltransferase (Bovine α-1,3-Galactosyltransferase), mouse α-1,3-galactosyltransferase (Murine α-1,3-galactosyltransferase), Neisseria meningitidis α-1,4-galactosyltransferase (Neisseriameningitidis α-1,4-galactosyltransferase, NmLgtC), Helicobacter pylori β-1,4-galactosyltransferase (Helicobacter pylori β-1,4-galactosyltransferase, pyloriβ-1,4-galactosyltransferase), humanβ-1,4-galactosyltransferase7, bovineβ-1,4-galactosyltransferase, Escherichia coliO55:H7β-1,3-galactosyltransferase, Campylobacterjejuniβ-1,3-galactosyltransferase, Chromobacterium violaceumβ-1,3-galactosyltransferase, GlcAT-P enzyme, Pasteurella multocida heparosan synthase 2, PmHS2), Pasteurella multocida Type F Chondroitin synthase (PmCS), Pasteurella multocida HA synthase (PmHAS), human α-1,3-N-acetylgalactosaminyl transferase, Family 6 glycosyltransferase 1 of Bacteroides,One or more of the following: BoGT6a), Helicobacter mustelae α-1,3-N-acetylgalactosaminyl transferase (BgtA), Campylobacter jejuni β-1,4-acetylgalactosaminyl transferase (CgtA), and Bovine β-1,4-acetylgalactosaminyl transferase (Bovine β-1,4-Nacetylgalactosaminyl transferase).

[0046] In one embodiment of the present invention, when the substrate is a disaccharide, the glycosyltransferases are AtGlcAK, AtUSP, PmPPA and GlcAT-P to obtain a trisaccharide initiator.

[0047] In another embodiment of the present invention, when the substrate is a trisaccharide, the glycosyltransferase or polymerase is one or more of Pasteurella multocida heparosan synthase 2 (PmHS2), Pasteurella multocida HA synthase (PmHAS), Pasteurella multocida or chondroitin synthase (PmCS) and Escherichia coli K4 from Chondroitin synthase (KfoC) to obtain a tetrasaccharide initiator.

[0048] In one embodiment of the present invention, when the substrate is a tetrasaccharide, the glycosyltransferase or polymerase is one or more of Pasteurella multocida heparosan synthase 2 (PmHS2), Pasteurella multocida HA synthase (PmHAS), Pasteurella multocida or chondroitin synthase (Chondroitin synthase from Pasteurellamultocida, PmCS) and Escherichia coli K4 from Chondroitin synthase (E.coliK4 from Chondroitin synthase, KfoC), to obtain a pentasaccharide initiator.

[0049] In another embodiment of the present invention, when the substrate is a pentasaccharide, the glycosyltransferase or polymerase is one or more of Pasteurella multocida heparosan synthase 2 (PmHS2), Pasteurella multocida HA synthase (PmHAS), Pasteurella multocida or chondroitin synthase (PmCS) and Escherichia coli K4 from Chondroitin synthase (KfoC) to obtain a hexasaccharide initiator.

[0050] In another embodiment of the present invention, when the substrate is a hexasaccharide, the glycosyltransferase or polymerase is one or more of Pasteurella multocida heparosan synthase 2 (PmHS2), Pasteurella multocida HA synthase (PmHAS), Pasteurella multocida or chondroitin synthase (PmCS) and Escherichia coli K4 from Chondroitin synthase (KfoC), to obtain a heptasaccharide initiator.

[0051] In another embodiment of the present invention, when the substrate is a heptasaccharide, the glycosyltransferase or polymerase is one or more of Pasteurella multocida heparosan synthase 2 (PmHS2), Pasteurella multocida HA synthase (PmHAS), Pasteurella multocida or chondroitin synthase (Chondroitin synthase from Pasteurellamultocida, PmCS) and Escherichia coli K4 from Chondroitin synthase (E.coliK4 from Chondroitin synthase, KfoC), to obtain an octasaccharide initiator.

[0052] In a fifth aspect, the present invention provides a reaction package for generating chondroitin of different molecular weights through a polymerization reaction, wherein the reaction package comprises an initiator, a polymerase, and a comonomer.

[0053] The initiator is selected from one or more of the following: GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (trisaccharide), GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (tetrasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (pentasaccharide) , GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (hexasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalN Acβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (heptasaccharide), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3 Galβ1-4GlcNAcαorβProN3 (octasaccharide), GalNAcβ1-4Galβ1-4GlcNAcαorβProN3 (GN-1), GalNAcβ1-4Galβ1-3Galβ1-4GlcN AcαorβProN3(GN-2), GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc(CH4) and GalNAcβ1-4GlcAβ1-3GalNAcαorβProN3(CH3).

[0054] Furthermore, the polymerase is selected from one or more of: Pasteurella multicida heparosan synthase 2 (PmHS2), Escherichia coli K4 from Chondroitin synthase (KfoC), Pasteurella multicida HA synthase (PmHAS) and Pasteurella multocida from Chondroitin synthase (PmCS).

[0055] Furthermore, the comonomer is connected by acetylated hexosamine and uronic acid through a glycosidic bond.

[0056] Furthermore, the acetylated hexosamine is selected from N-acetylgalactosamine, UDP-GalNAc (uridine-5'-diphosphate-N-acetyl-galactosamine sodium salt), UDP-GalNAz (uridine-5'-diphosphate-N-azidoacetylgalactosamine), UDP-GalNTFA (uridine-5'-diphosphate-N-trifluoroacetylgalactosamine), UDP-GalN (uridine-5'-diphosphate-galactosamine), UDP-GalNAalk (uridine-5'-diphosphate-N-alkynylacetylgalactosamine), One or more of UDP-GlcNAc (uridine-5'-diphosphate-N-acetyl-glucosamine sodium salt), UDP-GlcNAz (uridine-5'-diphosphate-N-azidoacetylglucosamine), UDP-GlcNTFA (uridine-5'-diphosphate-N-trifluoroacetylglucosamine), UDP-GlcN (uridine-5'-diphosphate-glucosamine) and UDP-GlcNAalk (uridine-5'-diphosphate-N-alkynyl acetylglucosamine).

[0057] Furthermore, the uronic acid can be selected from glucuronic acid and / or UDP-GlcA (uridine-5'-diphosphoglucuronic acid trisodium salt).

