CN118146353A - Method for removing glycosaminoglycan in collagen by using elastase Myroilysin - Google Patents
Method for removing glycosaminoglycan in collagen by using elastase Myroilysin Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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Abstract
A method for removing glycosaminoglycan from collagen by elastase Myroilysin is provided. The amino acid sequence number of the elastase Myroilysin is GenBank: ACG59772.1. The method comprises the steps of preparing the cod skin into acid-soluble collagen, and treating the acid-soluble collagen by using elastase Myroilysin. The inventor surprisingly discovers that the elastase Myroilysin has a new function of removing glycosaminoglycan, and the enzyme can loosen the protein structure but does not destroy the triple helix structure of the protein, so that the application further treats acid-soluble collagen by using the enzyme, effectively reduces the content of the glycosaminoglycan in the collagen, does not introduce other miscellaneous proteins, and keeps the high purity of the collagen, so that the purity of the collagen prepared by the method is up to 99.66%, the application range of the protein is enlarged, and the application is beneficial to the preparation of related products in the fields of medicine, cosmetics, tissue engineering and the like. Meanwhile, the utilization rate and the added value of fish offal are improved, and good environmental benefit and social benefit are obtained.
Description
Technical Field
The invention relates to a method for removing glycosaminoglycan from collagen by using elastase Myroilysin, belonging to the technical field of biotechnology.
Background
The sea area of China is wide, the sea resources are rich, and the method is not only a large country for producing the aquatic products, but also a main export country for the aquatic products. The statistics of fishery in 2020 shows that: in 2019, the aquaculture amount of China is 5079.07 ten thousand tons, and the fish aquaculture amount is 2708.61 ten thousand tons. The current aquatic product cultures in international trade account for about 1/4 of the total amount of trade and 1/3 of the total amount of trade. In 2018, the cod trade amount accounts for about 10% of the international trade total amount of the aquatic products. The huge processing amount and export amount of the aquatic products can inevitably generate a large amount of offal such as fish skin, and even the aquatic products with 40 to 50 percent of the fishing amount are discarded and even cause environmental pollution although partial byproducts rich in protein are processed into cultivation feeds, crop fertilizers and the like for the second time. In addition, marine animal-derived collagen is considered as an important alternative source of land-animal-derived collagen for the reasons of outbreaks of Bovine Spongiform Encephalopathy (BSE), transmissible Spongiform Encephalopathy (TSE), and foot-and-mouth disease (FMD), among other factors. Collagen is an important constituent of cod skin, accounting for about 60%, and has been widely used in the fields of foods, medicines, tissue engineering, cosmetics, etc. due to its excellent biocompatibility, low antigenicity, low allergy, etc.
Collagen is the most abundant protein in animals, and 29 types have been found, the most predominant of which is type I collagen. The structure of Collagen fibers (Collagen fibers) in animal tissues is complex. First, the procollagen molecules (protocollagen) form collagen fibrils (Collagen fibril) through intermolecular charge effects and various covalent crosslinks; then, many Collagen fibrils are stacked in parallel in the lateral direction, and by interaction with components such as proteoglycan in the extracellular matrix, a Collagen fiber (Collagen fiber) having a compact structure and a certain mechanical strength is formed. Thus, the collagen fibers extracted from animal tissues contain not only collagen but also proteoglycan and other components. Proteoglycans (proteoglycan, PG) are polymers formed by the core protein together with glycosaminoglycan (GAG) chains attached thereto. Proteoglycans adhere to the surface of collagen fibrils through electrostatic adsorption of adjacent glycosaminoglycan chains and the binding of core proteins to collagen, which plays an important role in maintaining the compact structure and strength of collagen fibers.
