A method for preparing a specific n-acetyl chitosan

Abstract

The application belongs to the technical field of marine biological engineering, and particularly relates to a preparation method of specific N-acetylated chitosan (N-acetylated chitosan with different acetylation degrees). Specifically, the method comprises the following steps: taking fully deacetylated chitosan as a substrate, and performing reaction on the substrate in sodium acetate buffer solution through chitin deacetylase (PesCDA) with a specific deacetylation mode; and after the reaction, salt is removed, concentrated and freeze-dried, thereby obtaining chitosan with a specific N-acetylation degree. Compared with a traditional method for preparing chitosan with a specific N-acetylation degree, the method is green, environmentally friendly and pollution-free.

Description

Technical Field

[0001] This invention belongs to the field of marine bioengineering technology, specifically relating to a method for preparing chitosan with a specific degree of N-acetylation (N-acetylated chitosan with different degrees of acetylation). Background Technology

[0002] Chitosan is a linear polysaccharide composed of D-glucosamine (GlcN) and N-acetyl-D-glucosamine (GlcNAc) linked by β-1,4 glycosidic bonds. Chitosan has been found to possess various physiological activities, such as antitumor, antibacterial, anti-inflammatory, antioxidant, hemostatic, blood glucose and lipid regulation, and immune enhancement. The activity of chitosan is closely related to its structure; the degree of acetylation refers to the proportion of acetylated glucosamine units to the total number of glucosamine units, and is an important structural parameter affecting the properties and activity of chitosan. Currently, the preparation of chitosan with a specific degree of acetylation typically employs chemical methods, such as the acetic anhydride method to add acetyl groups or the sodium hydroxide method to remove acetyl groups. These methods are difficult to control in terms of reaction rate, produce complex products with poor reproducibility, and are not environmentally friendly. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing chitosan with a specific degree of N-acetylation. To achieve the above objective, the technical solution adopted by this invention is as follows:

[0004] A method for preparing chitosan with a specific degree of N-acetylation is characterized by: using fully deacetylated chitosan as a substrate, reacting it with a chitin deacetylase (PesCDA) in a sodium acetate buffer solution via a specific deacetylation mode, followed by desalting, concentration, and lyophilization to obtain chitosan with a specific degree of N-acetylation.

[0005] The chitin deacetylase (PesCDA) and fully deacetylated chitosan were reacted in sodium acetate buffer at 37-60°C for 40-55 h; wherein the mass ratio of PesCDA to fully deacetylated chitosan was 1:25-1:100.

[0006] The reaction system has a pH of 5-7.

[0007] The sodium acetate buffer solution is a 2-3M sodium acetate solution, with the pH adjusted to 5-7 using a 0.1-0.5M acetic acid solution.

[0008] The pH of the system after the reaction was adjusted to 9-11 with NaOH solution, and the polysaccharide precipitated out. The salt was removed by repeated washing with water and centrifugation.

[0009] The fully deacetylated chitosan is a fully deacetylated chitosan with a molecular weight of 100 kDa-3 kDa (preferably 50 kDa-5 kDa) and a degree of deacetylation >99%.

[0010] The fully deacetylated chitosan is prepared by soaking chitosan with a degree of deacetylation of 60%-90% in a 20-40 wt% NaOH solution, and then reacting it at a pressure of 0.11-0.12 MPa and a temperature of 115-125°C for 2-3 hours. This step is repeated once. After the reaction is complete, the chitosan is washed with water until neutral and dried to obtain a fully deacetylated chitosan with a degree of deacetylation >99%.

[0011] The chitin deacetylase (PesCDA) was obtained by heterologous expression of the PesCDA plasmid in E. coli and low-temperature induction to obtain purified PesCDA protein.

[0012] The PesCDA plasmid is based on PMAL-C5x, into which a chitin deacetylase from the genus *Polytrichum* (GenBank acc. No. KY024221) is inserted.

[0013] The heterologous expression of *E. coli* was performed by adding the PesCDA plasmid obtained above to BL21(DE3) competent cells and culturing at 37°C with shaking until OD. 600 The value was around 0.6. The temperature was lowered to 16-18℃ for low-temperature induction culture. The precipitate was collected by centrifugation and then purified by nickel column to obtain purified PesCDA protein.

