Preparation method and application of chitooligosaccharide uronic acid

Abstract

The application discloses a preparation method and application of chitooligosaccharide alduronic acid and belongs to the technical field of marine bioengineering. The preparation method comprises the following steps: after beta-chitin and a cleaving polysaccharide monooxygenase SmLPMO10 are dissolved in a Tris-hydrochloric acid buffer solution, a reducing agent is added, and an enzymatic reaction is carried out; after the reaction is carried out for 24 hours, the reducing agent is supplemented, the reaction is continuously carried out, the reducing agent is supplemented once every 24 hours, after the reaction is carried out for 96 hours, supernatant is obtained by filtration, and the chitooligosaccharide alduronic acid is obtained after inactivation, centrifugal removal of supernatant, desalination and freeze-drying. The chitin can be cleaved by using a single cleaving polysaccharide monooxygenase, without additional addition of chitinase, and the reaction condition is mild, so that the generation of by-products and environmental pollution are avoided. The application provides an environment-friendly preparation method of chitooligosaccharide alduronic acid, and the research and development of a green conversion technology of chitin and chitin-like biomass resources are greatly promoted.

Description

Technical Field

[0001] This invention relates to the field of marine bioengineering technology, and in particular to a method for preparing and applying a chitosan oligosaccharide uronic acid. Background Technology

[0002] Chitin, also known as chitosan, is a natural linear polysaccharide widely found in the shells of marine crustaceans and the cartilage of cephalopods. Its abundance in nature is second only to cellulose, making it the second largest natural macromolecule and a recyclable resource. The chemical structure of chitin is N-acetylglucosamine (GlcNAc) linked by β-1,4 glycosidic bonds. Chitin can be further deacetylated to prepare chitosan, and both can be degraded by specific chitinases and chitosanases to produce chitosan oligosaccharides (chitosan oligosaccharides and chitosan oligosaccharides), thus generating a series of diversified high-value derivatives. Chitosan polysaccharides / oligosaccharides have been reported to possess various physiological activities, including antioxidant, antibacterial, immunomodulatory, and plant growth-promoting effects, attracting widespread attention in the fields of medical materials, functional foods, and biopesticides.

[0003] Chitin is one of the most stubborn biopolymers, with numerous hydrogen bonds forming a tightly packed crystalline fibrous network within and between its molecules. This makes it insoluble in water, dilute acids, dilute alkalis, and common organic solvents, necessitating deacetylation with concentrated alkali to prepare chitosan for further development and utilization. Even with specific chitinase degradation, it is still necessary to first prepare colloidal chitin by swelling with concentrated hydrochloric acid beforehand to use as the initial enzymatic hydrolysis substrate. The reaction conditions are also very demanding, and a truly green conversion has not yet been achieved.

