Process for co-producing chondroitin sulfate and glucosamine through enzymolysis

Abstract

The invention relates to the technical field of biological fermentation, in particular to a process for co-producing chondroitin sulfate and glucosamine through enzymolysis. The method comprises the following steps: preparing a homogeneous slurry from a vegetable protein raw material; preparing a seed solution; carrying out enzymolysis on the vegetable protein slurry; inoculating a microbial seed solution for coupling culture; after the reaction is finished, carrying out solid-liquid separation, carrying out alcohol precipitation on the mixed supernate, and precipitating to obtain a chondroitin sulfate crude product; recovering the residual solution, and crystallizing to obtain glucosamine crystals; and refining the chondroitin sulfate crude product to obtain a high-purity product. According to the method, plant protein enzymolysis and microbial fermentation are innovatively coupled in the same system, an enzymolysis product is used for providing nutrition for fermentation, engineering bacteria are driven to efficiently synthesize non-animal-source chondroitin sulfate, and meanwhile, synchronous conversion and accumulation of a plant-source glucosamine precursor are achieved; and finally, high-purity chondroitin sulfate and glucosamine are simultaneously obtained from the same system.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation technology, and more specifically, to a process for the enzymatic hydrolysis of chondroitin sulfate and glucosamine. Background Technology

[0002] Bio-fermentation technology refers to a process that uses microbial metabolites, such as amino acids, keto acids, lactic acid, and acetic acid, to produce useful chemicals, pharmaceuticals, food, and energy from waste, wastewater, or inexpensive materials.

[0003] While existing bio-fermentation technologies can obtain glucosamine from plant proteins through enzymatic hydrolysis, they cannot simultaneously produce chondroitin sulfate. Traditional chondroitin sulfate extraction relies entirely on cartilage from animals such as cattle, pigs, and sharks, posing a risk of animal-derived diseases. Furthermore, enzymatic hydrolysis using a mixture of animal and plant materials is essentially a physical mixture, not a true biosynthesis, and inherently suffers from conflicting process conditions, complex impurities, and separation difficulties.

[0004] In recent years, the production of chondroitin sulfate via microbial fermentation has become an important development direction. The principle involves genetically engineering microorganisms to express key enzyme systems for chondroitin sulfate synthesis from animal sources, thereby directly synthesizing chondroitin sulfate using carbon sources such as sugars.

[0005] However, existing fermentation technologies mostly focus on the production of single products, and there are no reports of deep integration of the fermentation process for producing non-animal chondroitin sulfate with the process for producing glucosamine from plant enzymes, achieving efficient and economical co-production in the same system. Summary of the Invention

[0006] The purpose of this invention is to solve the problem in the prior art that it is impossible to efficiently co-produce chondroitin sulfate and glucosamine in a single, non-animal source system.

[0007] The purpose of this invention is to provide a process for the enzymatic hydrolysis and co-production of chondroitin sulfate and glucosamine, constructing an integrated system that couples plant protein hydrolysis with microbial fermentation. First, through enzymatic hydrolysis of plant protein, glucosamine precursor and nitrogen source required for microbial growth are produced. Simultaneously, genetically engineered microbial strains capable of synthesizing chondroitin sulfate are introduced, and fermentation is carried out using nutrients in the enzymatic hydrolysate and an added carbon source to directly synthesize non-animal-derived chondroitin sulfate. Finally, two high-purity products are separated and purified from the same reaction system.