The preparation method of the collagen mainly comprises acid extraction, alkali extraction, hot water extraction, enzymolysis extraction and the like. The collagen obtained by the acid dissolution method can keep a complete triple helix structure, and has higher extraction purity, but the glycosaminoglycan is not removed; chemical bonds of collagen extracted by an alkaline method can be destroyed, so that the collagen structure becomes loose, and the complete structure of the collagen can be destroyed by peptide bond hydrolysis; the three-helix structure of the collagen is easy to change and is denatured into gelatin when hot water is extracted; enzymatic extraction is of interest because of its mild conditions of action, and the three-helix structure of the collagen obtained by extraction is complete. However, the collagen extracted by the existing enzymatic method does not remove proteoglycan, so that the extracted collagen contains proteoglycan and has lower purity. Because glycosaminoglycan chains in proteoglycan are easy to serve as antigens to cause immune response of organisms, products such as cell growth scaffolds, sutures and hemostatic sponges prepared by utilizing the collagen are likely to cause immune response of organisms after being implanted into human bodies. Thus, there is currently no method for removing glycosaminoglycans from collagen.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for removing glycosaminoglycan in collagen by using elastase Myroilysin. The method firstly utilizes an acid extraction method to extract collagen, then utilizes elastase Myroilysin to further treat acid-soluble collagen, obtains collagen with low glycosaminoglycan content and high purity, and improves the added value of fish skin which is a byproduct of aquatic product processing.
The technical scheme of the invention is as follows:
Use of elastase Myroilysin for removing glycosaminoglycans from collagen and preparing collagen with low glycosaminoglycan content, wherein the amino acid sequence number of elastase Myroilysin is GenBank: ACG59772.1.
This elastase Myroilysin is a metalloprotease previously discovered by the inventors to be secreted by psychrophilic bacteria (Myroides profundi) D25. The enzyme can efficiently degrade elastin and has obvious expansion effect on collagen. Then, after further intensive researches of the inventor, the inventor unexpectedly finds that the elastase Myroilysin has a new function of removing glycosaminoglycan in collagen, and the enzyme can make a protein structure loose, but can not damage a triple helix structure of the protein, so that the application further treats acid-soluble collagen by using the enzyme, effectively reduces the content of glycosaminoglycan in the collagen, can not introduce other hybrid proteins, and keeps the high purity of the collagen, so that the purity of the collagen prepared by the method is up to 99.66%, the application range of the protein is enlarged, and the application is beneficial to the preparation of related products in the fields of medicine, cosmetics, tissue engineering and the like. Meanwhile, the utilization rate and the added value of fish offal are improved, and good environmental benefit and social benefit are obtained.
A method for removing glycosaminoglycans from collagen by elastase Myroilysin, comprising the steps of:
(1) Soaking the cod skin in NaOH solution for 24-48 h, washing with distilled water to neutrality, then continuously soaking in n-butanol solution for 24h, washing with distilled water to neutrality, and obtaining pretreated cod skin;
(2) Adding acetic acid solution into the pretreated cod skin, soaking and extracting for 60-72 h to obtain collagen-containing solution; adding NaCl into the collagen-containing solution, standing for 1-3 h, and centrifuging to obtain collagen precipitate; re-dissolving the collagen precipitate, dialyzing, and freeze-drying to obtain acid-soluble collagen;
(3) Soaking acid-soluble collagen in ultrapure water, then adding elastase Myroilysin to make the final concentration of the collagen be 0.5-1 mg/ml, reacting for 20-30 h at 37 ℃, removing glycosaminoglycan in the collagen, washing with ultrapure water after the reaction is finished, and freeze-drying to obtain the collagen with low glycosaminoglycan content.
According to the invention, in the step (1), the feed liquid ratio of the cod skin to the NaOH solution is 1:10, and the concentration of the NaOH solution is 0.1M; the temperature at which the NaOH solution was soaked was 4 ℃, and the NaOH solution was changed every 12 hours.
According to the invention, in the step (1), the feed liquid ratio of the cod skin to the n-butanol solution is 1:10, and the concentration of the n-butanol solution is 10%; the temperature at which the n-butanol solution was immersed was 4℃and the n-butanol solution was replaced every 12 hours.
According to the invention, in the step (2), the feed liquid ratio of the cod skin to the acetic acid solution is 1:10, and the concentration of the acetic acid solution is 0.5M; the soaking and extracting temperature is 4 ℃, and the soaking and extracting time is 72 hours.
According to a preferred embodiment of the present invention, in step (2), naCl is added to a final concentration of 0.9M and then allowed to stand at 4℃for 2 hours.
