Chitotetraose with anti-inflammatory activity as well as preparation method and application of chitotetraose
Through the selective knockout and enzymatic reaction of chitin deacetylase, chitotetrasaccharides were accurately prepared, solving the problem of complexity of chitooligosaccharide separation and uncertain anti-inflammatory activity in the prior art, and obtaining high purity and anti-inflammatory The highly active chitosaccharide is suitable for the pharmaceutical field.
Patent Information
- Application Number
- CN202411579328.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-07
- Publication Date
- 2024-12-27
AI Technical Summary
The prior art is difficult to accurately isolate and prepare chitooligosaccharides with specific sequences, resulting in unclear structure-activity relationships of their anti-inflammatory activity and complex and uncertain separation process.
Selective knockout of specific sites of chitin deacetylase is used to accurately prepare chitosaccharide with anti-inflammatory activity through alcohol precipitation, gel column separation and enzymatic reactions, avoiding by-products and environmental pollution.
The preparation of chitosaccharides with high purity and specific sequences has been achieved, which significantly improves anti-inflammatory activity, is suitable for development in the pharmaceutical field, and has a gentle and environmentally friendly process.
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Figure CN119192258A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of marine bioengineering, and particularly relates to chitotetraose with anti-inflammatory activity and a preparation method and application thereof. Background Art
[0002] Chitin is a natural polysaccharide widely found in the shells of marine crustaceans. Its stock in nature is second only to cellulose, and it is the second largest natural polymer compound. It is a recyclable resource. Chitosan is the product of N-deacetylation of chitin, and is composed of N-acetylglucose (GlcNAc, A) and glucosamine (GlcN, D) units. Chitooligosaccharides are degradation products of chitosan, which can be produced by enzymatic hydrolysis, separation and chemical synthesis, and the degree of polymerization is generally 2-20. Chitooligosaccharides have excellent properties such as good water solubility, easy absorption by the human body, and high biological activity. These unique properties make it widely used in the fields of plant stimulants, functional food development and biomedicine.
[0003] At present, the biological activities of chitosan oligosaccharides include anti-cancer, anti-inflammatory, antioxidant, anti-diabetic, anti-hypertensive effects, and promotion of wound healing. Therefore, it has great potential for application development. Among the many biological activities of chitosan oligosaccharides, anti-inflammatory activity has attracted extensive attention from scholars at home and abroad. Chitosan oligosaccharides have been shown to have good anti-inflammatory activity in various inflammation models in vivo and in vitro, and are expected to become a good natural anti-inflammatory drug.
[0004] In previous studies on the anti-inflammatory activity of chitosan oligosaccharides, complex chitosan oligosaccharide mixtures were used as test samples. Their structural parameters such as degree of polymerization (DP), degree of acetylation (DA), and acetylation pattern (DP) will affect their anti-inflammatory activity. The disorder of the structure leads to unclear structure-activity relationship. Studies have shown that chitosan oligosaccharides with DA = 12% have better anti-inflammatory activity. However, chitosan oligosaccharides of many sequences are still mixed in this DA = 12%. It is difficult to separate these sequences one by one with current separation technology, and there is a certain degree of blindness. Summary of the invention
[0005] In order to solve the above technical problems, the present invention provides a chitosan with anti-inflammatory activity and a preparation method and application thereof, and utilizes the specific site selectivity of chitin deacetylase (CDA) to achieve the knockout of acetyl groups at specific sites in the sugar chain, thereby accurately preparing chitosan oligosaccharides of specific sequences, which is of great significance for identifying the active center sequence of chitosan oligosaccharides that exert anti-inflammatory activity.
[0006] To achieve the above object, the technical solution of the present invention is as follows:
[0007] A chitotetraose with anti-inflammatory activity, the structural formula of which is as follows:
[0008]
[0009] Wherein, D represents a glucosamine unit and A represents an N-acetylglucosamine unit.
[0010] A method for preparing the chitotetraose having anti-inflammatory activity as described above comprises the following steps:
[0011] (1) subjecting chitosan oligosaccharides to alcohol precipitation to remove monosaccharides, thereby obtaining alcohol-precipitated chitosan oligosaccharides;
[0012] (2) separating the alcohol-precipitated chitosan oligosaccharide through a gel column Bio Rad P10 to obtain fully N-acetylated chitosan tetraose;
[0013] (3) reacting fully N-acetylated chitosan with chitin deacetylase VcCOD, followed by freeze drying to obtain chitosan ADAA;
[0014]
[0015] (4) Chitosan tetrasaccharide ADAA is reacted with chitin deacetylase NodB, and then desalted and freeze-dried to obtain chitosan tetrasaccharide DDAA with anti-inflammatory activity.