[0008] To achieve the above objectives, the present invention aims to provide a process for the enzymatic hydrolysis of chondroitin sulfate and glucosamine, comprising the following steps: Step S1: Prepare the plant protein raw material into a homogeneous slurry; at the same time, activate and culture genetically engineered microorganisms with the ability to synthesize chondroitin sulfate to prepare a high-density seed liquid; Formulate proteases for the hydrolysis of plant proteins; Step S2: Place the homogenized plant protein slurry in a temperature- and pH-controlled reactor, add protease for enzymatic hydrolysis, and provide a nitrogen source and glucosamine precursor for microbial fermentation; when the enzymatic hydrolysis reaches a specific stage, inoculate the above-mentioned microbial seed liquid into the system and supplement the fermentation carbon source, adjusting the conditions to suitable fermentation conditions for microbial growth and chondroitin sulfate synthesis, and carry out coupled fermentation; at this stage, the microorganisms use the substrate in the system to synthesize and secrete chondroitin sulfate, while the enzymatic hydrolysis of plant protein and microbial metabolism continue; Step S3: After the coupling process is completed, sterilization is performed to terminate all biological reactions. Then, solid-liquid separation is performed to remove bacterial cells and incompletely hydrolyzed solids, resulting in a mixed supernatant containing chondroitin sulfate, glucosamine, peptides, and sugars. Then, impurities and colloidal substances in the mixed supernatant are removed. Step S4: The mixed supernatant after preliminary purification is subjected to gradient alcohol precipitation to obtain crude chondroitin sulfate. The remaining alcohol solution is recycled to obtain a mother liquor rich in glucosamine and its precursors. The mother liquor is then decolorized with activated carbon, desalted with ion exchange resin, concentrated and crystallized to obtain glucosamine crystals. The crude chondroitin sulfate is purified by dissolving, reprecipitating and drying to obtain the final product.

[0009] As a further improvement to this technical solution, in step S1, the plant protein raw material is crushed and hydrated to prepare a homogeneous slurry, and the plant protein raw material is one or more of soybean, wheat, peanut, cottonseed or rapeseed protein powder; the genetically engineered microorganism is an engineered Escherichia coli, Bacillus subtilis or Pichia pastoris with an animal-derived chondroitin sulfate synthase gene introduced into it.

[0010] As a further improvement to this technical solution, in step S1, the protease is one or more of alkaline protease, neutral protease, or complex plant hydrolase.

[0011] As a further improvement to this technical solution, in step S2, the temperature of the enzymatic hydrolysis stage is 45-55℃, the pH is 6.5-7.5, and the time is controlled to be 1-3h. The temperature during the coupled fermentation stage is 30-37℃ or 28-30℃, the pH is maintained at 6.8-7.4, and the fermentation time is controlled at 24-72h.

[0012] As a further improvement to this technical solution, in step S2, the fermentation carbon source is one of glucose, sucrose, or corn syrup.

[0013] As a further improvement to this technical solution, in step S3, the sterilization process involves heating the reaction system to 121°C and maintaining it for 15-30 minutes, or using pasteurization.

[0014] As a further improvement to this technical solution, in step S3, isoelectric point sedimentation, heat treatment, or membrane filtration techniques are used to remove impurities and colloidal substances from the mixed supernatant.

[0015] As a further improvement to this technical solution, in step S4, the gradient alcohol precipitation specifically involves adding ethanol to the mixed supernatant to make the final concentration of the system 50-70%, precipitating and separating the crude chondroitin sulfate.

[0016] As a further improvement to this technical solution, in step S4, after recovering ethanol from the remaining alcohol solution, a mother liquor is obtained. Then, the mother liquor is subjected to activated carbon decolorization, ion exchange resin desalting, and concentration crystallization steps to obtain glucosamine crystals.

[0017] As a further improvement to this technical solution, in step S4, the purification of crude chondroitin sulfate involves dissolving the crude product in water, removing small molecule impurities by ultrafiltration, followed by secondary precipitation with ethanol and freeze-drying to obtain a high-purity chondroitin sulfate product.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: In this enzymatic hydrolysis process for the co-production of chondroitin sulfate and glucosamine, plant protein slurry is first used as the starting material. Enzymatic hydrolysis is carried out by protease, which efficiently degrades the plant protein network into abundant peptides, amino acids, and key precursor substances such as N-acetylglucosamine under mild conditions. Subsequently, genetically engineered microbial seed liquid is introduced, and the precursor substances in the enzymatic hydrolysate and an external carbon source are used to carry out growth metabolism and de novo biosynthesis of chondroitin sulfate, which is then secreted into the extracellular space. This coupled process fundamentally solves the problem of low efficiency caused by the significant differences in the properties of animal and plant raw materials and the conflicting optimal reaction conditions in traditional processes. Furthermore, in the subsequent separation, the inherent differences in the physicochemical properties of chondroitin sulfate and glucosamine are utilized to achieve efficient separation and purification through steps such as gradient alcohol precipitation and crystallization. Ultimately, it enables the efficient and high-purity co-production of non-animal chondroitin sulfate and glucosamine from a single plant-based starting material within a single system, offering advantages such as pure raw material sources, high process integration, and wide product applicability. Attached Figure Description