[0058] In one embodiment of the present invention, the reaction package uses trisaccharide (GlcAβ1-3Galβ1-4GlcNAcαorβProN3) as an initiator; the polymerase can be Pasteurellamultocida heparosan synthase 2 (PmHS2), Escherichia coli K4 from Chondroitin synthase (KfoC), Pasteurellamultocida hyaluronic acid synthase (PmHAS) and Pasteurellamultocida chondroitin synthase (Chondroitinsynthase from Pasteurellamultocida multocida, PmCS) in one or more; the polymerization monomers are UDP-GlcA and UDP-GalNAc; wherein m includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000; as shown in I.

[0059]

[0060] In one embodiment of the present invention, the reaction package uses GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (tetrasaccharide) as an initiator; its polymerase is one or more of Escherichia coli K4 source chondroitin synthase (E.coliK4 fromChondroitin synthase, KfoC) and Pasteurella multocida source F type chondroitinase synthase (Pasteurellamultocida Type F Chondroitin synthase, PmCS); the polymerization monomers are UDP-GlcA and UDP-GalNAc, wherein m includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000; as shown in II.

[0061]

[0062] In one embodiment of the present invention, the reaction package uses (pentasaccharide) as an initiator; its polymerase is Pasteurella multocida heparosan synthase 2 (PmHS2), Escherichia coli K4 from Chondroitin synthase (KfoC), Pasteurella multocida HA synthase (PmHAS) and Pasteurella multocida chondroitin synthase (Chondroitin synthase from Pasteurella multocida, PmCS), the polymerization monomer is UDP-GlcA, UDP-GalNAc and UDP-GalNAz, wherein a includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000; b includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000, as shown in formula III.

[0063]

[0064] In one embodiment of the present invention, the reaction package uses (hexasaccharide) as an initiator; its polymerase is Pasteurella multocida heparosan synthase 2 (PmHS2), E. coli K4 from Chondroitin synthase (KfoC), Pasteurella multocida HA synthase (PmHAS) and Pasteurella multocida chondroitin synthase (Chondroitin synthase from Pasteurella multocida, PmCS), the polymerization monomer is UDP-GlcA, UDP-GalNAc and UDP-GalNNTFA, wherein c includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000; d includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000, as shown in formula IV.

[0065]

[0066] In one embodiment of the present invention, the reaction package uses (heptasaccharide) as an initiator; its polymerase is Pasteurella multocida heparosan synthase 2 (PmHS2), E. coli K4 from Chondroitin synthase (KfoC), Pasteurella multocida HA synthase (PmHAS) and Pasteurella multocida chondroitin synthase (Chondroitin synthase from Pasteurella multocida, PmCS), wherein the polymerization monomer is UDP-GlcA, UDP-GalNAc, UDP-GalNTFA and UDP-GalNAz, wherein e includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000; f includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000; g includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000, as shown in V formula.

[0067]

[0068] In one embodiment of the present invention, when GalNAcβ1-4Galβ1-4GlcNAcαorβProN3 (GN-1) is used as a chondroitin polymerization initiator, the catalytic enzyme used includes a combination of Escherichia coli K4 from Chondroitin synthase (E. coli K4 fromChondroitin synthase, KfoC) and Pasteurella multocida Type FChondroitin synthase (Pasteurella multocida Type FChondroitin synthase, PmCS), and the chondroitin that can be formed is shown in the following formula VI; wherein h includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000.

[0069]

[0070] In one embodiment of the present invention, when GalNAcβ1-4Galβ1-3Galβ1-4GlcNAcαorβProN3 (GN-2) is used as a chondroitin polymerization initiator, the catalytic enzyme used includes a combination of Escherichia coli K4-derived chondroitin synthase (E. coli K4from Chondroitin synthase, KfoC) and Pasteurella multocida Type F Chondroitin synthase (Pasteurella multocida Type F Chondroitin synthase, PmCS), and the chondroitin that can be formed is shown in Formula VII; wherein i includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000.

[0071]

[0072] In one embodiment of the present invention, when GalNAcβ1-4GlcAβ1-3GalNAcαorβProN3 (CH3) is used as a chondroitin polymerization initiator, the catalytic enzyme used includes a combination of Escherichia coli K4 source chondroitin synthase (E. coli K4 fromChondroitin synthase, KfoC) and Pasteurella multocida source F type chondroitinase synthase (Pasteurella multocida Type FChondroitin synthase, PmCS), and the chondroitin that can be formed is shown in Formula VIII; wherein j includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000.

[0073]

[0074] In one embodiment of the present invention, when GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc(CH4) is used as a chondroitin polymerization initiator, the catalytic enzyme used includes a combination of Escherichia coli K4-derived chondroitin synthase (E. coli K4 fromChondroitin synthase, KfoC) and Pasteurella multocida-derived Type F chondroitinase synthase (Pasteurella multocida Type FChondroitin synthase, PmCS), and the chondroitin that can be formed is shown in Formula IX; wherein k includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000.

[0075]

[0076] Beneficial effects

[0077] The chondroitin preparation technology of the present application can produce chondroitin with a linear molecular weight change, a wide controllable molecular weight range, and a narrow molecular weight distribution. That is, only the mother liquor of each raw material needs to be prepared to produce chondroitin with a specific molecular weight. The molecular weight range may include 1-5 million; 100,000-4 million, 250,000-3 million, 350,000-2 million, 500,000-1 million, 200,000-300,000, 1,000-70,000, 70,000-300,000, 350,000-660,000, and 380,000-900,000. The chondroitin prepared by this technology can achieve the advantages of convenient operation, high efficiency, and reliability. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 Polyacrylamide gel electrophoresis results of engineered sugar synthesis-related enzymes (1: Protein Maker; 2: BiGalK; 3: AtUSP; 4: PmPPA; 5: AtGlcAK; 6: BiNahK; 7: AGX1).

[0079] Figure 2 Polyacrylamide gel electrophoresis results of engineered sugar synthesis-related enzymes (1: Protein Maker; 2: NmLgtB; 3: GlcAT-P; 4: PmCS).

[0080] Figure 3 This is a process flow chart for receptor molecules and chondroitin.

[0081] Figure 4 GlcNAcαProN3 1 HNMR (D2O) spectrum.

