WO2025167469A1 - Method for preparing chondroitin - Google Patents

Abstract

A method for preparing chondroitin with a controllable molecular weight. The chondroitin is obtained by taking an initiator as an initial polymerization substrate; sequentially bonding n comonomers at a non-reducing end of the initiator, wherein the comonomers are formed from acetylated hexosamine and uronic acid by means of a glycosidic bond; and the comonomers and the initiator being polymerized under the action of catalysis of a polymerase to form a polysaccharide with a certain molecular weight, namely the chondroitin. The chondroitin preparation technology can prepare chondroitin for which the molecular weight varies in a linear manner, the controllable molecular weight range is wide, and the molecular weight distribution is narrow. The molecular weight range can include 10,000-5,000,000; 100,000-4,000,000; 250,000-3,000,000; 350,000-2,000,000; 500,000-1,000,000; 20,000-300,000; 1,000-70,000; 70,000-300,000; 350,000-660,000; 380,000-900,000.

Description

A preparation method of chondroitin Technical Field

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

Background Art

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.

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.

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.

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.

Enzymatic synthesis of chondroitin utilizes specific enzyme-catalyzed reactions to synthesize chondroitin. Currently, researchers have developed two chondroitin synthases: P. multocida chondroitin synthase (PmCS) and E. coli K4 strain chondroitin synthase (KfoC). These enzymes primarily produce chondroitin by simultaneously transferring UDP-GalNAc and UDP-GlcA to an initial oligosaccharide substrate, alternating these reactions. Unlike previously described methods of natural extraction and microbial fermentation, 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 concentration, allows chondroitin to be synthesized 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%.

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.

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

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;

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.

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

Further, the initiator is selected from one or more of the following: GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (trisaccharide), GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (tetrasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (pentasaccharide), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (hexasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (heptose), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (octasaccharide), GalNAcβ1-4Galβ1-4GlcNAcαorβProN ₃ (GN-1), GalNAcβ1-4Galβ1-3Galβ1-4GlcNAcαorβProN ₃ (GN-2), GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc (CH4), and GalNAcβ1-4GlcAβ1-3GalNAcαorβProN ₃ (CH3).

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).

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

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.

Furthermore, 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 from Chondroitin synthase (PmCS).

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

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

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

Further, the initiator is selected from one or more of the following: GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (trisaccharide), GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (tetrasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (pentasaccharide), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (hexasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (heptose), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4 GlcNAcαorβProN ₃ (octasaccharide), GalNAcβ1-4Galβ1-4GlcNAcαorβProN ₃ (GN-1), GalNAcβ1-4Galβ1-3Galβ1-4GlcNAcαorβProN ₃ (GN-2), GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc (CH4), and GalNAcβ1-4GlcAβ1-3GalNAcαorβProN ₃ (CH3).

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.

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.

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

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).

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

Furthermore, 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 or chondroitin synthase (PmCS).

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 , and the initiator is selected from one or more of the following: GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (trisaccharide), GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (tetrasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (pentasaccharide), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (Hexasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (Heptasaccharide), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4 GlcNAcαorβProN ₃ (octasaccharide), GalNAcβ1-4Galβ1-4GlcNAcαorβProN ₃ (GN-1), GalNAcβ1-4Galβ1-3Galβ1-4GlcNAcαorβProN ₃ (GN-2), GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc (CH4) and GalNAcβ1-4GlcAβ1-3GalNAcαorβProN ₃ (CH3).

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

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, add glycosyltransferase and acetylated hexosamine to synthesize octasaccharide initiator.

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.

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.

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.

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).

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

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

In one embodiment, the intermediate M2 generated by adding an azide compound to the synthesized intermediate M1 is GlcNAcαorβProN ₃ .