[0004] lytic polysaccharide monooxygenases (LPMOs) are a class of oxidases discovered in recent years that catalyze the degradation of polysaccharide biomass and are widely found in organisms such as bacteria, fungi, and viruses. LPMOs can directly act on the crystalline regions of stubborn chitin, oxidizing the C1 atom of the sugar ring in the polysaccharide chain, thereby breaking the glycosidic bond to generate chitosan oligosaccharide aldonic acid with N-acetylglucosinolate (GlcNAc1A) at the end. However, since the discovery of LPMOs, they have mainly been used to assist chitinase in the degradation of chitin to prepare chitin oligosaccharides. Researchers at home and abroad generally believe that LPMOs play an auxiliary role in the degradation of chitin, while chitinase is the key enzyme in the degradation process. This is mainly because in current studies, when LPMOs are used alone to treat chitin, only a very small amount of chitin oligosaccharide auronic acid products can be detected. The conversion rate of chitin oligosaccharide auronic acid is extremely low, basically between 1% and 5% or even lower. How to improve the preparation efficiency of chitin oligosaccharide auronic acid is crucial for the green conversion of chitin. At the same time, since chitin oligosaccharide auronic acid is currently difficult to obtain, there are no reports on its application research. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing and applying chitosan oligosaccharide uronic acid, thereby addressing the problems existing in the prior art. This invention utilizes a cleaving polysaccharide monooxygenase to directly degrade chitin (without requiring additional chitinase), optimizing the enzymatic hydrolysis process, thus establishing a new technology for preparing chitosan oligosaccharide uronic acid from chitin biomass. Furthermore, this invention has verified that chitosan oligosaccharide uronic acid possesses strong activity in regulating the body's immune response. The method provided by this invention will greatly advance the research and development of green conversion technology for chitin-based biomass resources, laying an important foundation for the discovery of novel chitosan oligosaccharide uronic acid immunomodulators and their application in the field of marine biomedicine.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for preparing chitosan oligosaccharide uronic acid, comprising the following steps: β-Chitin and the polysaccharide-cleaving monooxygenase SmLPMO10 were dissolved in Tris-hydrochloric acid buffer solution, and a reducing agent was added to carry out the enzymatic hydrolysis reaction. After 24 hours of reaction, the reducing agent was added and the reaction continued. The reducing agent was added every 24 hours. After 96 hours of reaction, the supernatant was collected by filtration, and after inactivation, centrifugation to remove the supernatant, desalting, and freeze-drying, chitin oligosaccharide aldonic acid was obtained. Alternatively, β-chitin and cleaving polysaccharide monooxygenase SmLPMO10 are dissolved in Tris-hydrochloric acid buffer solution, and a reducing agent is added for enzymatic hydrolysis. After 24 hours of reaction, a reducing agent is added, and the reaction continues. The reducing agent is added every 24 hours. After 96 hours of reaction, cleaving polysaccharide monooxygenase SmLPMO10 is added, and the above enzymatic hydrolysis reaction and the operation of adding a reducing agent every 24 hours are repeated. After 96 hours of reaction, the supernatant is filtered, inactivated, centrifuged to remove the supernatant, desalted, and freeze-dried to obtain chitin oligosaccharide aldonic acid. The addition of the cleaving polysaccharide monooxygenase SmLPMO10 involves repeating the above enzymatic hydrolysis reaction and the addition of a reducing agent every 24 hours 1-3 times.

[0007] Optionally, the concentration of the Tris-hydrochloric acid buffer solution is 20 mM and the pH is 8.0-9.0.

[0008] Optionally, the concentration of β-chitin in the Tris-hydrochloric acid buffer solution is 10 g / L, the concentration of the cleaving polysaccharide monooxygenase SmLPMO10 in the Tris-hydrochloric acid buffer solution is 1.0 μmol / L, and the concentration of the reducing agent in the Tris-hydrochloric acid buffer solution is 1 mM.

[0009] Optionally, the temperature of the enzymatic hydrolysis reaction is 37℃-50℃.

[0010] Optionally, the concentration of the reducing agent added to the reaction solution is 1 mM.

[0011] Optionally, the concentration of the cleaving polysaccharide monooxygenase SmLPMO10 added to the reaction solution is 1.0 μmol / L.

[0012] Optionally, the reducing agent includes ascorbic acid or gallic acid.

[0013] The present invention also provides chitin oligosaccharide aldonic acid prepared according to the preparation method described above.

[0014] The present invention also provides the application of the chitosan oligosaccharide aldonic acid in the preparation of immunomodulatory agents.

[0015] The present invention also provides an immunomodulatory agent comprising the aforementioned chitosan oligosaccharide aldonic acid; The chitosan oligosaccharide aldonic acid has immunomodulatory activity.

[0016] The present invention discloses the following technical effects: 1. This invention utilizes a strategy and mode for the direct degradation of stubborn chitin using cleaving polysaccharide monooxygenases (without the need for additional chitinase). It is the first discovery of a long-term stable mode for LPMOs under intermittent supplementation of reducing agents and high substrate concentrations, effectively avoiding the inactivation reaction during the catalytic conversion of chitin by LPMOs. This strategy of directly degrading stubborn chitin using cleaving polysaccharide monooxygenases will break through the barrier of existing chitin-chitin utilization technologies that rely on concentrated acids and alkalis, promoting technological innovation in the application and development of chitin-chitin resources. Furthermore, the enzyme reaction conditions in this invention are mild, avoiding the generation of byproducts and environmental pollution, making it an environmentally friendly method for preparing chitosan oligosaccharide aldonic acid.

[0017] 2. This invention significantly improves the conversion rate of chitin oligosaccharide uronic acid. Using only one LPMO to oxidize and degrade chitin, the conversion rate of chitin oligosaccharide uronic acid can reach more than 30%. With the addition of new enzymes to repeat the degradation of the same substrate, the conversion rate can reach more than 60%, which is far higher than the conversion rate of 1-5% reported in existing studies. This technical solution makes the direct and green degradation of chitin possible and will open up a new direction for the high-value utilization of chitin.