[0082] Figure 5Galβ1-4GlcNAcαProN3 1 H NMR (D2O) spectrum.

[0083] Figure 6 GlcAβ1-3Galβ1-4GlcNAcαProN3 1 H NMR (D2O) spectrum.

[0084] Figure 7 GalNAcβ1-4GlcAβ1-3GalNAcβProN3 1 H NMR (D2O) spectrum.

[0085] Figure 8 GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN3 1 H NMR (D2O) spectrum.

[0086] Figure 9 GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc 1 HNMR (D2O) spectrum.

[0087] Figure 10 This is an analysis of the differences in sugar pool binding energy.

[0088] Figure 11 The elution curves of chondroitin with different molecular weights prepared by polymerization with different concentrations of tetrasaccharide initiator at different feed ratios. a: [I0] = 20 μM, [M0] / [I0] = 50-400; b: [I0] = 10 μM, [M0] / [I0] = 50-800, linear correlation R 2 =0.998; c: [I0] = 1 μM, [M0] / [I0] = 1000-3500, linear correlation R 2 =0.997.

[0089] Figure 12 is the linear relationship diagram between feed ratio and output, linear correlation R 2 =0.990.

[0090] Figure 13 The elution curves of chondroitin with different molecular weights prepared by polymerization of trisaccharide initiator at different feed ratios and different concentrations are shown. a: [I0] = 10 μM, [M0] / [I0] = 50-500, linear correlation R 2 =0.990; b: [I0] = 1 μM, [M0] / [I0] = 800-3000, linear correlation R 2 =0.992.

[0091] Figure 14 Preparation of block chondroitin with different block patterns and molecular weights using different comonomers, along with their elution curves. a: Tetrablock with comonomers UDP-GalNAc and UDP-GalNAz; b: Tetrablock with comonomers UDP-GalNAc and UDP-GalNTFA; c: Mixed-block with comonomers UDP-GalNAc, UDP-GalNAz, and UDP-GalNTFA. Green: Comonomer UDP-GalNAc; Orange: Comonomer UDP-GalNAz; Blue: Comonomer UDP-GalNTFA. DETAILED DESCRIPTION

[0092] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below may be combined with each other as long as they do not conflict with each other.

[0093] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0094] The term "linking group" as used herein refers to the introduction of a linking group at the anomeric carbon of a monosaccharide molecule in the present invention, so that the functional group at the anomeric carbon of the monosaccharide molecule increases the flexibility of the chain, facilitates the exposure of the functional group (such as an azide group), reduces the adverse effects of steric hindrance, and thus increases the reaction efficiency. The linking functional group compounds herein include 3-chloro-1-propanol, 3-bromo-1-propanol, 2-bromoethanol, 2-chloroethanol, 3-chloro-2-propanol, 1-chloro-2-methyl-2-propanol, (S)-(+)-2-chloro-1-propanol, 2-[2-(2-chloroethoxy)ethoxy]ethanol or 2-(2-chloroethoxy)ethanol.

[0095] The term "azido group" as described herein refers to a class of small molecule compounds such as fluorescent chromophores and drug molecules introduced through a click reaction, and can also be bonded to macromolecules such as proteins and DNA, thereby performing complex and diverse biological functions. In this application, the introduction of an azide group at the linker group at the anomeric carbon of a monosaccharide molecule can make the monosaccharide or subsequent oligosaccharide molecule have click chemistry reaction characteristics, thereby achieving the above-mentioned special biological functions; the azide compounds described herein include but are not limited to sodium azide, potassium azide, lithium azide, lead azide, 3-azido-1-propanol, 2-azidoethanol, 2-azido-1-amine hydrobromide or 3-azido-1-propylamine.

[0096] In one embodiment of the present invention, when trisaccharide (GlcAβ1-3Galβ1-4GlcNAcαorβProN3) is used as an initiator for chondroitin synthesis, the catalytic enzyme used can be Pasteurella multocida heparosan synthase 2 (PmHS2), E. coli K4 from Chondroitin synthase (KfoC), Pasteurella multocida HA synthase (PmHAS) and Pasteurella multocida chondroitin synthase (PmHS1). multocida, PmCS); the chondroitin that can be formed is shown in the following formula I; wherein m includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000.

[0097]

[0098] In one embodiment of the present invention, when GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (tetrasaccharide) is used as a chondroitin polymerization initiator, the catalytic enzyme used includes a combination of Escherichia coli K4-derived chondroitin synthase (E. coli K4from Chondroitin synthase, KfoC) and Pasteurella multocida Type F Chondroitin synthase (Pasteurella multocida Type F Chondroitin synthase, PmCS), and the chondroitin that can be formed is shown in the following formula II; wherein n includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000.

[0099]

[0100] In one embodiment of the present invention, when GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (pentasaccharide) is used as a chondroitin polymerization initiator, the catalytic enzymes used include E. coli K4 from Chondroitin synthase (KfoC), Pasteurella multocida Type F Chondroitin synthase (Pasteurella multocida Type F Chondroitin synthase), synthase, PmCS) can form chondroitin as shown in the following formula III; wherein a includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000; b includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000.

[0101]

[0102] In one embodiment of the present invention, when GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (hexasaccharide) is used as a chondroitin polymerization initiator, the catalytic enzymes used include E. coli K4 from Chondroitin synthase (KfoC), Pasteurella multocida Type F Chondroitin synthase (Pasteurella multocida Type F Chondroitin synthase, PmCS), the chondroitin that can be formed is shown in formula IV; wherein c includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000; d includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000.

[0103]

[0104] In one embodiment of the present invention, when GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (heptasaccharide) is used as a chondroitin polymerization initiator, the catalytic enzymes used include E. coli K4 from Chondroitin synthase (KfoC), Pasteurella multocida Type F chondroitinase synthase (FChondroitinase), and GlcAβ1-3GalNAcβ1-4GlcNAcαorβProN3 (heptasaccharide). synthase, PmCS), the chondroitin that can be formed is shown in the following formula V; wherein e includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000; f includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000; g includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000.