[0019] Figure 1 This is a flowchart of the present invention; Figure 2 A schematic diagram showing the yield efficiency of co-producing chondroitin sulfate in the process when the temperature during the enzymatic hydrolysis stage is different. Figure 3This diagram illustrates the yield of glucosamine produced by the co-production process when the temperature during the enzymatic hydrolysis stage is different. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Please see Figure 1 As shown, the purpose of this invention is to provide a process for the enzymatic hydrolysis of chondroitin sulfate and glucosamine, comprising the following steps: Step S1: Raw material preparation and strain activation: The plant protein raw materials are crushed and hydrated to prepare a homogeneous slurry. The plant protein raw materials are derived from one or more of the protein powders such as soybean, wheat, peanut, cottonseed, and rapeseed. At the same time, genetically engineered microorganisms with the ability to synthesize chondroitin sulfate are activated and cultured to prepare a high-density seed liquid. Among them, the genetically engineered microorganisms are engineered Escherichia coli, Bacillus subtilis, or Pichia pastoris with the introduction of animal-derived chondroitin sulfate synthase genes.

[0022] Formulate a protease for the hydrolysis of plant proteins. Specifically, the protease is one or more of alkaline protease, neutral protease, or complex plant hydrolase. Step S2: Coupling Enzymatic Hydrolysis and Fermentation: The homogenized plant protein slurry is placed in a temperature- and pH-controlled reactor, and protease is added for enzymatic hydrolysis, providing a nitrogen source and glucosamine precursor for microbial fermentation. When the enzymatic hydrolysis reaches a specific stage, the above-mentioned microbial seed liquid is inoculated into the system, and a fermentation carbon source (one of glucose, sucrose, or corn syrup) is added. The conditions are adjusted to suitable fermentation conditions for microbial growth and chondroitin sulfate synthesis, and coupled fermentation is carried out. In this stage, the microorganisms use the substrate in the system to synthesize and secrete chondroitin sulfate, while the enzymatic hydrolysis of plant protein and microbial metabolism continue. It is worth noting that the temperature for the enzymatic hydrolysis stage is 45-55℃, the pH is 6.5-7.5, and the time is controlled at 1-3h; the temperature for the coupled fermentation stage is 30-37℃ (for bacteria) or 28-30℃ (for yeast), the pH is maintained at 6.8-7.4, and the fermentation time is controlled at 24-72h.

[0023] Step S3: Termination of reaction and preliminary separation: After the coupling process is completed, sterilization is performed to terminate all biological reactions. Then, solid-liquid separation is performed to remove bacterial cells and incompletely hydrolyzed solids, resulting in a mixed supernatant containing chondroitin sulfate, glucosamine, peptides and sugars. Then, isoelectric point sedimentation, heat treatment or membrane filtration technology is used to remove impurities and colloidal substances from the mixed supernatant. Sterilization is performed by heating the reaction system to 121°C and holding it for 15-30 minutes, or by pasteurization.

[0024] Step S4: Separation and purification of the product: The mixed supernatant after preliminary purification is subjected to gradient alcohol precipitation. Specifically, ethanol is added to the mixed supernatant to make the final concentration of the system 50-70%, and crude chondroitin sulfate is precipitated and separated. The crude chondroitin sulfate is obtained by precipitation first. After recovering the ethanol from the remaining alcohol solution, a mother liquor rich in glucosamine and its precursor is obtained. Then, the mother liquor is subjected to activated carbon decolorization, ion exchange resin desalting, concentration and crystallization steps to obtain glucosamine crystals. The purification of crude chondroitin sulfate involves dissolving the crude product in water, removing small molecule impurities by ultrafiltration, followed by secondary precipitation with ethanol and freeze drying to obtain high-purity chondroitin sulfate product.