[0018] 3. Currently, chitin oligosaccharide products obtained by degrading chitin with chitinase have poor water solubility. Generally, chitin oligosaccharides with a degree of polymerization greater than six have poor or even no solubility in water. The chitin oligosaccharide aldonic acid obtained by this invention has high purity and excellent water solubility. Products with a degree of polymerization greater than eight of chitin oligosaccharide aldonic acid can be clearly detected in the water-soluble products (chitin oligosaccharides with the same degree of polymerization are insoluble in water). At the same time, chitin oligosaccharide aldonic acid also has good immunostimulatory activity and can be used for development in the pharmaceutical field. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The mass spectrum of chitosan oligosaccharide aldonic acid provided by the present invention; Figure 2 The image shows the screening results of the immunostimulatory macrophage NO release activity of the chitin oligosaccharide aldonic acid prepared in this invention. Figure 3 The results show the TNF-α release activity of macrophages stimulated by the chitin oligosaccharide aldonic acid prepared in this invention. Figure 4 The results show the IL-6 release activity of macrophages stimulated by the chitin oligosaccharide aldonic acid prepared in this invention. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Preparation of the cleavable polysaccharide monooxygenase SmLPMO10: Plasmids containing the sequence of the cleavable polysaccharide monooxygenase SmAA10SmLPMO10 (GenBank™, accession number AY665558) (source: pRSETB, Invitrogen Corporation) were added to competent BL21(DE3) cells and cultured with shaking at 37°C for 16 h. The cells were collected by centrifugation, and the protein was collected by osmotic shock. The supernatant was separated by chitin affinity chromatography to purify the protein, thus obtaining the purified cleavable polysaccharide monooxygenase SmLPMO10 protein.

[0027] Example 1: A method for preparing chitosan oligosaccharide uronic acid At 37°C, β-chitin and the cleaving polysaccharide monooxygenase SmLPMO10 were dissolved in 20 mM Tris-hydrochloric acid buffer solution at pH 8.0, with a final chitin concentration of 10 g / L and a final SmLPMO10 concentration of 1.0 μmol / L. Then, ascorbic acid, a reducing agent, was added to the reaction solution, with an ascorbic acid concentration of 1 mM. The enzymatic hydrolysis reaction was started by shaking. After 24 hours of reaction, ascorbic acid was added to the reaction solution to a concentration of 1 mM, and the reaction was continued. Ascorbic acid was added every 24 hours. After 96 hours of reaction, the supernatant was filtered, boiled to inactivate the enzyme, centrifuged to remove the supernatant, desalted using Sephadex G10, and then freeze-dried to obtain chitin oligosaccharide aldonic acid.

[0028] Mass spectrometry characterization spectrum (see) Figure 1 It mainly contains chitin tetrasulphuronic acid, chitin pentasulphuronic acid, chitin hexasulphuronic acid, chitin heptasulphuronic acid, chitin octasulphuronic acid, chitin nonasulphuronic acid, and chitin decasulphuronic acid. High-performance liquid chromatography (HPLC) was used for quantitative detection of chitin oligosulphuronic acid products, and the results showed that the conversion rate of chitin oligosulphuronic acid reached 35%.

[0029] Example 2: A method for preparing chitosan oligosaccharide uronic acid Same as Example 1, except that the pH of the Tris-hydrochloric acid buffer solution was adjusted to 9.0.

[0030] At 37°C, β-chitin and the cleaving polysaccharide monooxygenase SmLPMO10 were dissolved in 20 mM Tris-hydrochloric acid buffer solution at pH 9.0, with a final chitin concentration of 10 g / L and a final SmLPMO10 concentration of 1.0 μmol / L. Then, ascorbic acid, a reducing agent, was added to the reaction solution, with an ascorbic acid concentration of 1 mM. The enzymatic hydrolysis reaction was started by shaking. After 24 hours of reaction, ascorbic acid was added to the reaction solution to a concentration of 1 mM, and the reaction was continued. Ascorbic acid was added every 24 hours. After 96 hours of reaction, the supernatant was filtered, boiled to inactivate the polysaccharide, centrifuged to remove the supernatant, desalted using Sephadex G10, and then freeze-dried to obtain chitin oligosaccharide aldonic acid.