[0105]

[0106] In one embodiment of the present invention, when GalNAcβ1-4Galβ1-4GlcNAcαProN3 (GN-1) is used as a chondroitin polymerization initiator, the catalytic enzyme used includes a combination of Escherichia coli K4 fromChondroitin synthase (E. coli K4 fromChondroitin synthase, KfoC) and Pasteurella multocida type F chondroitinase synthase (Pasteurella multocida type FChondroitin synthase, PmCS), and the chondroitin that can be formed is shown in the following formula VI; wherein h includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000.

[0107]

[0108] In one embodiment of the present invention, when GalNAcβ1-4Galβ1-3Galβ1-4GlcNAcαProN3 (GN-2) is used as a chondroitin polymerization initiator, the catalytic enzyme used includes a combination of Escherichia coli K4 from Chondroitin synthase (E. coli K4 fromChondroitin synthase, KfoC) and Pasteurella multocida Type F Chondroitin synthase (Pasteurella multocida Type F Chondroitin synthase, PmCS), and the chondroitin that can be formed is shown in the following formula VII; wherein i includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000.

[0109]

[0110] In one embodiment of the present invention, when GalNAcβ1-4GlcAβ1-3GalNAcProN3 (CH3) is used as a chondroitin polymerization initiator, the catalytic enzyme used includes a combination of Escherichia coli K4 source chondroitin synthase (E. coli K4 fromChondroitin synthase, KfoC) and Pasteurella multocida source F type chondroitinase synthase (Pasteurella multocida Type FChondroitin synthase, PmCS), and the chondroitin that can be formed is shown in the following formula VIII; wherein j includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000.

[0111]

[0112] In one embodiment of the present invention, when GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc(CH4) is used as a chondroitin polymerization initiator, the catalytic enzyme used includes a combination of Escherichia coli K4 from Chondroitin synthase (E. coli K4 fromChondroitin synthase, KfoC) and Pasteurella multocida Type FChondroitin synthase (Pasteurella multocida Type FChondroitin synthase, PmCS), and the chondroitin that can be formed is shown in the following formula IX; wherein k includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000.

[0113]

[0114] The term "glycosyltransferase" in the present invention can refer to both glycosyltransferase and polymerase, and the expressions of glycosyltransferase and polymerase are interchangeable.

[0115] Example 1 Expression and purification of sugar synthesis related enzymes

[0116] 1.1 Expression and purification of sugar synthesis-related enzymes

[0117] The engineered BiNahK, AGX1, AtGlcAK, AtUSP, BiGalK, PmPPA, NmLgtB, GlcAT-P, and PmCS were expressed and purified. The specific implementation steps are as follows:

[0118] Among them, BlNahK is N-acetylhexosamine 1-kinase from Bifidobacterium longum;

[0119] AGX1 is a human UDP-N-acetylgalactosamine pyrophosphorylase (UDP-N-acetylgalactosaminepyrophosphorylase from Homo sapiens);

[0120] AtGlcAK is glucuronokinase from Arabidopsis thaliana;

[0121] AtUSP is UDP-sugar pyrophosphorylase from Arabidopsis thaliana;

[0122] BiGalK is galactokinase from Bifidobacterium infantis;

[0123] PmPPA is inorganic pyrophosphatase from Pasteurella multocida;

[0124] NmLgtB is β1-4 galactosyltransferase from Neisseria meningitidis;

[0125] GlcAT-P is murine glucuronyltransferase-P (Glucuronyltransferase-P from Mus musculus);

[0126] PmCS is chondroitin synthase from Pasteurella multocida.

[0127] (1) Construction of prokaryotic expression vector. Refer to the relevant sequence information published in NCBI. The specific restriction sites at both ends of the whole gene synthesis and the plasmid vector are shown in Table 1 below:

[0128] Table 1 Carriers and cleavage sites of sugar synthesis-related enzymes

[0129]

[0130]

[0131] (2) Induced expression and purification of sugar synthesis-related enzymes

[0132] After sequencing and verification, the relevant sugar synthase plasmids in Table 1 were transformed into BL21 (DE3) competent cells and plated onto culture dishes containing LB solid medium. After inverted culture at 37°C overnight in an incubator, single clones were picked and placed in 100 mL of LB liquid medium containing 100 μg / mL of the corresponding antibiotic for small-scale culture at 37°C and 250 rpm. Culture was continued for 8-12 h and the OD 600When the OD value is about 1, 10 mL of the turbid bacterial solution was transferred to 1 L of LB medium containing 100 μg / mL antibiotics to induce large-scale expression. The culture conditions were 37°C and 250 rpm. Culture at 37°C until the OD value reached 0. 600 After the pH value reaches 0.6-0.8, the temperature is lowered to 16°C, and appropriate IPTG is added to induce the culture for 20 hours.

[0133] (3) The above-mentioned induced bacterial solution was collected by centrifugation at 4000 rpm for 20 minutes, and then the collected bacterial solution was resuspended in 40 mL of NiA buffer (50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 7.5) and crushed by a high-pressure sterilizer at a pressure of 1000 bar. The crushed bacterial solution was centrifuged at 18000 rpm for 1 hour, and the supernatant was collected. The supernatant was purified by affinity chromatography on a 5 mL Ni-NTA column, and the target protein was eluted with NiB buffer (50 mM Tris-HCl, 300 mM NaCl, 300 mM imidazole, pH 7.5). The UV 280 The eluted sugar synthesis related enzymes were collected by absorbance. The purified samples were tested for purity by polyacrylamide gel electrophoresis (SDS-PAGE) and the protein concentration was determined by Bradford method.

[0134] 1.2 Experimental Results

[0135] like Figure 1 and Figure 2 As shown in the figure, the SDS-PAGE electrophoresis results show that the constructed series of engineered sugar synthesis-related enzymes are consistent with the theoretical molecular weight and the purity meets the purpose of use.

[0136] Example 2 Enzymatic Synthesis of Receptor Molecules

[0137] The relevant enzymes purified in Example 1 were used to further synthesize the following molecules, such as Galβ1-4GlcNAcProN3 (disaccharide), GlcAβ1-3Galβ1-4GlcNAcProN3 (trisaccharide), GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcProN3 (tetrasaccharide), pentasaccharide-octasaccharide; GalNAcβ1-4Galβ1-4GlcNAcαorβProN3(GN-1), GalNAcβ1-4Galβ1-3Galβ1-4GlcNAcαorβProN3(GN-2); GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc(CH4), GalNAcβ1-4GlcAβ1-3GalNAcαorβProN3(CH3). The trisaccharide and tetrasaccharide receptor molecules synthesized in this process are then used as initiators in the preparation of chondroitin, and chondroitin with a wide molecular weight range and a narrow molecular weight distribution can be synthesized. The process flow chart is shown in Figure 3 .