According to a preferred embodiment of the present invention, in step (3), the dialysis is specifically: the dialysis is carried out for 24 hours by using acetic acid solution with the concentration of 0.1M, then the dialysis is carried out for 3-5 days by using ultrapure water, and the dialysate is replaced every 12 hours.
According to the present invention, preferably, in the step (4), the pH of the ultrapure water is 8.5 to 9.0; the feed liquid ratio of the acid-soluble collagen to the ultrapure water is 5:1.
According to a preferred embodiment of the present invention, in step (4), the final concentration of elastase Myroilysin is 0.75mg/mL and the reaction time is 24 hours.
The invention has the technical characteristics and beneficial effects that:
1. The inventor of the application surprisingly discovers that elastase Myroilysin has a novel function of removing glycosaminoglycan in collagen, and the enzyme can make the protein structure loose, but does not damage the triple helix structure of the protein, so that the elastase can effectively remove the glycosaminoglycan in the collagen, expand the application range of the collagen, and is used for preparing related products in the fields of medicine, cosmetics, tissue engineering and the like.
2. The invention provides a method for removing glycosaminoglycan in collagen by using elastase Myroilysin, which effectively reduces the content of the glycosaminoglycan in the collagen, does not introduce other foreign proteins, and keeps the high purity of the collagen, so that the purity of the collagen prepared by the method is up to 99.66%.
3. The fish skin is used as a fish processing byproduct, a large amount of collagen is not fully utilized, and the cod skin is used as a raw material to extract the collagen therein, so that the utilization rate and the added value of fish offal are improved, and good environmental benefit and social benefit are obtained.
Drawings
FIG. 1 is a graph showing the change in glycosaminoglycan (GAGs) content in supernatant of bovine Achilles tendon type I collagen treated with elastase Myroilysin.
FIG. 2 is a SDS-PAGE gel electrophoresis of acid-soluble collagen, acid-soluble collagen after enzymatic hydrolysis of collagenase VhaC and collagenase VhaC;
In the figure, lane 1: acid-soluble collagen; lane 2: acid-soluble collagen obtained after enzymolysis of collagenase VhaC; lane 3: collagenase VhaC.
FIG. 3 is a graph showing the change in glycosaminoglycan (GAGs) content of supernatant obtained by treating acid-soluble collagen with elastase Myroilysin.
FIG. 4 is a circular dichroism spectrum of bovine Achilles tendon type I collagen and cod skin collagen after elastase Myroilysin treatment.
Detailed Description
The technical scheme of the invention is further described below by referring to examples, the scope of the invention is not limited thereto.
Biological material source
Cod skin is sold by Qingdao sea big biological group Co., ltd (China); bovine Achilles tendon type I collagen was purchased from Worthington Biochemical (USA), and bran, soybean meal, and corn meal were purchased from Shandong province department of agricultural sciences. Sea salt, fu Lin Fen were purchased from Sigma, na 2CO3, trichloroacetic acid, tyrosine, glacial acetic acid, n-butanol were purchased from the national drug group. The preparation raw materials of the culture medium are all common raw materials in the field and can be purchased in the market.
The deep sea psychrophilic bacteria (Myroides profundi) D25 strain in the examples is purchased from China center for type culture Collection, and the strain collection number is CCTCC M2012534. The elastase myroilysin used in the examples was obtained according to the method described in chinese patent document CN103589680 a.
The collagenase VhaC used was obtained as described in article "Structure of Vibrio collagenase VhaC provides insight into the mechanism of bacterial collagenolysis".
After collagen is treated by elastase Myriolysin, glycosaminoglycan can be released by degrading the cross-links between collagen and glycosaminoglycan, and the content of glycosaminoglycan in the supernatant can be increased accordingly, so that the removal degree of glycosaminoglycan in collagen can be judged by measuring the content of glycosaminoglycan in the supernatant.
Example 1
Bovine achilles tendon type I collagen was mixed with ultrapure water (pH 9.0) at a feed solution ratio of 5:1 (M/V, mg/ml), then elastase Myroilysin was added to a final concentration of 1mg/ml as a reaction group (bovine achilles tendon collagen + Myroilysin), and inactivated elastase Myroilysin and water were used as a control group 1 (bovine achilles tendon collagen +inactivated Myroilysin) and a control group 2 (bovine achilles tendon collagen +water), and then reacted at 37℃for 24 hours, and 20. Mu.L of reaction supernatant was taken at reaction times of 5 hours, 17 hours, 18.5 hours, 24 hours, respectively, and the glycosaminoglycan (GAGs) content in the reaction supernatant was measured, and the results are shown in FIG. 1. And repeatedly flushing protein precipitate by using ultrapure water after the reaction is finished, and freeze-drying again to obtain the bovine achilles tendon type I collagen.