Furthermore, the glycosyltransferase is selected from the group consisting of: 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 (Neisseria meningitidis α-1,4-galactosyltransferase, NmLgtC), Helicobacter pylori β-1,4-galactosyltransferase (Helicobacter pylori β-1 ,4-galactosyltransferase), human β-1,4-galactosyltransferase 7, bovine β-1,4-galactosyltransferase, Escherichia coli O55:H7 β-1,3-galactosyltransferase, Campylobacter jejuni β-1,3-galactosyltransferase, Chromobacterium violac eumβ-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, Bacteroides family 6 saccharide One or more of the following: α-1,3-N-acetylgalactosaminyl transferase from Helicobacter mustelae (α-1,3-N-acetylgalactosaminyl transferase, BgtA), β-1,4-acetylgalactosaminyl transferase from Campylobacter jejuni (β-1,4-Nacetylgalactosaminyl transferase, CgtA) and β-1,4-acetylgalactosaminyl transferase from bovine (β-1,4-Nacetylgalactosaminyl transferase).

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.

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.

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 (PmCS) and Escherichia coli K4 from Chondroitin synthase (KfoC), to obtain a pentasaccharide initiator.

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.

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.

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 (PmCS) and Escherichia coli K4 from Chondroitin synthase (KfoC) to obtain an octasaccharide initiator.

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.

The initiator is selected from one or more of the following: GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (trisaccharide), GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (tetrasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (pentasaccharide), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (hexasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (heptose), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (octasaccharide), GalNAcβ1-4Galβ1-4GlcNAcαorβProN ₃ (GN-1), GalNAcβ1-4Galβ1-3Galβ1-4GlcNAcαorβProN ₃ (GN-2), GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc (CH4), and GalNAcβ1-4GlcAβ1-3GalNAcαorβProN ₃ (CH3).

Furthermore, 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 from Chondroitin synthase (PmCS).

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

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).

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

In one embodiment of the present invention, the reaction package uses trisaccharide (GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ ) as an initiator; the polymerase 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) 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.

In one embodiment of the present invention, the reaction package uses GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (tetrasaccharide) as an initiator; its polymerase is one or more of Escherichia coli K4 from Chondroitin synthase (E. coli K4 from Chondroitin synthase, KfoC) and Pasteurella multocida Type F Chondroitin synthase (Pasteurella multocida 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.

In one embodiment of the present invention, the reaction package uses (pentasaccharide) 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-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.

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.

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.

In one embodiment of the present invention, when GalNAcβ1-4Galβ1-4GlcNAcαorβProN ₃ (GN-1) is used as a chondroitin polymerization initiator, the catalytic enzyme used includes a combination of E. coli K4 from Chondroitin synthase (KfoC) and 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.

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

In one embodiment of the present invention, when GalNAcβ1-4GlcAβ1-3GalNAcαorβProN ₃ (CH3) is used as a chondroitin polymerization initiator, the catalytic enzyme used includes a combination of E. coli K4 from Chondroitin synthase (KfoC) and Pasteurella multocida Type F Chondroitin synthase (PmCS), and the chondroitin that can be formed is represented by Formula VIII; wherein j includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000.

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 from Chondroitin synthase, KfoC) and Pasteurella multocida Type FChondroitin 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.

Beneficial effects

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

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

Figure 2 shows the results of polyacrylamide gel electrophoresis of engineered sugar synthesis-related enzymes (1: Protein Maker; 2: NmLgtB; 3: GlcAT-P; 4: PmCS).

FIG3 is a process flow chart of receptor molecules and chondroitin.

FIG4 is a GlcNAcαProN ₃ ¹ H NMR (D ₂ O) spectrum.

FIG5 is a graph showing ^{the 1H} _NMR ( _D2O ) spectrum of Galβ1-4GlcNAcαProN3.

FIG6 is a GlcAβ1-3Galβ1-4GlcNAcαProN ₃ ¹ H NMR (D ₂ O) spectrum.

FIG7 is a graph showing the N 3 1 H NMR (D 2 O) spectrum of GalNAcβ1-4GlcAβ1-3GalNAcβProN ₃ ¹ H NMR (D ₂ O) spectrum.

FIG8 is a graph showing the N 3 1 H NMR (D 2 O) spectrum of GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN ₃ ¹ H NMR (D ₂ O) spectrum.

FIG9 is ^{a 1} H NMR (D ₂ O) spectrum of GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc.

Figure 10 shows the difference analysis of sugar pool binding energy.