[0138] 2.1 Preparation of GlcNAcαProN3 receptor molecules

[0139] Dried N-acetyl-D-glucosamine (1 eq) loaded with sulfated silica gel powder was added to a 100 mL round-bottom flask. 10 eq of 3-bromo-1-propanol was then added. The atmosphere was replaced with nitrogen and the mixture was heated at 60°C with stirring overnight. The reaction was analyzed by TLC (dichloromethane:methanol = 5:1). After completion, the mixture was purified by silica gel column chromatography. Excess 3-bromo-1-propanol was removed by elution with dichloromethane, followed by elution with dichloromethane:methanol = 5:1 to obtain GlcNAcαProBr. The product was then spin-dried into a 50 mL round-bottom flask and weighed. To the round-bottom flask containing GlcNAcαProBr, 2 eq of sodium azide and a predetermined volume of DMF were added, stirred, and heated to 80°C for overnight reaction. Filter and remove salt, take the supernatant and separate and purify it by silica gel column chromatography, elute with polar dichloromethane: methanol = 5:1, collect the fractions containing GlcNAcαProN3, spin dry, and weigh. 1 HNMR (400 MHz, D2O) Figure 4 ).

[0140] 2.2 Enzymatic Synthesis of Galβ1-4GlcNAcαProN3 (Disaccharide)

[0141] UDP-Gal is prepared by BiGalK, AtUSP and PmPPA, and then Gal is transferred to the end of GlcNAcαProN3 by NmLgtB to obtain Galβ1-4GlcNAcαProN3 (disaccharide).

[0142] The specific reaction system is as follows: 100 mM Tris-HCl pH 8.0, 20 mM MgCl2, GlcNAcProN3, adenosine-5'-triphosphate (ATP, 1.3 eq), uracil-5'-triphosphate (UTP, 1.3 eq), galactose (Gal, 1.3 eq) adjusted to pH 7.5, incubated at 37°C for 15 min, and a certain amount of BiGalK, AtUSP, PmPPA, and NmLgtB were added. The final volume was adjusted to 30 mL, and the reaction was slowly stirred at 80 rpm at 37°C.

[0143] The reaction was analyzed by TLC spot plate. After the reaction was complete, an equal volume of icy ethanol was added to quench the reaction. The product was separated and purified by silica gel column chromatography using a gradient elution of ethyl acetate: methanol: water. The target fractions were pooled and dried, and purified by BioGel P-2 size exclusion column. The target fractions were pooled to obtain the final pure Galβ1-4GlcNAcαProN3. 1 HNMR (400 MHz, D2O) Figure 5 ).

[0144] 2.3 Enzymatic Synthesis of GlcAβ1-3Galβ1-4GlcNAcαProN3 (Trisaccharide)

[0145] UDP-GlcA is prepared by the enzymes AtGlcAK, AtUSP and PmPPA, and then GlcA is transferred to the end of Galβ1-4GlcNAcαProN3 by GlcAT-P to obtain GlcAβ1-3Galβ1-4GlcNAcαProN3.

[0146] The reaction system was as follows: 100 mM Tris-HCl (pH 8.0), 20 mM MgCl2, Galβ1-4GlcNAcαProN3, adenosine-5'-triphosphate (ATP, 1.3 eq), uracil-5'-triphosphate (UTP, 1.3 eq), and glucuronic acid (Gal, 1.3 eq) adjusted to pH 7.5. The mixture was incubated at 37°C for 15 min. The appropriate amounts of AtGlcAK, AtUSP, PmPPA, and GlcAT-P were added, and the final volume was adjusted to 30 mL. The reaction was stirred at 80 rpm and incubated at 37°C. The reaction was analyzed by TLC. Upon completion, an equal volume of glacial ethanol was added to quench the reaction. The product was separated and purified by silica gel column chromatography, with ethyl acetate: methanol: water as the elution components for gradient elution. The target fractions were pooled and dried, and then purified by BioGel P-2 size exclusion column. The target fractions were pooled to obtain the final pure product of GlcAβ1-3Galβ1-4GlcNAcαProN3. 1 HNMR (400 MHz, D2O) Figure 6 ).

[0147] 2.4 Enzymatic Synthesis of GalNAcβ1-4GlcAβ1-3GalNAcβProN3(CH3)

[0148] PmCS transfers GlcA to the end of GalNAcβProN3 to obtain GlcAβ1-3GalNAcβProN3. Similarly, PmCS transfers GalNAc to the end of GlcAβ1-3GalNAcβProN3 to obtain the final GalNAcβ1-4GlcAβ1-3GalNAcβProN3.

[0149] The specific reaction system is as follows:

[0150] Synthesis of GlcAβ1-3GalNAcβProN3: 100 mM Tris-HCl pH 8.0, 20 mM MgCl2, GalNAcProN3, UDP-glucuronic acid (UDP-GlcA, 1.3 eq), a certain amount of PmCS were added, and the reaction was slowly stirred at 80 rpm at 37°C.

[0151] The reaction was analyzed by TLC. Upon completion, an equal volume of glacial ethanol was added to quench the reaction. The product was separated and purified by silica gel column chromatography using a gradient elution of ethyl acetate:methanol:water. The target fractions were pooled and dried, and then purified on a BioGel P-2 size exclusion column to obtain GlcAβ1-3GalNAcβProN3.

[0152] Synthesis of GalNAcβ1-4GlcAβ1-3GalNAcβProN3: 100 mM Tris-HCl pH 8.0, 20 mM MgCl2, GlcAβ1-3GalNAcProN3, UDP-N-acetylgalactosamine (UDP-GalNAc, 1.3 eq), add a certain amount of PmCS, and react slowly with stirring at 80 rpm at 37°C.

[0153] The reaction was analyzed by TLC spot plate. After the reaction was complete, an equal volume of icy ethanol was added to quench the reaction. The product was separated and purified by silica gel column chromatography using a gradient elution of ethyl acetate: methanol: water. The target fractions were pooled and dried, and then purified by BioGel P-2 size exclusion column. The target fractions were pooled to obtain the final pure product of GalNAcβ1-4GlcAβ1-3GalNAcβProN3. 1 H NMR (400 MHz, D2O) Figure 7 ).