[0016] In the above scheme, the specific method of step (1) is as follows: chitosan oligosaccharide is put into anhydrous ethanol, allowed to stand overnight, centrifuged to obtain the supernatant, and freeze-dried to obtain alcohol-precipitated chitosan oligosaccharide, wherein the mass volume ratio of chitosan oligosaccharide to anhydrous ethanol is 1g:80-150mL.
[0017] In the above scheme, in step (2), the elution buffer solution of the gel column Bio Rad P10 is a 20-70 mM ammonium acetate solution.
[0018] In a further technical solution, the flow rate of the ammonium acetate solution is 0.2-0.8 min / mL.
[0019] In the above scheme, chitin deacetylase VcCOD and NodB are prepared by heterologously expressing chitin deacetylase VcCOD and NodB plasmids in Escherichia coli, and obtaining purified VcCOD and NodB proteins by low temperature induction at 15-20°C.
[0020] In the above scheme, the specific method of step (3) is as follows: reacting fully N-acetylated chitotetraose and chitin deacetylase VcCOD in an ammonium bicarbonate buffer solution with a concentration of 8-15mM at 34-40°C for 12-20h, and freeze-drying after the reaction to obtain chitotetraose ADAA; wherein the mass ratio of chitotin deacetylase VcCOD to N-acetylated chitotetraose is not less than 1:20, and each milligram of fully N-acetylated chitotetraose is added to 200-800uL of ammonium bicarbonate buffer solution.
[0021] In the above scheme, the specific method of step (4) is as follows:
[0022] A mixed buffer of 3-morpholinepropanesulfonic acid, dithiothreitol and manganese sulfate is prepared, chitosan ADAA and chitin deacetylase NodB are added to the mixed buffer, the pH is adjusted to 6-8, the mixture is reacted at 36-40°C for 12-20h, and after the reaction, the mixture is desalted and freeze-dried to obtain chitosan DDAA with anti-inflammatory activity; wherein, after adding chitosan ADAA and chitosan deacetylase NodB, the concentrations of 3-morpholinepropanesulfonic acid, dithiothreitol and manganese sulfate are 10-30mM, 7-15mM and 0.8-3mM, respectively, the mass ratio of chitosan deacetylase NodB to chitosan ADAA is not less than 1:20, and 200-800uL of the mixed buffer is added per milligram of chitosan ADAA.
[0023] A use of the chitotetraose having anti-inflammatory activity as described above in the preparation of anti-inflammatory drugs.
[0024] Through the above technical scheme, the present invention provides a chitotetraose with anti-inflammatory activity and a preparation method and application thereof, which have the following beneficial effects:
[0025] 1. The present invention uses a gel column to separate the substrate first and then perform enzymatic hydrolysis for fixed-point removal, thereby avoiding the complexity and uncertainty of directly separating chitosan oligosaccharides with different polymerization degrees and chitosan oligosaccharide isomer mixtures with different sequence arrangements, and can directly prepare a target chitosan tetraose product with a specific sequence structure.
[0026] 2. The enzyme reaction conditions in the present invention are mild, which avoids the generation of by-products and environmental pollution. It is an environmentally friendly method for preparing specific sequence chitotetraose.
[0027] 3. The low temperature used in the present invention to induce bacterial protein production avoids the existence of protein inclusion bodies and improves protein yield.
[0028] 4. The chitotetraose of a specific sequence obtained by the present invention does not contain any nucleic acid and protein, has high purity, good water solubility, and good anti-inflammatory activity, and can be used for the development of the medical field. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.
[0030] Figure 1 A flow chart of preparing chitotetraose DDAA provided in an embodiment of the present invention.
[0031] Figure 2 This is a separation elution curve of alcohol-precipitated chitosan oligosaccharides provided in an embodiment of the present invention.
[0032] Figure 3 This is the mass spectrum of fully N-acetylated chitotetraose provided in Example 1 of the present invention.