Figure 11 shows the polymerization curves of chondroitin with different molecular weights prepared by using different concentrations of tetrasaccharide initiators at different feed ratios. a: [I ₀ ] = 20 μM, [M ₀ ]/[I ₀ ] = 50-400; b: [I ₀ ] = 10 μM, [M ₀ ]/[I ₀ ] = 50-800, linear correlation R ² = 0.998; c: [I ₀ ] = 1 μM, [M ₀ ]/[I ₀ ] = 1000-3500, linear correlation R ² = 0.997.

FIG12 is a linear relationship diagram between feed ratio and output, with a linear correlation R ² =0.990.

Figure 13 shows the elution curves of chondroitin with different molecular weights prepared by polymerization with different concentrations of trisaccharide initiator at different feed ratios. a: [I ₀ ] = 10 μM, [M ₀ ]/[I ₀ ] = 50-500, linear correlation R ² = 0.990; b: [I ₀ ] = 1 μM, [M ₀ ]/[I ₀ ] = 800-3000, linear correlation R ² = 0.992.

Figure 14 shows the preparation of block chondroitin with different block patterns and molecular weights under different comonomers, as well as their elution curves. a: Comonomers UDP-GalNAc and UDP-GalNAz, tetrablock; b: Comonomers UDP-GalNAc and UDP-GalNTFA, tetrablock; c: Comonomers UDP-GalNAc, UDP-GalNAz, and UDP-GalNTFA, mixed block. Green: Comonomer UDP-GalNAc; Orange: Comonomer UDP-GalNAz; Blue: Comonomer UDP-GalNTFA.

DETAILED DESCRIPTION

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.

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.

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.

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.

In one embodiment of the present invention, when trisaccharide (GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ ) is used as the 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.

In one embodiment of the present invention, when GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (tetrasaccharide) is used as a chondroitin polymerization initiator, the catalytic enzyme used includes a combination of E. coli K4 from Chondroitin synthase (KfoC) and 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.

In one embodiment of the present invention, when GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (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), and the like. 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.

In one embodiment of the present invention, when GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (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), and the like. 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.

In one embodiment of the present invention, when GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (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 GlcAβ1-3GalNAcβ1-4GlcNAcαorβProN 3 (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.

In one embodiment of the present invention, when GalNAcβ1-4Galβ1-4GlcNAcαProN ₃ (GN-1) is used as a chondroitin polymerization initiator, the catalytic enzyme used includes a combination of E. coli K4 from Chondroitin synthase (KfoC) and 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.

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

In one embodiment of the present invention, when GalNAcβ1-4GlcAβ1-3GalNAcProN ₃ (CH3) is used as a chondroitin polymerization initiator, the catalytic enzyme used includes a combination of E. coli K4 from Chondroitin synthase (KfoC) and Pasteurella multocida Type FChondroitin synthase (PmCS), and the chondroitin that can be formed is represented by the following formula VIII; wherein j includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000.

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 from Chondroitin 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.

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

Example 1 Expression and purification of sugar synthesis related enzymes

1.1 Expression and purification of sugar synthesis-related enzymes

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

Among them, BlNahK is N-Acetylhexosamine 1-kinase from Bifidobacterium longum;

AGX1 is a human UDP-N-acetylgalactosamine pyrophosphorylase (UDP-N-acetylgalactosamine pyrophosphorylase from Homo sapiens);

AtGlcAK is glucuronokinase from Arabidopsis thaliana;

AtUSP is UDP-sugar pyrophosphorylase from Arabidopsis thaliana;

BiGalK is galactokinase from Bifidobacterium infantis;

PmPPA is inorganic pyrophosphatase from Pasteurella multocida;

NmLgtB is β1-4 galactosyltransferase from Neisseria meningitidis;

GlcAT-P is Glucuronyltransferase-P from Mus musculus;

PmCS is chondroitin synthase from Pasteurella multocida.

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

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

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

After sequencing verification of the relevant sugar synthase plasmids listed in Table 1, they were transformed into competent BL21(DE3) cells and plated onto culture dishes containing LB solid medium. After inverted culture overnight at 37°C in an incubator, single clones were selected and cultured on a small scale in 100 mL of LB liquid medium containing 100 μg/mL of the corresponding antibiotic at 37°C and 250 rpm. After 8-12 hours of culture and an _OD600 of approximately 1, 10 mL of the turbid bacterial solution was transferred to 1 L of LB medium containing 100 μg/mL of antibiotic for large-scale induction of expression at 37°C and 250 rpm. After incubation at 37°C to an _OD600 of 0.6-0.8, the temperature was lowered to 16°C, and the appropriate IPTG was added for induction for 20 hours.