As can be seen from fig. 1, the GAGs content in the supernatant of the reaction group (bovine achilles tendon collagen + Myroilysin) gradually increases and tends to be stable, which indicates that elastase Myroilysin can effectively remove glycosaminoglycans in bovine achilles tendon type i collagen and reduce collagen immunogenicity. The elastase Myroilysin was shown to have a novel function of removing glycosaminoglycans.
Example 2
The preparation method of the acid-soluble collagen comprises the following steps:
(1) Soaking cod skin for 24 hours according to a feed liquid ratio of 1:10 (M/V) with a NaOH solution with a concentration of 0.1M, replacing the solution every 12 hours, washing with distilled water to be neutral, then continuously soaking the cod skin for 24 hours according to a feed liquid ratio of 1:10 (M/V) with an n-butanol solution with a concentration of 10% at the temperature of 4 ℃, replacing the solution every 12 hours, and washing with distilled water to be neutral to obtain pretreated cod skin;
(2) Adding acetic acid solution with the concentration of 0.5M into the pretreated cod skin according to the feed liquid ratio of 1:10 (M/V) at the temperature of 4 ℃ for soaking and extracting for 72 hours to obtain collagen-containing solution; adding NaCl to the collagen-containing solution until the final concentration is 0.9M, standing for 1h, separating out collagen precipitate from the collagen-containing solution by a salting-out method, and centrifuging at 10000rpm for 15min to obtain collagen precipitate; re-dissolving the collagen precipitate with 0.5M acetic acid solution, dialyzing the re-sol crude protein solution with 0.1M acetic acid solution for 24h, continuing dialyzing with ultrapure water for 3d, replacing dialysate every 12h, dialyzing, and lyophilizing to obtain acid-soluble collagen;
The acid-soluble collagen prepared in step (2) of this example was analyzed for hydroxyproline content and non-denatured protein content, and the results are shown in table 1.
TABLE 1 hydroxyproline content, non-denatured protein content in acid-soluble collagen
The acid-soluble collagen prepared in this example was specifically degraded with collagenase VhaC, then the acid-soluble collagen, the acid-soluble collagen obtained by enzymolysis with collagenase VhaC, and collagenase VhaC were subjected to SDS-PAGE gel electrophoresis analysis, and the optical density value was analyzed by imageJ according to the detection method in the tissue engineering medical device collagen Standard, to obtain the purity of collagen, and the results are shown in Table 2 and FIG. 2.
TABLE 2 band optical Density values for lanes
As can be seen from table 2 and fig. 2, the purity of the acid-soluble collagen prepared in this example was 99.66%.
Example 3
1. A method for removing glycosaminoglycan from collagen by using elastase Myroilysin comprises the following steps:
The cod skin-derived acid-soluble collagen prepared in example 2 was mixed with ultrapure water (pH 9.0) at a feed solution ratio of 5:1 (M/V, mg/ml), then elastase Myroilysin was added to a final concentration of 0.75mg/ml as a reaction group (cod skin collagen+ Myroilysin), and inactivated elastase Myroilysin and water were used as a control group 1 (cod skin collagen+inactivated Myroilysin) and a control group 2 (cod skin collagen+water), and then reacted at 37 ℃ for 24 hours, respectively, 20 μl of reaction supernatant was taken at reaction times of 5 hours, 17 hours, 18.5 hours, 24 hours, and the glycosaminoglycan (GAGs) content in the reaction supernatant was measured, as shown in fig. 3. And repeatedly flushing protein sediment by using ultrapure water after the reaction is finished, and freeze-drying again to obtain the cod skin collagen with low glycosaminoglycan content.