[0025] The following specific examples further illustrate the process for the enzymatic hydrolysis and co-production of chondroitin sulfate and glucosamine provided by the present invention.

[0026] Example 1 This embodiment provides a process for the enzymatic hydrolysis of chondroitin sulfate and glucosamine, including the following steps: Step S1: Crush soybean protein powder, mix it with water and homogenize it to prepare a plant protein slurry. Simultaneously, activate engineered E. coli strains infused with the chondroitin sulfate synthase gene in LB medium, then transfer them to a seed tank for large-scale cultivation to prepare a high-density seed culture. Prepare an alkaline protease solution.

[0027] Step S2: The plant protein slurry was placed in a fermenter, and alkaline protease was added. Enzymatic hydrolysis was carried out at 50°C and pH 7.0 for 2 hours. Subsequently, *E. coli* seed culture was inoculated into the tank, and glucose was added as the main carbon source. The tank temperature was adjusted to 37°C, and the pH was controlled at 7.0 by adding ammonia water. Aerobic fermentation was carried out for 48 hours. During this process, microorganisms grew rapidly and synthesized chondroitin sulfate, which was secreted into the fermentation broth. Simultaneously, the plant protein was continuously degraded.

[0028] Step S3: After fermentation, heat the fermentation broth to 121°C and maintain for 20 minutes for sterilization. After cooling, remove the cells and residue by centrifugation to obtain the supernatant. Use an ultrafiltration membrane system to remove most of the impurities and proteins from the supernatant.

[0029] Step S4: Add ethanol to the supernatant to achieve a final concentration of 65%, and allow it to stand to precipitate. Centrifuge to obtain crude chondroitin sulfate. Distill the ethanol solution to recover the ethanol, obtaining the mother liquor. The mother liquor is then subjected to activated carbon decolorization, ion exchange resin desalting, vacuum concentration, and finally cooled crystallization to obtain glucosamine hydrochloride crystals. The crude chondroitin sulfate is then reconstituted, ultrafiltered to remove impurities, reprecipitated with ethanol, and freeze-dried to obtain high-purity chondroitin sulfate powder.

[0030] Example 2 This embodiment provides a process for the enzymatic hydrolysis of chondroitin sulfate and glucosamine, including the following steps: Step S1: Prepare homogenized slurry from peanut protein powder and wheat protein powder. Simultaneously, activate and culture genetically engineered Pichia pastoris capable of synthesizing chondroitin sulfate to prepare seed culture. Prepare neutral protease and complex plant hydrolase.

[0031] Step S2: Place the protein slurry in a reactor, add a compound hydrolytic enzyme, and hydrolyze for 1.5 hours at 48°C and pH 6.8. Then inoculate with Pichia pastoris seed culture, add corn syrup, adjust the temperature to 28°C and pH to 6.8, and ferment for 60 hours.

[0032] Step S3: After fermentation, the reaction was terminated by pasteurization (80℃, 30 min). After centrifugation, the supernatant was subjected to isoelectric point precipitation (adjusting the pH to 4.5) to remove impurities and proteins.

[0033] Step S4: Add ethanol to the clarified liquid to a final concentration of 55%, and precipitate to obtain crude chondroitin sulfate. Subsequent mother liquor treatment and product purification steps are the same as in Example 1.

[0034] Example 3 This embodiment provides a process for the enzymatic hydrolysis of chondroitin sulfate and glucosamine, including the following steps: Step S1: Prepare homogenized slurry from wheat protein powder, cottonseed protein powder, and rapeseed protein powder. Simultaneously, activate and culture genetically engineered Bacillus subtilis to prepare seed liquid. Prepare alkaline protease and neutral protease.

[0035] Step S2: Place the protein slurry in a reactor, add neutral protease, and enzymatically hydrolyze for 2.5 hours at 52°C and pH 7.2. Inoculate with Bacillus subtilis seed culture, add sucrose, adjust the temperature to 35°C and pH to 7.2, and ferment for 36 hours.

[0036] Step S3: After fermentation, heat to 95°C and maintain for 40 minutes for sterilization. After centrifugation, obtain a clear filtrate through precision filtration.

