WO2004020470A1

WO2004020470A1 - Process for producing collagen treated with cysteine protease and collagen treated with cysteine protease

Info

Publication number: WO2004020470A1
Application number: PCT/JP2003/002346
Authority: WO
Inventors: Koichi Morimoto; Ben'ichiro Tonomura; Takafumi Yoshikawa
Original assignee: Tissue Engineering Initiative Co., Ltd.; Kinki University
Priority date: 2002-08-28
Filing date: 2003-02-28
Publication date: 2004-03-11
Also published as: JPWO2004020470A1; JP4490268B2; AU2003211379A8; AU2003211379A1

Abstract

It is intended to provide a highly functional collagen which has a high biocompatibility and is excellent in humidifying performance, hemostatic effect and cell retention power. A process for producing a collagen having been treated with cysteine protease characterized by comprising bringing collagen or atelocollagen into contact with cysteine protease.

Description

Method for producing collagen treated with cysteine protease and collagen treated with cysteine protease

The present invention relates to collagen, a method for producing the same, and uses thereof. Specifically, it relates to cysteine protease-treated collagen, its production method, and its use. Background art

Collagen, which is a main protein constituting connective tissue and bone tissue between cells in animals, is widely used in fields such as cosmetics, food additives and medical materials.

Properties required of such a collagen material include, for example, good mechanical properties as a biomaterial in tissue regeneration, construction, etc., biocompatibility, rapid biological angle separation, and hemostatic power. Focusing on the properties that such collagen can exhibit, collagen-containing materials include, for example, food additives; cosmetic materials; solvents for preparing artificial skin, hemostatic sponges, bone reconstituting materials, and soft tissue fillers. Is being applied to medical materials.

As described above, collagen is becoming a very important biomaterial in industry, but on the other hand, further improvements in various functions such as compatibility with living bodies, moisturizing properties, and hemostatic power have been required.

Furthermore, collagen, which is widely used at present, is often extracted and purified from dermis or bones of calves and pigs. For example, skins such as calves contain hairs and fats, so pretreatment is required to remove them.The yield of acid-soluble collagen to be purified is not as high as about 5%. Complicated operations may be required in some cases, such as the necessity of a process for removing the causative substances that cause infection and disease among livestock and an analysis process. In addition, in the case of calves, there was a problem that the amount of collagen obtained from one animal was relatively small. For this reason, while maintaining excellent functions, it is possible to apply collagen to biomaterials and the like by a simple method that does not require complicated steps caused by infection between livestock and the like. Appearance was also required.

Therefore, the present invention has been made based on the background art as described above, and has excellent biocompatibility, excellent moisturizing effect, excellent hemostatic power, and excellent cell (embryo) culture technology in vitro. It is an object to provide a highly functional collagen having properties and a method for producing the same. Disclosure of the invention

MEANS TO SOLVE THE PROBLEM The present inventors researched hard in order to solve the said subject, The collagen obtained by contacting collagen or atelocollagen and cysteine protease (it may be hereafter called "cysteine protease-treated collagen") force Biocompatibility. In addition, the present invention was found to be excellent in moisture retention and hemostatic power, and completed the present invention. Such cysteine protease-treated collagen has a unique network-like aggregate structure, and fibrosis is inhibited.

That is, the outline of the present invention is as follows.

The method for producing collagen treated with cysteine protease according to the present invention is characterized by contacting collagen or atelocollagen with cysteine protease.

The collagen treated with cysteine protease according to the present invention is obtained by contacting collagen or atelocollagen with cysteine protease.

The collagen or atelocollagen is preferably fish or bird or mammal-derived collagen or atelocollagen, and more preferably fish-derived collagen or atelocollagen.

The cysteine protease-treated collagen has a triple helical structure in which one cysteine protease-treated collagen molecule is composed of three polypeptide chains, and a network-like aggregate structure is formed by a plurality of cysteine protease-treated collagen molecules. Preferably. It is preferable that the cysteine protease-treated collagen inhibits fibrosis into one fiber by a plurality of cysteine protease-treated collagen molecules.

The temperature at which the cysteine protease-treated collagen is denatured by heat is preferably 25 ° C. or higher.