[0154] 2.5 Enzymatic Synthesis of GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN3 (Tetrasaccharide)

[0155] UDP-GalNAc is transferred to the end of GlcAβ1-3Galβ1-4GlcNAcαProN3 by PmCS to obtain GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN3.

[0156] The specific reaction system is as follows: 100 mM Tris-HCl pH 8.0, 20 mM MgCl2, GlcAβ1-3Galβ1-4GlcNAcαProN3, UDP-N-acetyl-galactosamine (UDP-GalNAc, 1.3 eq), a certain amount of PmCS was added, and the reaction was slowly stirred at 80 rpm at 37°C.

[0157] The reaction was analyzed by TLC spot plate. After the reaction was complete, an equal volume of icy ethanol was added to quench the reaction. The product was separated and purified by silica gel column chromatography using a gradient elution of ethyl acetate: methanol: water. The target fractions were pooled and dried, and purified by BioGel P-2 size exclusion column. The target fractions were pooled to obtain the final pure product of GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN3. 1 H NMR (400 MHz, D2O) Figure 8 ).

[0158] 2.6 Preparation of GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc(CH4) by Enzymatic Degradation

[0159] Chondroitin sulfate A was dissolved in a methanol solution containing acetyl chloride, stirred at room temperature, filtered and the precipitate was collected. The solution was replaced with acidic methanol solution on days 1, 3, 5 and 7 to obtain a crude product of chondroitin methyl ester. The crude product was demethylated in 0.1 M NaOH for 1 day and then treated with H + The resin is neutralized and filtered, concentrated, precipitated and separated in an ethanol solution, and vacuum-dried to obtain chondroitin.

[0160] Chondroitin was resuspended in sodium acetate buffer (pH = 5.0), and bovine testicular hyaluronidase (2.5%) was added and reacted at 37 ° C for 7 days. The solution was heated to reflux for 15 minutes, cooled on ice and filtered using diatomaceous earth. The filtrate was mixed with ethanol and concentrated by rotary evaporation. Ethanol was gradually added dropwise to the concentrate, and the white precipitate formed was filtered and vacuum dried overnight to obtain a crude product. It was separated by Sephadex LH-20 and AG 1-X4 resin (200-400 mesh, analytical grade), and the purification was analyzed by TLC spot plate. The target fractions were pooled, desalted and lyophilized to obtain the final pure GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc(CH4). 1 H NMR (400 MHz, D2O) Figure 9 ).

[0161] 2.7 Enzymatic synthesis of other receptor molecules

[0162] More receptor molecules were synthesized by the enzymatic method of the present invention as triggers for chondroitin. Other receptor molecules are as follows:

[0163] (1) Pentasaccharide

[0164] Glucuronic acid-β1-3-N-acetylglucosamine-β1-4-glucuronic acid-β1-3-galactosamine-β1-4-N-acetylglucosamine-α-propyl azide (GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN3); molecular weight 1021.886 g / mol; chemical structure shown in α:

[0165]

[0166] (2) Hexasaccharide

[0167] N-acetylgalactosamine-β1-4-glucuronic acid-β1-3-N-acetylgalactosamine-β1-4-glucuronic acid-β1-3-galactosamine-β1-4-N-acetylglucosamine-α-propyl azide (GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN3); molecular weight is 1021.886 g / mol; chemical structure is shown in β:

[0168]

[0169] (3) Seven sugars

[0170] Glucuronic acid-β1-3-N-acetylgalactosamine-β1-4-glucuronic acid-β1-3-N-acetylgalactosamine-β1-4-glucuronic acid-β1-3-galactosamine-β1-4-N-acetylglucosamine-α-propyl azide (GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN3); molecular weight is 1401.2 g / mol; chemical structure is shown in γ,

[0171]

[0172] (4) Octasaccharide

[0173] N-acetylgalactosamine-β1-4-glucuronic acid-β1-3-N-acetylgalactosamine-β1-4-glucuronic acid-β1-3-N-acetylgalactosamine-β1-4-glucuronic acid-β1-3-galactosamine-β1-4-N-acetylglucosamine-α-propyl azide (GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN3); molecular weight 1604.398 g / mol, chemical structure shown in δ;

[0174]

[0175] (5)GN-1

[0176] N-acetylgalactosamine-β1-4-galactosamine-β1-4-N-acetylglucosamine-α-propyl azide (GalNAcβ1-4Galβ1-4GlcNAcαProN3); molecular weight 669.63 g / mol; chemical structure shown in ε;

[0177]

[0178] (6)GN-2

[0179] N-acetylgalactosamine-β1-4-galactosamine-β1-3-galactosamine-β1-4-N-acetylglucosamine-α-propyl azide (GalNAcβ1-4Galβ1-3Galβ1-4GlcNAcαProN3); molecular weight 831.77 g / mol; chemical structure shown in θ.

[0180]

[0181] 2.8 Receptor molecule screening

[0182] This technology uses Autodock molecular docking software to preliminarily analyze the binding energy of a series of receptor molecules as initiators. Through screening, it was found that the receptor molecules such as trisaccharides and tetrasaccharides used in this technology have lower binding energy (-5kcal·mol -1 ), which will be beneficial to linear aggregation.

[0183] like Figure 10 As shown, when the binding energy is ±30 kcal·mol -1 It is suitable as an initiator for chondroitin synthesis.

[0184] Example 3 Controllable Chondroitin Linear Polymerization

[0185] The polymerization reaction of chondroitin is catalyzed by PmCS polymerase, using GlcAβ1-3Galβ1-4GlcNAcαProN3 (trisaccharide) or GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN3 (tetrasaccharide) receptor molecules or other receptors synthesized by the present invention as initiators, UDP-GlcA and UDP-GalNAc as comonomers, and sequentially bonding GlcA and GalNAc to the non-reducing end of the initiator to form chondroitin polysaccharides. The reaction process is shown in the following R1 synthesis route;

[0186]

[0187]

[0188] GlcAβ1-3Galβ1-4GlcNAcαProN3 (trisaccharide) and GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN3 (tetrasaccharide) are used as initiators (I0), and uridine 5′-diphospho-glucuronic acid (UDP-GlcA) and uridine 5′-diphospho-N-acetylgalactosamine (UDP-GalNAc) are used as comonomers (M0). By controlling the ratio of the above two comonomers to the initiator ([M0] / [I0]), the ratio range mainly includes 50 to 4000, so as to accurately prepare chondroitin with different molecular weights.