[0033] Figure 4 This is the mass spectrum of chitotetraose ADAA prepared in Example 1 of the present invention, (a) is the primary mass spectrum, and (b) is the secondary mass spectrum.
[0034] Figure 5 The mass spectrum of the chitotetraose DDAA prepared in Example 1 of the present invention, (a) is the primary mass spectrum, and (b) is the secondary mass spectrum.
[0035] Figure 6 The mass spectrum of chitotetraose DAAA prepared in Comparative Example 4 of the present invention, (a) is the primary mass spectrum, and (b) is the secondary mass spectrum.
[0036] Figure 7 This is the mass spectrum of chitotetraose DDDA prepared in Comparative Example 5 of the present invention, (a) is the primary mass spectrum, and (b) is the secondary mass spectrum.
[0037] Figure 8 This is a screening diagram for the anti-inflammatory activity of four types of chitotetraosides prepared in the present invention. DETAILED DESCRIPTION
[0038] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention.
[0039] The present invention provides a method for preparing chitotetraose having anti-inflammatory activity, and the preparation process is as follows: Figure 1 As shown, the specific embodiments are as follows:
[0040] Example 1
[0041] 1. Preparation of chitin deacetylase VcCOD, NodB, ArCE4:
[0042] Chitin deacetylase VcCOD, NodB, and ArCE4 plasmids were added to the competent cells BL21 (DE3) respectively, and the cells were cultured with shaking at 37°C for 3 hours. The OD600 value was measured to be about 0.6, the temperature was lowered to 16°C, IPTG (isopropanol-β-D-thiogalactoside) was added to a final concentration of 0.1 mM, and the cells were induced for 15 hours. The bacteria were collected by centrifugation, the cells were broken by ultrasound, and the cells were centrifuged at 4°C and 10000 rpm for 20 minutes. The supernatant was collected and the protein was purified by nickel column to obtain purified chitin deacetylase VcCOD, NodB, and ArCE4 proteins.
[0043] 2. Separation of fully N-acetylated chitotetraose
[0044] 1 g of chitosan oligosaccharide was added to 100 mL of anhydrous ethanol, allowed to stand overnight, centrifuged, and the supernatant was taken out and freeze-dried to obtain alcohol-precipitated chitosan oligosaccharide.
[0045] Prepare a 100 mg / mL alcohol-precipitated chitosan oligosaccharide solution, adhere it to the wall and slowly add it to the Bio Rad P10 gel column. Use 50 mM ammonium acetate buffer and elute at a rate of 0.2 mL / min. After elution, detect the eluate by NanoDrop (micro-spectrophotometer), and collect and detect 6 eluate components (see Figure 2 ), collect the eluate in tubes 89-95, and freeze-dry to obtain uniform molecular weight full N-acetylated chitotetraose (AAAA), with a molecular weight of 830Da. Characterization spectrum see Figure 3 .
[0046] 3. Preparation of Chitotetraose ADAA
[0047] At 37°C, fully N-acetylated chitosan and chitosan deacetylase VcCOD were reacted in a 10mM ammonium bicarbonate buffer for 12 hours, and then freeze-dried to obtain chitosan ADAA; wherein the mass ratio of chitosan deacetylase VcCOD to N-acetylated chitosan was 1:20, and 500uL of ammonium bicarbonate buffer was added per mg of fully N-acetylated chitosan. The primary mass spectrometry characterization of chitosan ADAA is shown in Figure 4 (a), secondary mass spectrometry characterization see Figure 4 In (b), its structural formula is as follows:
[0048]
[0049] 4. Preparation of chitotetraose DDAA
[0050] Prepare a mixed buffer of 3-morpholinepropanesulfonic acid (Mops), dithiothreitol (DTT) and manganese sulfate, add chitosan ADAA and chitin deacetylase NodB to the mixed buffer, adjust the pH to 6-8, react at 36-40°C for 16 hours, desalt and freeze-dry after the reaction to obtain chitosan DDAA with anti-inflammatory activity; wherein, after adding chitosan ADAA and chitosan deacetylase NodB, the concentrations of 3-morpholinepropanesulfonic acid, dithiothreitol and manganese sulfate are 20mM, 10mM and 1mM respectively, the mass ratio of chitosan deacetylase NodB and chitosan ADAA is 1:20, and 500uL of mixed buffer is added for each milligram of chitosan ADAA. Primary mass spectrometry characterization of chitosan DDAA is shown in Figure 5 (a), secondary mass spectrometry characterization see Figure 5 In (b), its structural formula is as follows:
[0051]
[0052] Comparative Example 1
[0053] During the preparation of chitin deacetylase VcCOD, NodB, and ArCE4, chitin deacetylase VcCOD, NodB, and ArCE4 plasmids were added to the competent cells BL21 (DE3), respectively, and cultured at 37°C with shaking for 3 hours. The OD600 value was measured to be about 0.6, the temperature was lowered to 25°C, IPTG (isopropanol-β-D-thiogalactoside) was added to a final concentration of 0.1 mM, induced for 15 hours, and the bacteria were collected by centrifugation. The cells were ultrasonically broken and centrifuged at 4°C and 10,000 rpm for 20 minutes. The supernatant was collected and the protein was purified by a nickel column, and the protein yield was greatly reduced.