(3) The above-mentioned induction bacterial solution was centrifuged at 4000 rpm for 20 minutes for collection, 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 high-pressure sterilization 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 eluted sugar synthesis-related enzymes were collected by monitoring UV ₂₈₀ absorbance. The purified samples were tested for purity by polyacrylamide gel electrophoresis (SDS-PAGE), and the protein concentration was determined by Bradford method.

1.2 Experimental Results

As shown in Figures 1 and 2, 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.

Example 2 Enzymatic Synthesis of Receptor Molecules

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 receptor molecules such as trisaccharides and tetrasaccharides 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 controllably synthesized. The process flow chart is shown in Figure 3.

2.1 Preparation of GlcNAcαProN3 _receptor molecules

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. The salt was removed by filtration, and the supernatant was separated and purified by silica gel column chromatography, eluted with polar dichloromethane:methanol = 5:1, and the fractions containing GlcNAcαProN ₃ were collected and spin-dried, and weighed. ¹ H NMR (400 MHz, D ₂ O) ( FIG4 ).

2.2 Enzymatic Synthesis of Galβ1-4GlcNAcαProN ₃ (Disaccharide)

UDP-Gal is prepared by BiGalK, AtUSP and PmPPA, and then transferred to the GlcNAcαProN ₃ end by NmLgtB to obtain Galβ1-4GlcNAcαProN ₃ (disaccharide).

The specific reaction system is as follows: 100 mM Tris-HCl pH 8.0, 20 mM MgCl ₂ , GlcNAcProN ₃ , 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.

The reaction was analyzed by TLC spot plate. 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 consisting of ethyl acetate:methanol:water. The target fractions were pooled and dried, and then purified on a BioGel P-2 size exclusion column. The target fractions were pooled to obtain the pure product _of Galβ1-4GlcNAcαProN3, as shown by ^1H NMR (400 MHz, _D2O ) (Figure 5).

2.3 Enzymatic Synthesis of GlcAβ1-3Galβ1-4GlcNAcαProN ₃ (Trisaccharide)

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

The specific reaction system is as follows: 100 mM Tris-HCl (pH 8.0), 20 mM MgCl ₂ , Galβ1-4GlcNAcαProN ₃ , 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. Incubate at 37°C for 15 min. Add the appropriate amounts of AtGlcAK, AtUSP, PmPPA, and GlcAT-P, and adjust the volume to 30 mL. Stir slowly at 80 rpm at 37°C. TLC analysis is performed. Upon completion, quench the reaction by adding an equal volume of glacial ethanol. The product was separated and purified by silica gel column chromatography using ethyl acetate: methanol: water as the eluents 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 GlcAβ1-3Galβ1-4GlcNAcαProN ₃ , ¹ H NMR (400 MHz, D ₂ O) ( FIG6 ).

2.4 Enzymatic Synthesis of GalNAcβ1-4GlcAβ1-3GalNAcβProN ₃ (CH3)

GlcA was transferred to the _3- terminus of GalNAcβProN by PmCS to obtain GlcAβ1-3GalNAcβProN _3. Similarly, GalNAc was transferred to the _3- terminus of GlcAβ1-3GalNAcβProN by PmCS to obtain the final GalNAcβ1-4GlcAβ1-3GalNAcβProN ₃ .

The specific reaction system is as follows:

Synthesis of GlcAβ1-3GalNAcβProN ₃ : 100 mM Tris-HCl pH 8.0, 20 mM MgCl ₂ , GalNAcProN ₃ , 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.

The reaction was analyzed by TLC spot plate. After completion, an equal volume of icy ethanol was added to quench the reaction. The residue 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 to obtain GlcAβ1-3GalNAcβProN ₃ .

Synthesis of GalNAcβ1-4GlcAβ1-3GalNAcβProN ₃ : 100 mM Tris-HCl pH 8.0, 20 mM MgCl ₂ , GlcAβ1-3GalNAcProN ₃ , UDP-N-acetylgalactosamine (UDP-GalNAc, 1.3 eq), a certain amount of PmCS were added, and the reaction was stirred slowly at 80 rpm at 37°C.