As can be seen from fig. 3, the GAGs content in the reaction supernatant gradually increases and tends to be stable, which indicates that elastase Myroilysin can not only remove glycosaminoglycan from bovine achilles tendon type i collagen, but also effectively remove glycosaminoglycan from cod skin-derived collagen, thereby reducing collagen immunogenicity.
2. Circular dichroism spectrum analysis was performed on bovine achilles tendon type I collagen obtained in example 1 and cod skin collagen obtained in this example, and the results are shown in FIG. 4.
As can be seen from FIG. 4, the cod skin collagen prepared in this example, which had a low glycosaminoglycan content, had a positive peak around 220nm and a negative peak around 200nm, and the positive/negative peak value was approximately equal to 0.12, which indicates that the triple helix structure of the collagen was complete.
In summary, the inventor surprisingly found that elastase Myroilysin has a new function of removing glycosaminoglycan, and the enzyme can make the protein structure loose, but does not damage the triple helix structure of the protein, so that the application further treats acid-soluble collagen by using the enzyme, effectively reduces the content of glycosaminoglycan in the collagen, does not introduce other miscellaneous proteins, and keeps the high purity of the collagen, so that the purity of the collagen prepared by the method of the application is up to 99.66%, the application range of the protein is enlarged, and the application is beneficial to the preparation of related products in the fields of medicine, cosmetics, tissue engineering and the like. Meanwhile, the utilization rate and the added value of fish offal are improved, and good environmental benefit and social benefit are obtained.
Claims (9)
1. The application of elastase Myroilysin in removing glycosaminoglycan in collagen and preparing collagen with low glycosaminoglycan content is characterized in that the amino acid sequence number of elastase Myroilysin is GenBank: ACG59772.1.
2. A method for removing glycosaminoglycans from collagen by elastase Myroilysin, comprising the steps of:
(1) Soaking the cod skin in NaOH solution for 24-48 h, washing with distilled water to neutrality, then continuously soaking in n-butanol solution for 24h, washing with distilled water to neutrality, and obtaining pretreated cod skin;
(2) Adding acetic acid solution into the pretreated cod skin, soaking and extracting for 60-72 h to obtain collagen-containing solution; adding NaCl into the collagen-containing solution, standing for 1-3 h, and centrifuging to obtain collagen precipitate; re-dissolving the collagen precipitate, dialyzing, and freeze-drying to obtain acid-soluble collagen;
(3) Soaking acid-soluble collagen in ultrapure water, then adding elastase Myroilysin to make the final concentration of the collagen be 0.5-1 mg/ml, reacting for 20-30 h at 37 ℃, removing glycosaminoglycan in the collagen, washing with ultrapure water after the reaction is finished, and freeze-drying to obtain the collagen with low glycosaminoglycan content.
3. The method of claim 2, wherein in step (1), the ratio of the cod skin to the NaOH solution is 1:10, and the concentration of the NaOH solution is 0.1M; the temperature at which the NaOH solution was soaked was 4 ℃, and the NaOH solution was changed every 12 hours.
4. The method of claim 2, wherein in step (1), the feed liquid ratio of the cod skin to the n-butanol solution is 1:10, and the concentration of the n-butanol solution is 10%; the temperature at which the n-butanol solution was immersed was 4℃and the n-butanol solution was replaced every 12 hours.
5. The method of claim 2, wherein in step (2), the ratio of the cod skin to the acetic acid solution is 1:10, and the concentration of the acetic acid solution is 0.5M; the soaking and extracting temperature is 4 ℃, and the soaking and extracting time is 72 hours.
6. The method according to claim 2, wherein in the step (2), naCl is added to a final concentration of 0.9M and then allowed to stand at 4℃for 2 hours.
7. The method of claim 2, wherein in step (3), the dialysis is specifically: the dialysis is carried out for 24 hours by using acetic acid solution with the concentration of 0.1M, then the dialysis is carried out for 3-5 days by using ultrapure water, and the dialysate is replaced every 12 hours.
8. The method according to claim 2, wherein in the step (4), the pH of the ultrapure water is 8.5 to 9.0; the feed liquid ratio of the acid-soluble collagen to the ultrapure water is 5:1.
9. The method of claim 2, wherein in step (4), the final concentration of elastase Myroilysin is 0.75mg/mL and the reaction time is 24 hours.
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