[0031] Mass spectrometry characterization spectrum and Figure 1 Similar to other compounds, it mainly contains chitin tetrasulphuronic acid, chitin pentasulphuronic acid, chitin hexasulphuronic acid, chitin heptasulphuronic acid, chitin octasulphuronic acid, chitin nonasulphuronic acid, and chitin decasulphuronic acid. High-performance liquid chromatography (HPLC) was used for quantitative detection of chitin oligosulphuronic acid products, and the results showed that the conversion rate of chitin oligosulphuronic acid reached 42%.

[0032] Example 3: A method for preparing chitosan oligosaccharide uronic acid Same as Example 1, except that after 96 hours of reaction, SmLPMO10 is added again to repeat the step.

[0033] At 37°C, β-chitin and the cleaving polysaccharide monooxygenase SmLPMO10 were dissolved in 20 mM Tris-hydrochloric acid buffer solution at pH 8.0, with a final chitin concentration of 10 g / L and a final SmLPMO10 concentration of 1.0 μmol / L. Then, ascorbic acid was added as a reducing agent to the reaction solution, resulting in an ascorbic acid concentration of 1 mM. The reaction was initiated by shaking. After 24 hours, ascorbic acid was added to the reaction solution to maintain a concentration of 1 mM, and the reaction continued. Ascorbic acid was added every 24 hours. After 96 hours, fresh SmLPMO10 was added to maintain a final concentration of 1.0 μmol / L in the reaction solution. This process of adding ascorbic acid every 24 hours was repeated once. After the reaction, the mixture was filtered, and the supernatant was boiled to inactivate the polysaccharide. The supernatant was then centrifuged to remove the supernatant, desalted using Sephadex G10, and finally freeze-dried to obtain the chitin oligosaccharide aldonic acid.

[0034] Mass spectrometry characterization spectrum and Figure 1 Similar to other compounds, it mainly contains chitin tetrasuluronic acid, chitin pentasuluronic acid, chitin hexasuluronic acid, chitin heptasuluronic acid, chitin octasuluronic acid, chitin nonasuluronic acid, and chitin decasuluronic acid. Quantitative detection of chitin oligosuluronic acid products was performed using high-performance liquid chromatography (HPLC), and the results showed that the conversion rate of chitin oligosuluronic acid reached 47%.

[0035] Example 4: A method for preparing chitosan oligosaccharide uronic acid Same as Example 2, except that after 96 hours of reaction, SmLPMO10 was added again and the steps were repeated three times.

[0036] At 37°C, β-chitin and the polysaccharide-cleaving monooxygenase SmLPMO10 were dissolved in 20 mM Tris-hydrochloric acid buffer solution at pH 9.0, resulting in a final chitin concentration of 10 g / L and a final SmLPMO10 concentration of 1.0 μmol / L. Ascorbic acid, a reducing agent, was then added to the reaction solution to a concentration of 1 mM. The enzymatic hydrolysis reaction was initiated with shaking. After 24 hours of reaction, ascorbic acid was added to the reaction solution to a concentration of 1 mM, and the reaction continued. Ascorbic acid was added every 24 hours. After 96 hours of reaction, fresh SmLPMO10 was added to a final concentration of 1.0 μmol / L in the reaction solution. This process of enzymatic hydrolysis and ascorbic acid addition every 24 hours was repeated. After 96 hours of reaction, fresh SmLPMO10 was added again to a final concentration of 1.0 μmol / L in the reaction solution. The above enzymatic hydrolysis reaction was repeated, with ascorbic acid added every 24 hours. After 96 hours of reaction, a third addition of fresh SmLPMO10 was made until the final concentration in the reaction solution was 1.0 μmol / L. The above enzymatic hydrolysis reaction was repeated, with ascorbic acid added every 24 hours. After 96 hours of reaction, the mixture was filtered, the supernatant was boiled to inactivate the enzyme, centrifuged to remove the supernatant, desalted using Sephadex G10, and then freeze-dried to obtain chitin oligosaccharide aldonic acid.

[0037] Mass spectrometry characterization spectrum and Figure 1 Similar to other compounds, it mainly contains chitin tetrasulphuronic acid, chitin pentasulphuronic acid, chitin hexasulphuronic acid, chitin heptasulphuronic acid, chitin octasulphuronic acid, chitin nonasulphuronic acid, and chitin decasulphuronic acid. High-performance liquid chromatography (HPLC) was used for quantitative detection of chitin oligosulphuronic acid products, and the results showed that the conversion rate of chitin oligosulphuronic acid reached 61%.