The reaction was analyzed by TLC spot plate. 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 consisting of ethyl acetate:methanol:water. The target fractions were pooled and dried, and then purified on a BioGel P-2 size exclusion column. The target fractions were pooled to obtain the final pure product of GalNAcβ1-4GlcAβ1-3GalNAcβProN ₃ , as shown by ¹ H NMR (400 MHz, D ₂ O) (Figure 7).

2.5 Enzymatic Synthesis of GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN ₃ (Tetrasaccharide)

UDP-GalNAc is transferred to the _3- terminus of GlcAβ1-3Galβ1-4GlcNAcαProN by PmCS to obtain GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN ₃ .

The specific reaction system is as follows: 100 mM Tris-HCl pH 8.0, 20 mM MgCl ₂ , GlcAβ1-3Galβ1-4GlcNAcαProN ₃ , 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.

The reaction was analyzed by TLC spot plate. 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 consisting of ethyl acetate:methanol:water. The target fractions were pooled and dried, and then purified on a BioGel P-2 size exclusion column. The target fractions were pooled to obtain the final pure product of GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN ₃ , as shown by ¹ H NMR (400 MHz, D ₂ O) (Figure 8).

2.6 Preparation of GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc(CH4) by enzymatic degradation

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

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, filtered through celite, and the filtrate was mixed with ethanol and concentrated by rotary evaporation. Ethanol was gradually added dropwise to the concentrate. The resulting white precipitate was filtered and vacuum-dried overnight to obtain the crude product. The product was then separated using Sephadex LH-20 and AG 1-X4 resin (200-400 mesh, analytical grade), respectively. 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). ^1H NMR (400 MHz, _D2O ) (Figure 9).

2.7 Enzymatic synthesis of other receptor molecules

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

(1) Pentasaccharide

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

(2) Hexasaccharide

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

(3) Seven sugars

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 (GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN ₃ ); molecular weight is 1401.2 g/mol; chemical structure is shown in γ,

(4) Octasaccharide

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 (GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN ₃ ); molecular weight 1604.398 g/mol, chemical structure shown in δ;

(5)GN-1

N-acetylgalactosamine-β1-4-galactosamine-β1-4-N-acetylglucosamine-α-propyl azide (GalNAcβ1-4Galβ1-4GlcNAcαProN ₃ ); molecular weight 669.63 g/mol; chemical structure shown in ε;

(6)GN-2

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

2.8 Receptor molecule screening

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

As shown in FIG10 , when the binding energy is ±30 kcal·mol ^-1 , it is suitable as an initiator for chondroitin synthesis.

Example 3 Controllable Chondroitin Linear Polymerization

The polymerization reaction of chondroitin is catalyzed by PmCS polymerase, using GlcAβ1-3Galβ1-4GlcNAcαProN ₃ (trisaccharide) or GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN ₃ (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 glycans. The reaction process is shown in the following R1 synthesis route;

GlcAβ1-3Galβ1-4GlcNAcαProN ₃ (trisaccharide) and GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN ₃ (tetrasaccharide) were used as initiators (I ₀ ), and uridine 5′-diphospho-glucuronic acid (UDP-GlcA) and uridine 5′-diphospho-N-acetylgalactosamine (UDP-GalNAc) were used as comonomers (M ₀ ). By controlling the ratio of the two comonomers to the initiator ([M ₀ ]/[I ₀ ]), the ratio range mainly included 50-4000, thereby accurately preparing chondroitin with different molecular weights.

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

Chondroitin with varying molecular weights was precisely prepared by controlling the ratio of the two comonomers to the initiator ([M 0 ]/[I _{0 ]) using 20 μM, 10 μM, and 1 μM GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN 3 as the initiator (I 0 ) ,} uridine 5′-diphospho-glucuronic acid (UDP-GlcA) and uridine _5′ -diphospho-N-acetylgalactosamine (UDP- _GalNAc ), respectively. The synthetic route is shown in R2. The molecular weight and dispersibility of the prepared chondroitin were analyzed by SEC-MALLS-RI. The analytical column was a TSKgel GMPWXL column (13 μm, 7.8 x 300 mm, Tosoh Corporation), and the mobile phase was 0.1 M NaNO ₃ and 0.05 M Na ₂ HPO ₄ . The prepared chondroitin was analyzed at 25°C at 0.6 mL/min, and the data were processed using ASTRA software.