[0014] Advantages of this invention:

[0015] 1. This invention uses a chitin deacetylase with a specific deacetylation pattern to specifically N-acetylate fully deacetylated chitosan. The enzyme performs deacetylation at different positions between the penultimate and non-reducing units of the glycan chain, with a deacetylation pattern of AAD. n-3 A. When the reaction buffer is a 2-3M sodium acetate solution, the enzyme can add an acetyl group to the deacetylation site, and specific N-acetylated chitosans can be prepared by controlling the reaction time or reaction temperature.

[0016] 2. The enzyme reaction conditions are mild, avoiding the generation of byproducts and environmental pollution, and it is a green preparation method for specific N-acetylated chitosan.

[0017] 3. This invention uses low-temperature induction, which avoids the presence of protein inclusion bodies, purifies the protein, and optimizes the optimal reaction conditions.

[0018] 4. This invention removes salt by adjusting the pH value and centrifuging with water washing, resulting in minimal product loss and is simple and rapid.

[0019] 5. Chitosan with different molecular weights has different applications. This invention screens out the molecular weights with the best N-acetylation effect within a specific range by N-acetylating fully deacetylated chitosan with different relative molecular weights. Specifically, within the molecular weight range of 100 kDa to 3 kDa, under the conditions of pH = 6, 45°C, and a reaction time of 48 h, 5 kDa shows the best N-acetylation effect. Adjusting the reaction conditions is necessary to increase or decrease the degree of N-acetylation at this molecular weight.

[0020] 5. This invention uses fully deacetylated chitosan as raw material. By N-acetylating fully deacetylated chitosan with different relative molecular weights (within a certain range) under specific conditions, chitosan with a specific degree of N-acetylation can be prepared with good reproducibility and is of great significance. Attached Figure Description

[0021] Figure 1 The fully deacetylated chitosan provided in the embodiments of the present invention 1 H-NMR spectrum.

[0022] Figure 2 This is a diagram illustrating the construction of the PesCDA plasmid for the chitin deacetylase provided in an embodiment of the present invention.

[0023] Figure 3 Electrophoresis diagram of purified PesCDA protein provided in an embodiment of the present invention.

[0024] Figure 4 The chitosan with a specific degree of N-acetylation provided in the embodiments of the present invention 1 H-NMR spectrum. Specific Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings, and the scope of protection of the present invention is not limited to the following embodiments.

[0026] This invention provides a method for preparing chitosan with a specific degree of N-acetylation using an enzymatic approach. It uses fully deacetylated chitosan as a substrate and leverages the N-acetylation effect of chitin deacetylase to provide technical support for the preparation of chitosan with a specific degree of N-acetylation.

[0027] Meanwhile, the enzyme used was expressed heterologously in E. coli, and the antibiotic used was ampicillin sodium. Low-temperature induction was used to avoid the formation of protein inclusion bodies, which improved efficiency in the entire protein preparation, expression and purification process.

[0028] Example 1

[0029] Chemical deacetylation was employed. Chitosan with a degree of deacetylation of 80% and a relative molecular weight of 120 kDa was used as the raw material. It was soaked in a 40 wt% sodium hydroxide solution and reacted at 0.11 MPa and 120 °C for 2 hours. This step was repeated once. After the reaction, the mixture was washed with water until neutral and dried to obtain fully deacetylated chitosan with a degree of acetylation of 0.7% and a relative molecular weight of 50 kDa (see [link to product]). Figure 1 ).

[0030] Example 2

[0031] Construction of chitin deacetylase plasmids with specific deacetylation patterns:

[0032] Construction of PesCDA plasmid:

[0033] Following existing methods, the *Polychaetes* fungus (GenBank acc. No. KY024221) and the restriction site Sacl / BamHl in the vector PMAL-C5x were digested. This vector inherently possesses ampicillin resistance. A 6×His tag was added during synthesis, and the tag was ligated via T4 to obtain the plasmid containing the chitin deacetase PesCDA derived from *Polychaetes* fungus (see [link to original text]). Figure 2 The expressed protein can act on the acetyl group between the penultimate or non-reducing ends of the glycan chain. The plasmid used in this invention was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0034] Add 5 μL of the PesCDA plasmid (GenBank acc. No. KY024221, vector PMAL-c5X, cloning site Sacl / BamHl, synthesized by Sangon Biotech (Shanghai) Co., Ltd.) obtained above to BL21(DE3) competent cells, and incubate at 37℃ with shaking for about 3 hours. Measure OD. 600 The value was around 0.6. The temperature was lowered to 16℃ and the rotation speed was reduced to 100 rpm. IPTG was added to bring the final concentration to 0.1 mM, and induction was performed for 15 h. The bacterial cells were collected by centrifugation, the cells were sonicated to disrupt the cell structure, and centrifuged at 12000 rpm for 20 min at 4℃. The supernatant was collected and purified through a nickel column to obtain purified PesCDA protein.