[0038] Example 5: A method for preparing a chitin oligosaccharide uronic acid Same as Example 2, except that the temperature is adjusted to 50°C.

[0039] At 50°C, β-chitin and the cleaving polysaccharide monooxygenase SmLPMO10 were dissolved in 20 mM Tris-hydrochloric acid buffer solution at pH 9.0, with a final chitin concentration of 10 g / L and a final SmLPMO10 concentration of 1.0 μmol / L. Then, ascorbic acid, a reducing agent, was added to the reaction solution, with an ascorbic acid concentration of 1 mM. The enzymatic hydrolysis reaction was started by shaking. After 24 hours of reaction, ascorbic acid was added to the reaction solution to a concentration of 1 mM, and the reaction was continued. Ascorbic acid was added every 24 hours for 96 hours. The supernatant was filtered, boiled to inactivate the enzyme, centrifuged to remove the supernatant, desalted using Sephadex G10, and then freeze-dried to obtain chitin oligosaccharide aldonic acid.

[0040] Mass spectrometry characterization spectrum and Figure 1Similar to other compounds, it mainly contains chitin tetrasuluronic acid, chitin pentasuluronic acid, chitin hexasuluronic acid, chitin heptasuluronic acid, chitin octasuluronic acid, chitin nonasuluronic acid, and chitin decasuluronic acid. High-performance liquid chromatography (HPLC) was used for quantitative detection of chitin oligosuluronic acid products, and the results showed that the conversion rate of chitin oligosuluronic acid reached 31%.

[0041] Example 6: A method for preparing a chitin oligosaccharide uronic acid Same as Example 2, except that the reducing agent is changed to gallic acid.

[0042] At 37°C, β-chitin and the cleaving polysaccharide monooxygenase SmLPMO10 were dissolved in 20 mM Tris-hydrochloric acid buffer solution at pH 9.0, with a final chitin concentration of 10 g / L and a final SmLPMO10 concentration of 1.0 μmol / L. Gallic acid, a reducing agent, was then added to the reaction solution, with a gallic acid concentration of 1 mM. The enzymatic hydrolysis reaction was initiated by shaking. After 24 hours of reaction, gallic acid was added to the reaction solution to a concentration of 1 mM, and the reaction was continued. Gallic acid was added every 24 hours. After 96 hours of reaction, the supernatant was filtered, boiled to inactivate the enzyme, centrifuged to remove the supernatant, desalted using Sephadex G10, and then freeze-dried to obtain chitin oligosaccharide aldonic acid.

[0043] Mass spectrometry characterization spectrum and Figure 1 Similar to other compounds, it mainly contains chitosan tetrasuluronic acid, chitosan pentasuluronic acid, chitosan hexasuluronic acid, chitosan heptasuluronic acid, chitosan octasuluronic acid, chitosan nonasuluronic acid, and chitosan decasuluronic acid. High-performance liquid chromatography (HPLC) was used for quantitative detection of chitosan oligosuluronic acid products, and the results showed that the conversion rate of chitosan oligosuluronic acid reached 35%.

[0044] Comparative Example 1: A method for preparing a chitin oligosaccharide uronic acid Same as Example 1, except that the reducing agent ascorbic acid is not added during the reaction.

[0045] At 37°C, β-chitin and the cleaving polysaccharide monooxygenase SmLPMO10 were dissolved in 20 mM Tris-hydrochloric acid buffer solution at pH 8.0, with a final chitin concentration of 10 g / L and a final SmLPMO10 concentration of 1.0 μmol / L. Then, ascorbic acid, a reducing agent, was added to the reaction solution, with an ascorbic acid concentration of 1 mM. The enzymatic hydrolysis reaction was initiated by shaking. After 96 hours of reaction, the mixture was filtered, and the supernatant was boiled to inactivate the enzyme. The supernatant was then centrifuged to remove the supernatant, desalted using Sephadex G10, and finally freeze-dried to obtain the chitin oligosaccharide aldonic acid.

[0046] High performance liquid chromatography (HPLC) was used to quantitatively detect the chitin oligosaccharide aldonic acid product, and the results showed that the conversion rate of chitin oligosaccharide aldonic acid reached 5%.

[0047] Comparative Example 2: A method for preparing a chitin oligosaccharide uronic acid Same as Example 1, except that the pH of the Tris-hydrochloric acid buffer solution was adjusted to 10.0.