[0054] Comparative Example 2
[0055] In the separation process of fully N-acetylated chitosan, 1g of chitosan oligosaccharide is put into 100mL of anhydrous ethanol, left to stand overnight, centrifuged to obtain the supernatant, and freeze-dried to obtain alcohol-precipitated chitosan oligosaccharide. A 100mg / mL alcohol-precipitated chitosan oligosaccharide solution is prepared, which is slowly added to the Bio Rad P10 gel column and eluted at a speed of 0.1mL / min using 50mM ammonium acetate buffer. After the elution is completed, the eluate flowing out is detected by NanoDrop (micro-spectrophotometer). The elution curve obtained has only two peaks, and the corresponding number of tubes are collected and freeze-dried. The molecular weight of the obtained product is uneven.
[0056] Comparative Example 3
[0057] In the separation process of fully N-acetylated chitosan, 1g of chitosan oligosaccharide was put into 100mL of anhydrous ethanol, allowed to stand overnight, centrifuged to obtain the supernatant, and freeze-dried to obtain alcohol-precipitated chitosan oligosaccharide. A 100mg / mL alcohol-precipitated chitosan oligosaccharide solution was prepared, which was slowly added to the Bio Rad P10 gel column and eluted at a speed of 0.2mL / min using 10mM ammonium acetate buffer. After the elution was completed, the eluate was detected by NanoDrop (micro-spectrophotometer), and the peak signal values of the obtained elution curve were very low.
[0058] Comparative Example 4
[0059] A mixed buffer of 3-morpholinepropanesulfonic acid (Mops), dithiothreitol (DTT) and manganese sulfate was prepared, fully N-acetylated chitotetraose and chitin deacetylase NodB were added to the mixed buffer, the pH was adjusted to 8, and the mixture was reacted at 37°C for 12 hours. After the reaction, the mixture was desalted and freeze-dried to obtain chitotetraose DAAA. After the addition of fully N-acetylated chitotetraose and chitin deacetylase NodB, the concentrations of 3-morpholinepropanesulfonic acid, dithiothreitol and manganese sulfate were 20mM, 10mM and 1mM, respectively, and the mass ratio of chitin deacetylase NodB to fully N-acetylated chitotetraose was 1:20. The primary mass spectrometry characterization of chitotetraose DAAA is shown in Figure 6 (a), secondary mass spectrometry characterization see Figure 6 In (b), its structural formula is as follows:
[0060]
[0061] Comparative Example 5
[0062] At 37°C, fully N-acetylated chitotetraose and chitin deacetylase ArCE4 were mixed with 50 mM Tris-HCl and 10 uM CoCl 2 The reaction was carried out in a buffer solution, the pH was adjusted to 8, the reaction was carried out for 40 hours, and after the reaction, the salt was removed and freeze-dried to obtain chitosan tetraose DDDA; wherein the mass ratio of chitosan deacetylase ArCE4 to fully N-acetylated chitosan tetraose was 1:160. The primary mass spectrometry characterization of chitosan tetraose DDDA is shown in Figure 7 (a), secondary mass spectrometry characterization see Figure 7 In (b), its structural formula is as follows:
[0063]
[0064] Anti-inflammatory activity test:
[0065] In RAW264.7 cells, the five chitosan tetrasaccharides (AAAA, ADAA, DDAA, DAAA, DDDA) samples prepared above were prepared into 10mM mother solution, which was diluted to 1mM with cell culture medium. Lipopolysaccharide LPS1ug / ml was used as a positive control, and the blank group (culture medium) was used as a negative control. The supernatant was taken after incubation for 24h. The NO release was determined by the Griess method, and the chitosan tetrasaccharide with the best anti-NO burst effect was selected. The results are shown in Figure 8 As shown, it can be seen that chitotetraose DDAA has good anti-inflammatory activity.