[0189] 3.1 Synthesis of chondroitin using GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN3 (tetrasaccharide) as an initiator and analysis of its properties

[0190] 20μM, 10μM and 1μM GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN3 were used as initiators (I0), and uridine 5′-diphospho-glucuronic acid (UDP-GlcA) and uridine 5′-diphospho-N-acetylgalactosamine (UDP-GalNAc) were used as comonomers (M0). By controlling the ratio of the above two comonomers to the initiator ([M0] / [I0]), chondroitin with different molecular weights was accurately prepared. The synthesis route is shown in R2. The molecular weight and dispersity of the prepared chondroitin were analyzed by SEC-MALLS-RI. The analytical column was a TSKgel GMPWXL column (13 μm, 7.8*300 mm, Tosoh Corporation), and the mobile phase was 0.1 M NaNO3, 0.05 M Na2HPO4. The prepared chondroitin was analyzed at 0.6 mL / min at 25°C. The data were processed using ASTRA software.

[0191]

[0192] The experimental results are as follows Figure 11 As shown, when I0 = 20 μM, as the feed ratio increases, the molecular weight has a smaller linear correlation (R 2 =0.922), the maximum molecular weight can only reach 315kDa, and then the molecular weight will not continue to increase; when I0 = 10μM, the linear correlation is significantly enhanced (R 2 =0.998), the molecular weight can only reach 290kDa; when I0 = 1μM, under the premise of ensuring strong linear correlation, the molecular weight of chondroitin reaches 660kDa, at this time the molecular weight dispersion Very narrow distribution,

[0193] Depend on Figure 12 It can be seen that the yield of the prepared chondroitin increases linearly with the increase of feed ratio.

[0194] Table 1 Synthesis of chondroitin using tetrasaccharide (GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN3) as initiator

[0195]

[0196]

[0197] a:M n,实测 and The average result of four parallel experiments

[0198] 3.3 Preparation of chondroitin using GlcAβ1-3Galβ1-4GlcNAcαProN as the receptor molecule

[0199] A series of molecular weight chondroitin was prepared using GlcAβ1-3Galβ1-4GlcNAcαProN3 (trisaccharide molecule) as an initiator using the same preparation and analysis methods as GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN3 (tetrasaccharide molecule).

[0200] 20μM, 10μM and 1μM GlcAβ1-3Galβ1-4GlcNAcαProN3 were used as initiators (I0), and uridine 5′-diphospho-glucuronic acid (UDP-GlcA) and uridine 5′-diphospho-N-acetylgalactosamine (UDP-GalNAc) were used as comonomers (M0). By controlling the ratio of the above two comonomers to the initiator ([M0] / [I0]), the ratio range mainly included 50 to 4000, so as to accurately prepare chondroitin with different molecular weights. The synthesis route is shown in R3.

[0201] The molecular weight and dispersity of the prepared chondroitin were analyzed by SEC-MALLS-RI. The analytical column was a TSKgel GMPWXL column (13 μm, 7.8*300 mm, Tosoh Corporation), and the mobile phase was 0.1 M NaNO3, 0.05 M Na2HPO4. The prepared chondroitin was analyzed at 0.6 mL / min at 25°C. The data were processed using ASTRA software.

[0202]

[0203] like Figure 13 As shown in the figure, when the concentration of trisaccharide initiator is 10 μM, as the feed increases, the number average molecular weight (M n ) increases linearly, with the molecular weight increasing from 350kDa to 575kDa; when the concentration of trisaccharide initiator is 1μM, the molecular weight will increase linearly, up to 632kDa. At the same time, the molecular weight dispersion of the above chondroitin It has a narrow distribution, close to 1 and not higher than 1.1.

[0204] 3.4 Preparation of multi-block chondroitin using GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN3 (tetrasaccharide) as initiator

[0205] A preparation method and analysis method similar to GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN3 (tetrasaccharide molecule) were used to prepare a series of chondroitins with different block patterns by changing the type of comonomer and using UDP-GalNAc and its derivatives as comonomers, including UDP-GalNAz and UDP-GalNTFA, without changing UDP-GlcA.

[0206] 10 μM GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN3 was used as the initiator (I0), and uridine 5′-diphospho-glucuronic acid (UDP-GlcA) and uridine 5′-diphospho-N-acetylgalactosamine (UDP-GalNAc) or its derivatives (UDP-GalNAz (uridine-5`-diphospho-N-azidoacetylgalactosamine) or UDP-GalNTFA (uridine-5`-diphospho-N-trifluoroacetylgalactosamine)) were used as comonomers (M0).

[0207] like Figure 14 As shown in a, the ratio of comonomers (UDP-GlcA and UDP-GalNAc) to initiator was controlled to 200, and chondroitin (M) with GlcA and GalNAc as the backbone was obtained. n =76.8kDa). After ensuring that the above UDP-GalNAc reaction is complete, the comonomer UDP-GalNAz is added so that the ratio of UDP-GalNAz to the initiator is controlled to be 50, thus obtaining a block chondroitin (M n =92.8kDa). Using a similar strategy, we can obtain chondroitin with GalNAz tetrablocks (M n =236.8kDa). Similar strategies can also be used to prepare other multi-block chondroitin. Figure 14 b is GalNTFA-blocked chondroitin, and Figure 14 c is chondroitin blocked with GalNAz and GalNTFA.

Claims

1. A chondroitin, wherein the chondroitin uses an initiator as an initial polymerization substrate, and n comonomers are sequentially bonded to the non-reducing end of the initiator, wherein the comonomers are formed by acetylated hexosamine and uronic acid through glycosidic bonds; the comonomers and initiator are polymerized under the catalysis of a polymerase to form a polysaccharide of a certain molecular weight, namely the chondroitin; the range of n includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000.