The experimental results are shown in Figure 11. When I ₀ = 20 μM, as the feed ratio increases, the linear correlation of the molecular weight is smaller (R ² = 0.922), and the molecular weight can only reach a maximum of 315 kDa, and then the molecular weight will not continue to increase; when I ₀ = 10 μM, the linear correlation is significantly enhanced (R ² = 0.998), but the molecular weight can only reach 290 kDa; when I ₀ = 1 μM, while ensuring a strong linear correlation, the molecular weight of chondroitin reaches 660 kDa, and the molecular weight dispersity (D) is extremely narrow, D = 1.002.

As shown in FIG12 , the yield of the prepared chondroitin increases linearly with the increase of the feed ratio.

Table 1 Synthesis of chondroitin using tetrasaccharide (GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN ₃ ) as initiator

a:M _{n, measured} and The average result of four parallel experiments

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

A series of chondroitin molecular weights were prepared using GlcAβ1-3Galβ1-4GlcNAcαProN ₃ (trisaccharide molecule) as an initiator using the same preparation and analysis methods as those for GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN ₃ (tetrasaccharide molecule).

20 μM, 10 μM and 1 μM GlcAβ1-3Galβ1-4GlcNAcαProN ₃ were used as initiators (I ₀ ), uridine 5′-diphospho-glucuronic acid (UDP-GlcA) and uridine 5′-diphospho-N-acetylgalactosamine (UDP-GalNAc) were used as comonomers (M ₀ ), and the ratio of the two comonomers to the initiator ([M ₀ ]/[I ₀ ]) was controlled, with the ratio ranging from 50 to 4000, to accurately prepare chondroitin with different molecular weights. The synthetic route is shown in R3.

The molecular weight and dispersity of the prepared chondroitin were analyzed by SEC-MALLS-RI. The analytical column was TSKgel GMPWXL column (13 μm, 7.8*300 mm, Tosoh Corporation). The mobile phase was 0.1 M NaNO ₃ , 0.05 M Na ₂ HPO ₄ . The prepared chondroitin was analyzed at 0.6 mL/min at 25°C. The data were processed using ASTRA software.

As shown in Figure 13, when the trisaccharide initiator concentration is 10 μM, the number average molecular weight (M _n ) increases linearly with increasing feed, from 350 kDa to 575 kDa. When the trisaccharide initiator concentration is 1 μM, the molecular weight increases linearly, reaching a maximum of 632 kDa. Furthermore, the molecular weight dispersity (D) of the chondroitin exhibits a narrow distribution, close to 1 and no higher than 1.1.

3.4 Preparation of multi-block chondroitin using GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN ₃ (tetrasaccharide) as initiator

Similar to the preparation and analysis methods of GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN ₃ (tetrasaccharide molecule), by changing the type of comonomer, without changing UDP-GlcA, UDP-GalNAc and its derivatives, including UDP-GalNAz and UDP-GalNTFA, were used as comonomers to prepare a series of chondroitins with different block patterns.

10 μM GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαProN ₃ was used as the initiator (I ₀ ), 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 (M ₀ ).

As shown in Figure 14a, the ratio of comonomers (UDP-GlcA and UDP-GalNAc) to initiator was controlled to 200, yielding a chondroitin with a GlcA and GalNAc backbone (M _n = 76.8 kDa). After ensuring the complete reaction of UDP-GalNAc, the comonomer UDP-GalNAz was added at a ratio of 50 to initiator, yielding a monoblock chondroitin (M _n = 92.8 kDa). Using a similar strategy, a chondroitin tetrablock with GalNAz (M _n = 236.8 kDa) was obtained. Similar strategies can also be used to prepare other multiblock chondroitins. Figure 14b shows a chondroitin with a GalNTFA block, while Figure 14c shows a chondroitin with both GalNAz and GalNTFA blocks.