[0066] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A chitotetraose having anti-inflammatory activity, characterized in that: Its structural formula is as follows: Wherein, D represents a glucosamine unit and A represents an N-acetylglucosamine unit.
2. A method for preparing chitotetraose having anti-inflammatory activity as claimed in claim 1, characterized in that: The steps include: (1) subjecting chitosan oligosaccharides to alcohol precipitation to remove monosaccharides, thereby obtaining alcohol-precipitated chitosan oligosaccharides; (2) separating the alcohol-precipitated chitosan oligosaccharide through a gel column Bio Rad P10 to obtain fully N-acetylated chitosan tetraose; (3) reacting fully N-acetylated chitosan with chitin deacetylase VcCOD, followed by freeze drying to obtain chitosan ADAA; (4) Chitosan tetrasaccharide ADAA is reacted with chitin deacetylase NodB, and then desalted and freeze-dried to obtain chitosan tetrasaccharide DDAA with anti-inflammatory activity.
3. The method for preparing chitotetraose with anti-inflammatory activity according to claim 2, characterized in that: The specific method of step (1) is as follows: chitosan oligosaccharide is put into anhydrous ethanol, allowed to stand overnight, centrifuged to obtain the supernatant, and freeze-dried to obtain alcohol-precipitated chitosan oligosaccharide, wherein the mass volume ratio of chitosan oligosaccharide to anhydrous ethanol is 1g:80-150mL.
4. The method for preparing chitotetraose having anti-inflammatory activity according to claim 2, characterized in that: In step (2), the elution buffer solution of the gel column Bio Rad P10 is a 20-70 mM ammonium acetate solution.
5. The method for preparing chitotetraose having anti-inflammatory activity according to claim 4, characterized in that: The flow rate of the ammonium acetate solution is 0.2-0.8 min / mL.
6. The method for preparing chitotetraose having anti-inflammatory activity according to claim 2, characterized in that: Chitin deacetylase VcCOD and NodB are prepared by heterologously expressing chitin deacetylase VcCOD and NodB plasmids in Escherichia coli and inducing them at low temperature of 15-20°C to obtain purified VcCOD and NodB proteins.
7. The method for preparing chitotetraose having anti-inflammatory activity according to claim 2, characterized in that: The specific method of step (3) is as follows: reacting fully N-acetylated chitosan and chitin deacetylase VcCOD in an ammonium bicarbonate buffer solution with a concentration of 8-15mM at 34-40°C for 12-20h, freeze-drying after the reaction, and obtaining chitosan ADAA; wherein the mass ratio of chitosan deacetylase VcCOD to N-acetylated chitosan is not less than 1:20, and each milligram of fully N-acetylated chitosan is added to 200-800uL of ammonium bicarbonate buffer solution.
8. The method for preparing chitotetraose with anti-inflammatory activity according to claim 2, characterized in that: The specific method of step (4) is as follows: A mixed buffer of 3-morpholinepropanesulfonic acid, dithiothreitol and manganese sulfate is prepared, chitosan ADAA and chitin deacetylase NodB are added to the mixed buffer, the pH is adjusted to 6-8, the mixture is reacted at 36-40°C for 12-20h, and after the reaction, the mixture is desalted and freeze-dried to obtain chitosan DDAA with anti-inflammatory activity; wherein, after adding chitosan ADAA and chitin deacetylase NodB, the concentrations of 3-morpholinepropanesulfonic acid, dithiothreitol and manganese sulfate are 10-30mM, 7-15mM and 0.8-3mM, respectively, the mass ratio of chitin deacetylase NodB to chitosan ADAA is not less than 1:20, and 200-800uL of the mixed buffer is added per milligram of chitosan ADAA.
9. Use of the chitotetraose with anti-inflammatory activity as claimed in claim 1 in the preparation of anti-inflammatory drugs.