The cosmetic according to the present invention is characterized by containing the cysteine protease-treated collagen.

The medical material according to the present invention is characterized by containing the cysteine protease-treated collagen.

The food additive according to the present invention is characterized by containing the cysteine protease-treated collagen.

The culture material for cells, tissues or embryos according to the present invention is characterized in that it contains the cysteine protease-treated collagen. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a photograph showing an example of an electron micrograph image (60,000 times, 50 mmol ZL acetate buffer, H 4.0) of atelocollagen.

FIG. 2 is a photograph showing an example of an electron micrograph image (60,000 times, 50 mmol / L acetate buffer, H 4.0) of cysteine protease-treated collagen.

FIG. 3 is a photograph showing an example of an electron micrograph image (40,000 times) of gelatin. FIG. 4 is a schematic diagram showing an example of type I collagen that forms fibers existing between cells and type IV collagen that exists near the cell membrane. In FIG. 4, 1 indicates cells, 2 indicates cells, 3 indicates type I collagen, and 4 indicates type IV collagen.

FIG. 5 is a photograph showing the results of analysis of purified cysteine protease-treated collagen by 7.5% SDS polyacrylamide gel electrophoresis.

FIG. 6 is a chart showing peaks of _α1 and α2 components of cysteine protease-treated collagen and α1 and a2 components of atelocollagen measured by cation exchange chromatography.

Figure 7 shows the optical microscope of the collagen-containing solution treated with cysteine protease after air drying. It is a photograph by a mirror. (Magnification: 100 times)

FIG. 8 is a photograph taken by an optical microscope after the air-collagen-containing solution is air-dried. (Magnification: 100 times)

FIG. 9 is a photograph showing an example of an electron micrograph image (60,000-fold, 50 mmol ZL phosphate buffer, H7.4) of cysteine protease-treated collagen.

FIG. 10 is a photograph showing an example of an electron micrograph image (60,000 times, 50 mmol / L phosphate buffer, ρΗ7.4) of atelocollagen.

FIG. 11 is a graph showing the relative solubilities of atelocollagen and cysteine protease-treated collagen in ethanol.

FIG. 12 is a photograph showing the results of 5% SDS-polyacrylamide gel electrophoresis of atelocollagen and its cysteine protease-treated collagen from the tail of the rabbit's ear.

FIG. 13 is a photograph showing the results of 5% SDS-polyacrylamide gel electrophoresis of chicken cartilage-derived atelocollagen and its cysteine protease-treated collagen. BEST MODE FOR CARRYING OUT THE INVENTION

The cysteine protease-treated collagen according to the present invention (hereinafter sometimes referred to as “CP-collagen”) can be obtained by contacting collagen or atelocollagen with cystine protease.

Hereinafter, the collagen, atelocollagen, cysteine protease, collagen treated with cysteine protease used in the present invention, and a method for producing the same will be described in detail.

Collagen or atelocollagen

The collagen used in the present invention includes, for example, mammals such as sea lions, pigs, and egrets, birds such as chickens, animal dermis, tendons, bones, fascia, etc., and fish such as sharks, koi, and tuna. Collagen derived from the skin, scales and the like.

The atelocollagen used in the present invention includes, for example, dermis, tendons, bones, and fascia of animals such as mammals such as horses, pigs, and egrets, and birds such as chickens. Five

Atelocollagen, which uses collagen-rich tissue such as fish skin and scales such as shark, carp and tuna as raw materials, and removes the telopeptide region at the amino-terminal and carboxyl-terminal of the molecule with pepsin etc. Is mentioned.

Among these, fish-derived collagen or atelocollagen can be preferably used. In addition, among collagen and atelocollagen, atrate collagen can be preferably used.

By using fish-derived collagen or atelocollagen, raw materials can be obtained simply, safely and in large quantities, and cysteine protease-treated collagen that is safer to humans can be obtained.

Among the fish, specifically, for example, tuna such as yellowfin tuna, carp, and eel are mentioned, and of these, tuna can be more preferably used.

Among such atelocollagen, those having a heat denaturation temperature of preferably 20 ° C. or higher, more preferably 25 ° C. or higher are desirable.