[0035] The nickel column packing material is Ni-agarose gel (HP), a purification medium used for purifying 6×His-tagged recombinant proteins. It is prepared by coupling a tetradentate chelating agent, NTA, with 6% cross-linked Sepharose. NTA has four chelating regions that better bind Ni. 2+ 6×His can be combined with Ni 2+Chelation allows the His-tagged protein to bind to the Ni-NTA medium, while unbound proteins are washed away. Proteins bound to the medium are gently eluted with a certain concentration of imidazole or a low-pH buffer to obtain the target protein in high purity.

[0036] The nickel column purification conditions are as follows: Before purification, the supernatant is filtered using a 0.22 μM filter. 20 column volumes of binding buffer are injected to equilibrate the nickel column. The filtered supernatant is then injected, followed by 10 column volumes of washing buffer to wash away impurities. 2.5 column volumes of elution buffer are injected, and the effluent is collected as the target protein, with a molecular weight of approximately 75 kDa. (See...) Figure 3 ).

[0037] in:

[0038] Binding buffer: 50mM Tris, 150mM NaCl, pH 8.0;

[0039] Washing Buffer: 50mM Tris, 150mM NaCl, 20mM imidazole, pH=8.0;

[0040] Elution Buffer: 50mM Tris, 150mM NaCl, 250mM imidazole, pH=8.0.

[0041] Example 3

[0042] Take 1 mg of PesCDA protein prepared in Example 2 and 50 mg of fully deacetylated chitosan obtained in Example 1, and react them in 20 ml of 2M sodium acetate buffer at 37°C for 48 h at pH 5. After the reaction, adjust the pH to alkaline, wash with water, and centrifuge to remove salt. Concentrate the sample and freeze-dry to obtain specific N-acetylated chitosan.

[0043] Take 20 mg of the product obtained in this example, dissolve it in 90 μL of deuterated water and 10 μL of deuterated hydrochloric acid, and proceed with the reaction. 1 ¹H-NMR analysis of degree of acetylation (DA). The peak at 2.8–3.2 ppm represents the hydrogen atom on the second carbon of GlcN, and the peak at 1.9 ppm represents the hydrogen atom on the acetyl group of GlcNAc. The peak intensity at 1.9 ppm increases linearly with increasing DA. Calculation formula. The integral result shows an acetylation degree of 12% (see...). Figure 4 ).

[0044] Example 4

[0045] Chitosan with a degree of deacetylation of 80% and a relative molecular weight of 120 kDa was used as the raw material. It was soaked in 40 wt% sodium hydroxide solution and reacted at 0.11 MPa and 120 °C for 2 h. After the reaction, it was washed with water until neutral and dried to obtain a product with a degree of acetylation of 0.9% and a relative molecular weight of 100 kDa. 1 mg of the PesCDA protein prepared above and 50 mg of the product with a relative molecular weight of 100 kDa obtained in this example were reacted in 20 ml of 2 M sodium acetate buffer at 45 °C for 48 h, pH = 6. After the reaction, the pH was adjusted to alkaline, and the sample was washed with water and centrifuged to remove salt. The sample was concentrated, freeze-dried, and N-acetylated chitosan with a degree of acetylation of 13% was obtained according to the above method.

[0046] Example 5

[0047] 1 mg of PesCDA protein prepared in Example 2 and 50 mg of fully deacetylated chitosan (relative molecular mass 50 kDa) obtained in Example 1 were reacted in 20 mL of 2 M sodium acetate buffer at 45 °C for 48 h, pH = 6. After the reaction, the pH was adjusted to alkaline, and the sample was washed with water and centrifuged to remove salt. The sample was concentrated, freeze-dried, and N-acetylated chitosan with a degree of acetylation of 15% was determined according to the above method.