[0037] Step S4: Add ethanol to the filtrate to a final concentration of 60% to precipitate chondroitin sulfate. Subsequent steps are the same as in Example 1.

[0038] The co-production of chondroitin sulfate and glucosamine was carried out according to the processes provided in Examples 1-3, and the yields and contents of the two products were then determined. The quantitative detection methods for all products were uniformly performed according to GB / T 20365-2006 "Determination of Chondroitin Sulfate and Glucosamine Content by Liquid Chromatography". Specific detection methods are as follows: 1. Detection of chondroitin sulfate yield and content: Test method: Refer to GB / T 20365-2006. Take the refined chondroitin sulfate product obtained in process step S4, place it in a vacuum drying oven at 60℃ and dry it to constant weight. Accurately weigh the dry weight of the finished product (denoted as W_cs). Determine the actual content of chondroitin sulfate in the finished product (denoted as C_cs, %) using the liquid chromatography method specified in GB / T 20365-2006.

[0039] Yield Calculation: Since chondroitin sulfate is synthesized through microbial fermentation, its yield is characterized by "product production efficiency." The calculation formula is: Chondroitin sulfate production efficiency (%) = (W_cs × C_cs / V_fermentation) × 100%, where V_fermentation is the total volume (L) of the fermentation broth after step S3. This indicator reflects the weight of pure chondroitin sulfate produced per liter of fermentation broth. The results are recorded in Table 1.

[0040] 2. Detection of glucosamine yield and content: Test method: Refer to GB / T 20365-2006. Take the glucosamine crystals obtained in process step S4, place them in a desiccator until constant weight, and accurately weigh the crystals (recorded as W_glcn). Determine the actual content of glucosamine hydrochloride in the crystals (recorded as C_glcn, %) using the liquid chromatography method specified in GB / T 20365-2006.

[0041] Yield Calculation: Glucosamine is mainly derived from the enzymatic conversion of plant protein. Based on the input plant protein raw material, the calculation formula is: Glucosamine yield (%) = (W_glcn × C_glcn / W_protein) × 100%, where W_protein is the dry weight of the plant protein raw material input in step S1. The results are recorded in Table 1.

[0042] Table 1 Product yield of Examples 1-3 As shown in Table 1, in the processes provided in Examples 1-3, the chondroitin sulfate production efficiency is not less than 2.2 g / L, the chondroitin sulfate content is not less than 92.1%, the glucosamine yield is not less than 20.4%, and the glucosamine hydrochloride content is not less than 98.4%, indicating that the process provided by the present invention has a good co-production yield of chondroitin sulfate and glucosamine.

[0043] The core of this invention lies in constructing a completely non-animal-derived enzymatic hydrolysis-fermentation coupled system, achieving the single-raw-source conversion of plant protein resources and the efficient co-production of two high-value products within the same reactor. First, plant protein slurry is used as the starting material, and enzymatic hydrolysis, primarily driven by proteases, efficiently degrades the plant protein network into abundant peptides, amino acids, and key precursors such as N-acetylglucosamine under mild conditions. This stage not only provides a directly usable high-quality nitrogen source for subsequent microbial fermentation but also reserves the core building blocks for the final production of glucosamine.

[0044] Subsequently, genetically engineered microbial seed culture was precisely inoculated into the optimized reaction matrix of the enzymatic hydrolysate. These engineered bacteria, acting as highly efficient "cell factories," have internally introduced and expressed an animal-derived chondroitin sulfate synthase system. Under suitable fermentation conditions, the engineered bacteria utilize precursor substances in the enzymatic hydrolysate and an added carbon source to perform growth metabolism and de novo biosynthesis of chondroitin sulfate, which is then secreted extracellularly. This process completely eliminates dependence on animal cartilage tissue, achieving the entirely non-animal-derived, fermentation-based production of chondroitin sulfate. Simultaneously, the enzymatic hydrolysis of plant protein and the fermentation metabolism of microorganisms continue in parallel and mutually promote each other, with the hydrolysate continuously providing nutrients for fermentation, forming a highly efficient integrated coupled process.