For this reason, among fish, it is preferable to use tuna such as yellowfin tuna and atelocollagen such as carp as raw materials having a heat denaturation temperature of 25 ° C or higher. The denaturation temperature of cysteine protease-treated collagen can be adjusted to preferably 20 ° C. or higher, more preferably 25 ° C. or higher, by using such a substance. It is possible to obtain collagen whose denaturation temperature is significantly higher than the denaturation temperature of collagen (about 20 ° C). Therefore, atelocollagen derived from tuna is excellent in storage stability and utility value. Furthermore, in the case of a mug, it is possible to obtain a large amount and uniform performance of cysteine protease-treated atelocollagen in a large amount and with uniform performance due to the large catches and uniformity of the catching period.

The atelocollagen used in the present invention is usually one fibrous association with atelocollagen molecules, as shown in the photograph taken with an electron microscope (600,000 magnification) in FIG. It is a fibrillated atelocollagen that forms a coalescence, with little intermolecular space. In FIG. 1, the atelocollagen molecule is a portion having a stripe pattern in an electron microscope image. Further, the atelocollagen is usually composed of various components, and the composition ratio thereof is not limited because it differs depending on the raw material of the atelocollagen.

Such atelocollagen usually contains at least four kinds of components, i.e., <2 components, i.e. and gamma component. In addition, the constituent unit of the triple helix structure, << 1 component and 2 components are both single-stranded left-handed spirals, have different molecular weights, and β component has two α components covalently bonded between molecules. The γ component is a component of a dimer structure, and the γ component is a component of a trimeric structure in which three α components are covalently bonded between molecules. Generally, these components of the atelocollagen molecule form a right-handed triple helical structure.

The molecular weight of the α component is usually preferably 90,000 to 130,000, the molecular weight of the component is usually 180,000 to 260,000, and the molecular weight of the γ component is usually 270,000 to 390,000. 000 is desirable.

Each component can be easily separated by a known method such as electrophoresis.

The collagen or atelocollagen used in the present invention is usually insoluble in a neutral ρΗ aqueous solution.

Such collagen or atelocollagen can be extracted from animals or fish by a known method. For example, a tissue rich in collagen, such as the dermis, tendons, bones, and fascia of the mammal, bird or fish, is put into an acidic solution of about ρ 溶液 2 to 4, eluted, and pepsin is added thereto to add Remove the telopeptide region. Thereafter, a salt such as sodium chloride is added to the acidic solution of atelocollagen to obtain a precipitate containing atelocollagen.

Cysteine protease

The cysteine protease that can be used in the present invention is a known cystine protease, and the type thereof is not particularly limited. Among them, for example, cysteine protease has a larger amount of acidic amino acids than a basic amino acid. Among them, those which are active at the hydrogen ion concentration in the acidic region can be preferably used.

Actinidyne [EC 3.4.22. 2346

7

14], papain [EC 3.4.22.2], fusin [EC 3.4.22.3], bromelain [EC 3.4.22.32], cathepsin B [EC 3.4.22.1], cathepsin L [EC 3.4.22.15], cathepsin S [EC 3.4.22.27], cathepsin K [EC 3. 4.22.38], cathepsin H [EC 3.4.22.16], alloline, calcium-dependent protease and the like. Among these, it is preferable to use actinidyne, alloline, bromelain, and more preferably, actinidyne.

The use of Acti-Dyne enables effective contact reaction, especially with atelocollagen derived from tuna, and significantly reduces or substantially reduces the content of i3 component and γ component contained in the resulting cysteine protease-treated collagen. ] 3, τ / component can be removed. As a result, it is possible to obtain uniform cysteine protease-treated collagen with a large amount of α component and high purity. It is also presumed that the covalent bond between the components of the cysteine protease-treated collagen molecule is lacking.

Such cysteine proteases can be obtained by known methods, for example, chemical synthesis, extraction from cells or tissues of bacteria and fungi, or various animals and plants, cultured cells thereof, and cysteine derived therefrom. It can be obtained from recombinant protein of protease by genetic engineering means.