Claims (11)

A chondroitin is disclosed. 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 from acetylated hexosamine and uronic acid via 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. The range of n includes 25-15000, 50-12000, 50-800, 100-10000, 150-8000, 200-6000, 250-3000 and 1000-4000. The chondroitin according to claim 1, characterized in that the initiator is selected from one or more of the following: GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (trisaccharide), GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (tetrasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (pentasaccharide), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (hexasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (heptose), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4 GlcNAcαorβProN ₃ (octasaccharide), GalNAcβ1-4Galβ1-4GlcNAcαorβProN ₃ (GN-1), GalNAcβ1-4Galβ1-3Galβ1-4GlcNAcαorβProN ₃ (GN-2), GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc (CH4), and GalNAcβ1-4GlcAβ1-3GalNAcαorβProN ₃ (CH3). The chondroitin according to claim 1, characterized in that 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 -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 acetylglucosamine) One or more. The chondroitin according to claim 1, wherein the uronic acid can be selected from glucuronic acid and/or UDP-GlcA (uridine-5'-diphosphoglucuronic acid trisodium salt). The chondroitin according to claim 1, characterized in that the molecular weight range of the chondroitin includes 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. The chondroitin according to claim 1, characterized in that the polymerase is selected from one or more of Pasteurella multocida heparosan synthase 2 (PmHS2), Escherichia coli K4-derived chondroitin synthase (KfoC), Pasteurella multocida-derived hyaluronic acid synthase (PmHAS) and Pasteurella multocida-derived chondroitin synthase (PmCS). A method for synthesizing chondroitin; the method comprises the following steps: S1. Acetylated hexosamine and uronic acid bonded nucleotides form comonomers; S2. The comonomer and the initiator generate polysaccharide, namely the chondroitin of the present invention, through polymerization reaction catalyzed by polymerase. Wherein, the initiator is selected from one or more of the following: GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (trisaccharide), GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (tetrasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (pentasaccharide), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (hexasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (heptose), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4 GlcNAcαorβProN ₃ (octasaccharide), GalNAcβ1-4Galβ1-4GlcNAcαorβProN ₃ (GN-1), GalNAcβ1-4Galβ1-3Galβ1-4GlcNAcαorβProN ₃ (GN-2), GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc (CH4), and GalNAcβ1-4GlcAβ1-3GalNAcαorβProN ₃ (CH3). 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. An initiator for synthesizing chondroitin, wherein the binding energy of the initiator is ±30 kcal·mol ^-1 , and the initiator is selected from one or more of the following: GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (trisaccharide), GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (tetrasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (pentasaccharide), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (hexasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN 3 ( ₃ (heptose), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4 GlcNAcαorβProN ₃ (octasaccharide), GalNAcβ1-4Galβ1-4GlcNAcαorβProN ₃ (GN-1), GalNAcβ1-4Galβ1-3Galβ1-4GlcNAcαorβProN ₃ (GN-2), GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc (CH4), and GalNAcβ1-4GlcAβ1-3GalNAcαorβProN ₃ (CH3). A method for preparing an initiator, comprising the following steps: 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, add glycosyltransferase and acetylated hexosamine to synthesize octasaccharide initiator. 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, wherein the initiator is selected from one or more of the following: GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (trisaccharide), GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (tetrasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (pentose), GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (hexasaccharide), GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4GlcNAcαorβProN ₃ (heptasaccharide) and GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3Galβ1-4 GlcNAcαorβProN ₃ (octasaccharide); GalNAcβ1-4Galβ1-4GlcNAcαorβProN ₃ (GN-1), GalNAcβ1-4Galβ1-3Galβ1-4GlcNAcαorβProN ₃ (GN-2); GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc(CH4), GalNAcβ1-4GlcAβ1-3GalNAcαorβProN ₃ (CH3); the polymerase is selected from: Pasteurella multocida heparosan synthase 2 2, PmHS2), Escherichia coli K4 from Chondroitin synthase (E. coli K4 from Chondroitin synthase, KfoC), Pasteurella multocida HA synthase (Pasteurella multocida HA synthase, PmHAS) and Pasteurella multocida from chondroitin synthase (Chondroitin synthase from Pasteurella multocida, PmCS) One or more; the comonomer is composed of acetylated hexosamine and uronic acid connected by a glycosidic bond.