[0045] This coupled process fundamentally solves the problem of low efficiency caused by the significant differences in the properties of animal and plant raw materials and the conflicting optimal reaction conditions in traditional processes. Furthermore, in the subsequent separation, the inherent differences in the physicochemical properties of chondroitin sulfate (a macromolecular polysaccharide) and glucosamine (a monosaccharide derivative) are utilized to achieve efficient separation and purification through steps such as gradient alcohol precipitation and crystallization. This invention ultimately achieves the efficient and high-purity co-production of non-animal-derived chondroitin sulfate and glucosamine from a single plant-based starting material within a single system, offering advantages such as pure raw material sources, high process integration, and wide product applicability.

[0046] Test case In the process of this invention, the temperature is controlled at 45-55℃ during the enzymatic hydrolysis stage and at 30-37℃ (for bacteria) or 28-30℃ (for yeast) during the coupled fermentation stage. This precise temperature control in each stage is a key process parameter for achieving efficient co-production of the two products. As a core control condition, this temperature design first ensures that each biocatalyst (enzyme and microorganism) works efficiently within its optimal activity range.

[0047] During the enzymatic hydrolysis stage, the mild conditions of 45-55℃ ensure that the protease can efficiently and fully hydrolyze the plant protein network, while maximally protecting the chemical integrity of the released precursor substances such as N-acetylglucosamine. This accumulates a high concentration of substrate for the subsequent production of glucosamine and also provides an easily usable nitrogen source for microbial fermentation.

[0048] Subsequently, during the coupled fermentation stage, the system temperature was adjusted and strictly controlled at 30-37℃ (bacteria) or 28-30℃ (yeast). This temperature range is the optimal range for the growth, metabolism, and expression of exogenous synthetic enzyme systems of the genetically engineered microorganisms used, effectively driving the microorganisms to efficiently synthesize and secrete non-animal-derived chondroitin sulfate using precursor substances. This temperature is clearly distinguished from the previous enzymatic hydrolysis stage, aiming to provide independent and optimal thermodynamic environments for the two core biological processes (catalytic hydrolysis and microbial fermentation), thereby ensuring the efficient synergy and non-interference of the two core processes of plant protein conversion and chondroitin sulfate biosynthesis.

[0049] Therefore, this controlled switching strategy from enzymatic hydrolysis temperature to fermentation temperature is not a simple change of conditions, but rather a precise matching of the sequential requirements of substrate preparation and product synthesis in the time dimension by creating the optimal thermal environment for the two biological processes with different properties, namely enzyme-catalyzed hydrolysis and microbial fermentation. Ultimately, it achieves efficient synergy between the synthesis of non-animal chondroitin sulfate and the release of glucosamine precursor.

[0050] To verify that the 45-55℃ temperature during the enzymatic hydrolysis stage is one of the important factors contributing to the high efficiency of the co-production of non-animal chondroitin sulfate and glucosamine provided by the present invention, the following verification experiment was designed.

[0051] First, based on Example 1 (using engineered E. coli), this experimental example only changed the temperature of the enzymatic hydrolysis stage, setting it to 25℃, 35℃, 45℃, 50℃, 55℃, 65℃, 75℃, or 85℃. Then, the entire process was carried out for co-production. The yield efficiency of chondroitin sulfate and the yield of glucosamine were tested according to the detection method of GB / T 20365-2006. The results are as follows: Figure 2 , Figure 3 As shown.

[0052] according to Figure 2 , Figure 3 It can be seen that when the enzymatic hydrolysis temperature is 45-55℃, the co-production efficiency of chondroitin sulfate and the yield of glucosamine are relatively high. Therefore, it can be concluded that the enzymatic hydrolysis temperature of 45-55℃ is one of the important factors for achieving efficient co-production of non-animal-derived chondroitin sulfate and glucosamine in the process of this invention.