[0048] Example 6

[0049] The fully deacetylated chitosan prepared in Example 1 was reacted with chitosanase at a sugar-to-enzyme ratio of 5:1, pH=7, and 37℃ for 10 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes, respectively, to obtain fully deacetylated chitosan with molecular weights of 20 kDa, 10 kDa, 8 kDa, 5 kDa, and 3 kDa (acetylation degree of 0.7%).

[0050] 1 mg of the prepared PesCDA protein and 50 mg of the product obtained in this example were reacted separately in 20 ml of 2M sodium acetate buffer at 45°C for 48 h, pH = 6. After the reaction, the pH was adjusted to alkaline, and the samples were washed with water and centrifuged to remove salt. The samples were concentrated, freeze-dried, and N-acetylated chitosan with acetylation degrees of 19%, 21%, 21%, 30%, and 13% were determined according to the above method.

[0051] Examples 4, 5, and 6 show that within the relative molecular weight range of 100 kDa to 3 kDa, the molecular weight around 5 kDa has the most acetyl groups.

[0052] Example 7

[0053] 1 mg of PesCDA protein prepared in Example 2 and 50 mg of fully deacetylated chitosan (relative molecular mass 50 kDa) obtained in Example 1 were respectively reacted in 20 mL of 2M sodium acetate buffer at 45 °C for 48 h, with pH values ​​of 4, 5, 6, 7, and 8. After the reaction, the pH was adjusted to alkaline, and the samples were washed with water and centrifuged to remove salt. The samples were concentrated, freeze-dried, and N-acetylated chitosan with acetylation degrees of 11%, 38%, 43%, 25%, and 10% were determined according to the above method.

[0054] Example 8

[0055] Comparative Example 7: 1 mg of the prepared PesCDA protein and 50 mg of the product with a relative molecular weight of 50 kDa obtained in Example 1 were reacted in 20 ml of 2M sodium acetate buffer at 45°C for 48 h, pH = 6. After the reaction, the pH was adjusted to alkaline, and the sample was washed with water and centrifuged to remove salt. The sample was concentrated, freeze-dried, and N-acetylated chitosan with a degree of acetylation of 45% was determined according to the above method.

[0056] Therefore, the N-acetylation method of the present invention has good reproducibility.

Claims

1. A method for preparing a chitosan with a specific degree of N-acetylation, characterized in that: Chitosan with a specific degree of N-acetylation was obtained by reacting fully deacetylated chitosan with a chitin deacetylase (PesCDA) in sodium acetate buffer via a specific deacetylation mode. After the reaction, the chitosan was desalted, concentrated, and lyophilized to obtain chitosan with a specific degree of N-acetylation. The PesCDA is a chitin deacetylase from the fungus *Plasmodium* genus, GenBank acc. No. KY024221; the chitin deacetylase (PesCDA) and fully deacetylated chitosan are reacted in sodium acetate buffer at 37-60°C for 40-55 h; wherein the mass ratio of PesCDA to fully deacetylated chitosan is 1:25-1:100; The pH of the system after the reaction was adjusted to 9-11 with NaOH solution, and the polysaccharide precipitated out. The salt was removed by repeated washing with water and centrifugation. The fully deacetylated chitosan has a molecular weight between 100 kDa and 3 kDa and a degree of deacetylation > 99%.

2. The method for preparing chitosan with a specific N-acetylation degree according to claim 1, characterized in that: The reaction system has a pH of 5-7.

3. The method for preparing chitosan with a specific N-acetylation degree according to claim 1, characterized in that: The sodium acetate buffer solution is a 2-3M sodium acetate solution, with the pH adjusted to 5-7 using a 0.1-0.5M acetic acid solution.

4. The method for preparing chitosan with a specific N-acetylation degree according to claim 1, characterized in that: The chitin deacetylase (PesCDA) was obtained by heterologous expression of the PesCDA plasmid in Escherichia coli and induction at a low temperature of 16-18℃ to obtain the purified PesCDA protein.

5. The method for preparing chitosan with a specific N-acetylation degree according to claim 4, characterized in that: The fully deacetylated chitosan was prepared by soaking chitosan with a degree of deacetylation of 60%-90% in a 20wt%-40wt% NaOH solution, and then reacting it at a pressure of 0.11-0.12MPa and a temperature of 115-125℃ for 2-3 hours. This step was repeated once. After the reaction was completed, the chitosan was washed with water until neutral and dried to obtain a fully deacetylated chitosan with a degree of deacetylation >99%.

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