2. The chondroitin according to claim 1, wherein The initiator is selected from one or more of the following: GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (trisaccharide), GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (tetrasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (pentasaccharide), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (hexasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (tetrasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (pentasaccharide), GlcNAcαorβProN3 (heptose), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (octasaccharide), GalNAcβ1-4Galβ1-4GlcNAcαorβPro N3(GN-1), GalNAcβ1-4Galβ1-3Galβ1-4GlcNAcαorβProN3(GN-2), GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc(CH4) and GalNAcβ1-4GlcAβ1-3GalNAcαorβProN3(CH3).

3. The chondroitin according to claim 1, wherein The acetylated hexosamine is selected from N-acetylgalactosamine, UDP-GalNAc (uridine-5'-diphosphate-N-acetyl-galactosamine sodium salt), UDP-GalNAz (uridine-5'-diphosphate-N-azidoacetylgalactosamine), UDP-GalNTFA (uridine-5'-diphosphate-N-trifluoroacetylgalactosamine), UDP-GalN (uridine-5'-diphosphate-galactosamine), UDP-GalNAalk (uridine-5'-diphosphate-N-alkynylacetylgalactosamine) ), N-acetylglucosamine, UDP-GlcNAc (uridine-5'-diphosphate-N-acetyl-glucosamine sodium salt), UDP-GlcNAz (uridine-5'-diphosphate-N-azidoacetylglucosamine), UDP-GlcNTFA (uridine-5'-diphosphate-N-trifluoroacetylglucosamine), UDP-GlcN (uridine-5'-diphosphate-glucosamine) and UDP-GlcNAalk (uridine-5'-diphosphate-N-alkynyl acetylglucosamine) One or more.

4. The chondroitin according to claim 1, wherein The molecular weight ranges of the chondroitin include 100,000-5,000,000; 100,000-4,000,000, 250,000-3,000,000, 350,000-2,000,000, 500,000-1,000,000, 200,000-300,000, 1,000-70,000, 70,000-300,000, 350,000-660,000 and 380,000-900,000.

5. The chondroitin according to claim 1, wherein The polymerase is selected from one or more of Pasteurella multocida heparosan synthase 2 (PmHS2), Escherichia coli K4 from Chondroitin synthase (KfoC), Pasteurella multocida HA synthase (PmHAS) and Pasteurella multocida chondroitin synthase (PmCS).

6. A method for synthesizing chondroitin; the method comprising the following steps: S1. Acetylated hexosamine and uronic acid bonded nucleotides form comonomers; S2. The comonomer and the initiator are catalyzed by a polymerase to produce a polysaccharide, namely, the chondroitin of the present invention, by polymerization reaction; in, The initiator is selected from one or more of the following: GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (trisaccharide), GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (tetrasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (pentasaccharide), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (hexasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (tetrasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (pentasaccharide), GlcNAcαorβProN3 (heptose), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (octasaccharide), GalNAcβ1-4Galβ1-4GlcNAcαorβPro N3(GN-1), GalNAcβ1-4Galβ1-3Galβ1-4GlcNAcαorβProN3(GN-2), GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc(CH4) and GalNAcβ1-4GlcAβ1-3GalNAcαorβProN3(CH3).

7. A method for synthesizing chondroitin according to claim 6, characterized in that: The feeding ratio of the comonomer and the initiator includes 25-5000:1; 50-4000:1; 100-3000:1; 200-2000:1; 25-400:1; 50-1600:1 and 1000-7000:

1.

8. An initiator for synthesizing chondroitin, wherein the binding energy of the initiator is ±30 kcal·mol -1 , the initiator is selected from one or more of the following: GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (trisaccharide), GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (tetrasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (pentasaccharide), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (hexasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (tetrasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (pentasaccharide), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (hexasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4 GlcNAcαorβProN3 (heptose), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (octasaccharide), GalNAcβ1-4Galβ1-4GlcNAcαorβPro N3(GN-1), GalNAcβ1-4Galβ1-3Galβ1-4GlcNAcαorβProN3(GN-2), GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc(CH4) and GalNAcβ1-4GlcAβ1-3GalNAcαorβProN3(CH3).

9. A method for preparing an initiator, comprising the steps of: S01. Synthesizing intermediate M1 by reacting a monosaccharide with a compound containing a halogen functional group; S02. Adding an azide compound to the synthesized intermediate M1 to generate intermediate M2; S03. Using M2 as a substrate, a glycosyltransferase, a monosaccharide or its derivative is added to synthesize a disaccharide initiator; S04. Using disaccharide as substrate, glycosyltransferase and uronic acid are added to synthesize trisaccharide initiator; S05. Using trisaccharide as substrate, glycosyltransferase and acetylated-hexosamine were added to synthesize tetrasaccharide initiator; S06. Using tetrasaccharide as substrate, glycosyltransferase and uronic acid were added to synthesize pentasaccharide initiator; S07. Using pentasaccharide as substrate, glycosyltransferase and acetylated hexosamine were added to synthesize hexasaccharide initiator; S08. Using hexasaccharide as substrate, glycosyltransferase and uronic acid were added to synthesize heptasaccharide initiator; S09. Using heptasaccharide as substrate, glycosyltransferase and acetylated hexosamine were added to synthesize octasaccharide initiator.

10. A reaction package for producing chondroitin of different molecular weights through a polymerization reaction, wherein the reaction package comprises an initiator, a polymerase, and a comonomer, wherein the initiator is selected from one or more of the following: GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (trisaccharide), GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (tetrasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (pentasaccharide), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (hexasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (heptasaccharide) and GalNAcβ1-4GlcAβ1-3GalNAc β1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN3 (octasaccharide); GalNAcβ1-4Galβ1-4GlcNAcαorβProN3 (GN-1), GalNAcβ1-4Galβ1-3Galβ1-4GlcNAcαorβProN3(GN-2); GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc(CH4), GalNAcβ1-4GlcAβ1-3GalNAcαorβProN3(CH3); the polymerase is selected from: Pseudomonas multocida heparin precursor synthase 2 (Pasteurella multocidaheparosan synthase 2, PmHS2), Escherichia coli K4 chondroitin synthase (E.coliK4 fromChondroitin synthase, KfoC), Pasteurella multocida hyaluronic acid synthase (Pasteurellamultocida HA synthase, PmHAS) and Pasteurella multocida chondroitin synthase (Chondroitinsynthase fromPasteurella multocida, PmCS); the comonomers are connected by acetylated hexosamine and uronic acid through glycosidic bonds.

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