[0048] At 37°C, β-chitin and the cleaving polysaccharide monooxygenase SmLPMO10 were dissolved in 20 mM Tris-hydrochloric acid buffer solution at pH 10.0, with a final chitin concentration of 10 g / L and a final SmLPMO10 concentration of 1.0 μmol / L. Then, ascorbic acid, a reducing agent, was added to the reaction solution, with an ascorbic acid concentration of 1 mM. The enzymatic hydrolysis reaction was started by shaking. After 24 hours of reaction, ascorbic acid was added to the reaction solution to a concentration of 1 mM, and the reaction was continued. Ascorbic acid was added every 24 hours. After 96 hours of reaction, the supernatant was filtered, boiled to inactivate the enzyme, centrifuged to remove the supernatant, desalted using Sephadex G10, and then freeze-dried to obtain chitin oligosaccharide aldonic acid.

[0049] High performance liquid chromatography (HPLC) was used to quantitatively detect the chitosan oligosaccharide aldonic acid product, and the results showed that the conversion rate of chitosan oligosaccharide aldonic acid reached 10%.

[0050] Comparative Example 3: A method for preparing a chitin oligosaccharide uronic acid Same as Example 2, except that α-chitin is used.

[0051] At 37°C, α-chitin and the cleaving polysaccharide monooxygenase SmLPMO10 were dissolved in 20 mM Tris-hydrochloric acid buffer solution at pH 9.0, with a final chitin concentration of 10 g / L and a final SmLPMO10 concentration of 1.0 μmol / L. Then, ascorbic acid, a reducing agent, was added to the reaction solution, with an ascorbic acid concentration of 1 mM. The enzymatic hydrolysis reaction was initiated by shaking. After 24 hours of reaction, ascorbic acid was added to the reaction solution to a concentration of 1 mM, and the reaction was continued. Ascorbic acid was added every 24 hours. After 96 hours of reaction, the supernatant was filtered, boiled to inactivate the enzyme, centrifuged to remove the supernatant, desalted using Sephadex G10, and then freeze-dried to obtain chitin oligosaccharide aldonic acid.

[0052] High performance liquid chromatography (HPLC) was used to quantitatively detect the chitosan oligosaccharide aldonic acid product, and the results showed that the conversion rate of chitosan oligosaccharide aldonic acid reached 7%.

[0053] Comparative Example 4: A method for preparing a chitin oligosaccharide uronic acid Same as Example 2, except that the temperature is adjusted to 60°C.

[0054] At 60℃, β-chitin and the cleaving polysaccharide monooxygenase SmLPMO10 were dissolved in 20 mM Tris-hydrochloric acid buffer solution at pH 9.0, with a final chitin concentration of 10 g / L and a final SmLPMO10 concentration of 1.0 μmol / L. Then, ascorbic acid, a reducing agent, was added to the reaction solution, with an ascorbic acid concentration of 1 mM. The enzymatic hydrolysis reaction was started by shaking. After 24 hours of reaction, ascorbic acid was added to the reaction solution to a concentration of 1 mM, and the reaction was continued. Ascorbic acid was added every 24 hours. After 96 hours of reaction, the supernatant was filtered, boiled to inactivate the polysaccharide, centrifuged to remove the supernatant, desalted using Sephadex G10, and then freeze-dried to obtain chitin oligosaccharide aldonic acid.

[0055] High performance liquid chromatography (HPLC) was used to quantitatively detect the chitosan oligosaccharide aldonic acid product, and the results showed that the conversion rate of chitosan oligosaccharide aldonic acid reached 2%.

[0056] Comparative Example 5: A method for preparing a chitin oligosaccharide uronic acid Same as Example 2, except that the final concentration of SmLPMO10 was adjusted to 0.3 μmol / L.

[0057] At 37°C, β-chitin and the cleaving polysaccharide monooxygenase SmLPMO10 were dissolved in 20 mM Tris-hydrochloric acid buffer solution at pH 9.0, with a final chitin concentration of 10 g / L and a final SmLPMO10 concentration of 0.3 μmol / L. Then, ascorbic acid, a reducing agent, was added to the reaction solution, with an ascorbic acid concentration of 1 mM. The enzymatic hydrolysis reaction was started by shaking. After 24 hours of reaction, ascorbic acid was added to the reaction solution to a concentration of 1 mM, and the reaction was continued. Ascorbic acid was added every 24 hours. After 48 hours of reaction, the supernatant was filtered, boiled to inactivate the enzyme, centrifuged to remove the supernatant, desalted using Sephadex G10, and then freeze-dried to obtain chitin oligosaccharide aldonic acid.