-Zeta-treated collagen, its production method

The cysteine protease-treated collagen according to the present invention can be obtained by contacting the collagen or atelocollagen with the cysteine protease.

The contact between the cysteine protease and collagen or atelocollagen can be performed in a solvent. When the contact is performed in a solvent, for example, water can be used as the solvent. The amount of the solvent is preferably in the range of 1 to 1000 parts by mass with respect to 1 part by mass of collagen or atelocollagen.

The amount of cysteine protease used for the contact is preferably 0.1 to 10 parts by mass, more preferably 0.5 to 5 parts by mass, based on 100 parts by mass of collagen or atelocollagen. The conditions for contacting the cysteine protease with collagen or atelocollagen are not limited to pH, temperature, and treatment time, and the contact can be carried out in the same manner as a normal enzyme treatment.For example, pH is preferably 2. It is desirable to carry out in the range of 0 to 7.0, more preferably in the range of 3.0 to 5.0. In order to keep the pH constant, it is preferable to carry out the reaction in a buffer solution. By setting the pH within the above range, collagen or mouth-mouth collagen is uniformly dissolved, and there is an effect that the enzyme reaction proceeds efficiently.

The contact temperature is, for example, preferably in the range of about 15 to 4 ° C. The contact time is, for example, preferably in the range of about 1 hour to 5 days.

After the completion of the reaction, a step of readjusting the pH and a step of inactivating the enzyme may be performed, if necessary. Further, a purification step for removing impurities may be performed. In this case, it is possible to go through the usual steps for protein or peptide purification, fractionation, etc., for example, dialysis, gel filtration chromatography, isoelectric focusing, ion-exchange chromatography, hydrophobic interaction. It can go through steps such as action chromatography.

The cysteine protease-treated collagen thus obtained preferably has a triple helical structure in which one cysteine protease-treated collagen molecule is composed of three polypeptide chains, and is formed into a network by a plurality of cysteine protease-treated collagen molecules. It is desirable that an aggregate structure is formed. The cysteine protease-treated collagen of the present invention preferably inhibits fibrosis into a single fiber by a plurality of cysteine protease-treated collagen molecules, and preferably has a fibrous form such as the atelocollagen. Has substantially no aggregate structure. Such cysteine protease-treated collagen has a stable three-dimensional network structure irrespective of changes in pH.

In this specification, the term “network” refers to a structure in which molecules are connected to form a three-dimensional network by chemical bonding or Van der Waals bonding, and a gap is formed therebetween.

Also, in this specification, an association is defined as a case where the same kind of molecule is not formed by a covalent bond. More than one molecule interacts and binds to form one structural unit.

Specifically, for example, FIG. 2 is an electron micrograph (6000 × magnification) showing an example of the cysteine protease-treated collagen of the present invention. The collagen of the cysteine protease-treated collagen of the present invention is a thin filamentous molecule. Is a structure that shows a three-dimensional network-like aggregate formed by irregularly binding, similar to collagen called fabril (Fabril associated collagens with interrupted triple helix), which inhibits the fibrosis of the three-dimensional structure. The structure is similar to the type IV collagen present in the basement membrane morphologically, that is, it has many spatial gaps. In addition, the fact that collagen has a triple helical structure can be easily confirmed by a known method, for example, a circularly polarized two-color spectrum.

On the other hand, as described above, as shown in FIG. 1, the raw material atelocollagen is fibrotic atelocollagen in which atherocollagen molecules form a single fibrous aggregate, and the intermolecular space There is almost no.

For this reason, the cysteine protease-treated collagen according to the present invention has a high water retention effect because water molecules are easily taken into the gaps between the cysteine protease-treated collagen, and also has an excellent hemostatic effect because it is easy to take in cells such as red blood cells and platelets. In addition, it has a unique property that it is highly effective as a material for culturing various cells and embryos in vitro.