[0053] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A process for the enzymatic hydrolysis and co-production of chondroitin sulfate and hyaluronic acid, characterized in that, Includes the following steps: Step S1: Prepare the plant protein raw material into a homogeneous slurry; at the same time, activate and culture genetically engineered microorganisms with the ability to synthesize chondroitin sulfate to prepare a high-density microbial seed liquid; Formulate proteases for the hydrolysis of plant proteins; Step S2: Place the homogenized plant protein slurry in a temperature- and pH-controlled reactor, and add protease for enzymatic hydrolysis; The microbial seed liquid was introduced into the system and a fermentation carbon source was added. The conditions were adjusted to suitable fermentation conditions for microbial growth and chondroitin sulfate synthesis, and coupled fermentation was carried out. Step S3: After the coupling process is completed, sterilization is performed, followed by solid-liquid separation to remove bacterial cells and incompletely hydrolyzed solids, resulting in a mixed supernatant. Then remove any extraneous proteins and colloidal substances from the mixed supernatant; Step S4: Perform alcohol precipitation on the preliminarily purified mixed supernatant to precipitate and separate crude chondroitin sulfate; The remaining solution after alcohol precipitation was used to recover ethanol to obtain mother liquor. The mother liquor was then subjected to decolorization, desalting and crystallization steps to obtain glucosamine crystals. The crude chondroitin sulfate is refined to obtain the final product.

2. The process for the enzymatic hydrolysis and co-production of chondroitin sulfate and hyaluronic acid according to claim 1, characterized in that: In step S1, the plant protein raw material is crushed and hydrated to prepare a homogeneous slurry, and the plant protein raw material is one or more of soybean, wheat, peanut, cottonseed or rapeseed protein powder. The genetically engineered microorganisms are engineered Escherichia coli, Bacillus subtilis, or Pichia pastoris with the introduction of animal-derived chondroitin sulfate synthase genes.

3. The process for the enzymatic hydrolysis and co-production of chondroitin sulfate and hyaluronic acid according to claim 1, characterized in that: In step S1, the protease is one or more of alkaline protease, neutral protease, or complex plant hydrolase.

4. The process for the enzymatic hydrolysis and co-production of chondroitin sulfate and hyaluronic acid according to claim 1, characterized in that: In step S2, the enzymatic hydrolysis stage is carried out at a temperature of 45-55℃, a pH of 6.5-7.5, and a time of 1-3 hours. The temperature during the coupled fermentation stage is 30-37℃ or 28-30℃, the pH is maintained at 6.8-7.4, and the fermentation time is controlled at 24-72h.

5. The process for the enzymatic hydrolysis and co-production of chondroitin sulfate and hyaluronic acid according to claim 1, characterized in that: In step S2, the fermentation carbon source is one of glucose, sucrose, or corn syrup.

6. The process for the enzymatic hydrolysis and co-production of chondroitin sulfate and hyaluronic acid according to claim 1, characterized in that: In step S3, sterilization is performed by heating the reaction system to 121°C and maintaining it for 15-30 minutes, or by pasteurization.

7. The process for the enzymatic hydrolysis and co-production of chondroitin sulfate and hyaluronic acid according to claim 1, characterized in that: In step S3, isoelectric point sedimentation, heat treatment, or membrane filtration techniques are used to remove impurities such as proteins and colloidal substances from the mixed supernatant.

8. The process for the enzymatic hydrolysis and co-production of chondroitin sulfate and hyaluronic acid according to claim 1, characterized in that: In step S4, the gradient alcohol precipitation specifically involves adding ethanol to the mixed supernatant to make the final concentration of the system 50-70%, precipitating and separating the crude chondroitin sulfate.

9. The process for the enzymatic hydrolysis and co-production of chondroitin sulfate and hyaluronic acid according to claim 1, characterized in that: In step S4, the remaining alcohol solution is treated with ethanol recovery to obtain mother liquor. The mother liquor is then subjected to activated carbon decolorization, ion exchange resin desalting, and concentration crystallization steps to obtain glucosamine crystals.

10. The process for the enzymatic hydrolysis and co-production of chondroitin sulfate and hyaluronic acid according to claim 1, characterized in that: In step S4, the purification of crude chondroitin sulfate includes: dissolving the crude product in water, removing small molecule impurities by ultrafiltration, followed by secondary precipitation with ethanol and freeze-drying to obtain the chondroitin sulfate product.

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