[0058] High performance liquid chromatography (HPLC) was used to quantitatively detect the chitosan oligosaccharide aldonic acid product, and the results showed that the conversion rate of chitosan oligosaccharide aldonic acid reached 10%.

[0059] Experimental Example 1: Immunostimulatory Activity Test In mouse monocyte / macrophage RAW264.7 cells, the chitosan oligouronic acid sample prepared in Example 1 was used to prepare a 100 mg / L stock solution, which was then diluted to different concentrations with cell culture medium. Lipopolysaccharide (LPS) 1 μg / mL was used as a positive control, and the blank group (culture medium) was used as a negative control. After incubation for 24 h, the supernatant was collected. The NO release was measured using the Griess method to evaluate the effect of chitosan oligouronic acid on stimulating NO secretion in macrophages. Simultaneously, the effects of chitosan oligouronic acid on stimulating TNF-α and IL-6 secretion in macrophages were evaluated using TNF-α and IL-6 kits. The results are as follows: Figures 2-4 As shown, chitosan oligouronic acid has good immunomodulatory activity.

[0060] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing chitosan oligosaccharide aldonic acid, characterized in that, Includes the following steps: β-Chitin and the polysaccharide-cleaving monooxygenase SmLPMO10 were dissolved in Tris-hydrochloric acid buffer solution, and a reducing agent was added to carry out the enzymatic hydrolysis reaction. After 24 hours of reaction, the reducing agent was added and the reaction continued. The reducing agent was added every 24 hours. After 96 hours of reaction, the supernatant was collected by filtration, and after inactivation, centrifugation to remove the supernatant, desalting, and freeze-drying, chitin oligosaccharide aldonic acid was obtained. Alternatively, β-chitin and cleaving polysaccharide monooxygenase SmLPMO10 are dissolved in Tris-hydrochloric acid buffer solution, and a reducing agent is added for enzymatic hydrolysis. After 24 hours of reaction, a reducing agent is added, and the reaction continues. The reducing agent is added every 24 hours. After 96 hours of reaction, cleaving polysaccharide monooxygenase SmLPMO10 is added, and the above enzymatic hydrolysis reaction and the operation of adding a reducing agent every 24 hours are repeated. After 96 hours of reaction, the supernatant is filtered, inactivated, centrifuged to remove the supernatant, desalted, and freeze-dried to obtain chitin oligosaccharide aldonic acid. The addition of the cleaving polysaccharide monooxygenase SmLPMO10 involves repeating the above enzymatic hydrolysis reaction and the addition of a reducing agent every 24 hours 1-3 times.

2. The preparation method according to claim 1, characterized in that, The concentration of the Tris-hydrochloric acid buffer solution is 20 mM, and the pH is 8.0-9.

0.

3. The preparation method according to claim 1, characterized in that, The concentration of β-chitin in the Tris-hydrochloric acid buffer solution is 10 g / L, the concentration of the cleaving polysaccharide monooxygenase SmLPMO10 in the Tris-hydrochloric acid buffer solution is 1.0 μmol / L, and the concentration of the reducing agent in the Tris-hydrochloric acid buffer solution is 1 mM.

4. The preparation method according to claim 1, characterized in that, The temperature for all enzymatic hydrolysis reactions was 37℃-50℃.

5. The preparation method according to claim 1, characterized in that, The reducing agent is added to the reaction solution at a concentration of 1 mM.

6. The preparation method according to claim 1, characterized in that, The concentration of the cleaving polysaccharide monooxygenase SmLPMO10 added to the reaction solution was 1.0 μmol / L.

7. The preparation method according to claim 1, characterized in that, The reducing agent includes ascorbic acid or gallic acid.

8. Chitosan oligosaccharide uronic acid prepared by the preparation method according to any one of claims 1-7.

9. The use of chitosan oligosaccharide aldonic acid as described in claim 8 in the preparation of immunomodulatory agents.

10. An immunomodulatory agent, characterized in that, It comprises the chitin oligosaccharide aldonic acid as described in claim 8; The chitosan oligosaccharide aldonic acid has immunomodulatory activity.

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