In addition, as shown in FIG. 4, usually, the force that collagen exists between cells 1 and 2 In this case, collagen (type I) that forms a fiber such as the above-mentioned atelocollagen in the center between cells In the vicinity of the basement membrane between cells, type IV collagen 4 having a three-dimensional structure that does not form a fiber aggregate is present. Until now, a method for extracting type I collagen from intercellular matrix has been established, but according to a study by the present inventors, it has a network-like aggregate structure like type IV collagen and inhibits fibrosis. An effective preparation method has not been established for the collagen thus prepared, and the present invention is extremely useful as a means for easily providing a novel type IV collagen-like aggregate. Further, the cysteine protease-treated collagen according to the present invention contains _α components such as α α component and α2 component as components, and does not substantially contain i3 component and γ component. The composition ratio of α ΐ component and _α 2 component depends on the raw collagen, and is not limited. However, for example, it is preferable that α 1: α 2 = 1: 1 to 3: 1.

Usually, the molecular weight of the al and a2 components is preferably 90,000 to 130,000.

The cysteine protease-treated collagen according to the present invention is preferably dissolved in a neutral ρΗ aqueous solution. This is significantly different from the fact that the raw material aterocollagen is insoluble in neutral ρΗ aqueous solution, and that various collagens including natural fasit ゃ type IV collagen are insoluble in neutral ρΗ aqueous solution. This is very different. In addition, the cysteine protease-treated collagen according to the present invention usually has a triple helical structure due to heat denaturation, but does not gel. This is very different from the fact that collagen generally undergoes thermal transformation into gelatin (having a triple helical structure) and gels when cooled. In addition, as shown in an electron micrograph (× 40000) showing an example of the structure of gelatin in FIG. 3, the fibrous structure or network structure of gelatin has collapsed.

In other words, collagen containing natural fasit and type IV collagen are different from the cysteine protease-treated collagen according to the present invention. Further, usually, the α1 component and the ct2 component of the cysteine protease-treated collagen according to the present invention are different in surface charge of each α component from those of the natural atelocollagen as a raw material. I have. For example, as shown in FIG. 6, the α1 component and the α2 component of the cysteine protease-treated collagen according to the present invention usually give a total of four peaks by cation exchange chromatography, and § _alpha 1 terrorist in collagen, _alpha 2 component is usually given a total of six peaks, elution time of these peaks are all different. Therefore, it can be inferred that the surface charges are different.

In addition, the cysteine protease-treated collagen according to the present invention has remarkably improved solubility in alcohol such as ethanol as compared with atelocollagen. For this reason, it has excellent solubility in cosmetics and the like to which ethanol is added, and it is possible to add cysteine protease-treated collagen at a high concentration.

The above results indicate that the cysteine protease-treated collagen of the present invention is a raw material. It has properties that are not found in collagen or atelocollagen, indicating that the function that is the starting point of interaction with biological materials has been significantly changed.

The cysteine protease-treated collagen according to the present invention preferably has a denaturation temperature of 25 ° C. or higher, more preferably 28 ° C. or higher, by heat. Therefore, handling at room temperature is easy. As described above, when the atelocollagen is derived from fish, it is preferable to use tuna or the like as the raw fish in order to obtain cysteine protease-treated collagen at such a denaturation temperature.

The cysteine protease-treated collagen of the present invention can be crosslinked and used as polymerized cysteine protease-treated collagen.

The crosslinking treatment can be performed by a conventionally known method. Examples of the method include a method using chemical crosslinking, a method of crosslinking by heat treatment, and a method of crosslinking by irradiation with radiation such as ultraviolet rays.

Examples of the crosslinking agent used in the chemical crosslinking include water-soluble carpoimide compounds such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride; epichlorohydrin; diepoxy conjugates such as bisepoxydiethylene glycolone; And the like. The concentration of the crosslinking agent with respect to cis Tin protease treatment collagen, preferably 1 0 one ^third to one approximately 0 wt%, preferably in the range of. 5 to 4 0 ° C, preferably about 3 to 4 8 hours, By contacting the cysteine protease-treated collagen with a crosslinking agent, crosslinked cysteine protease-treated collagen can be obtained.

In the case of crosslinking with ultraviolet rays, the cysteine protease-treated collagen of the present invention is irradiated with ultraviolet rays, for example, by an ultraviolet lamp, usually at room temperature for about 3 to 48 hours to obtain crosslinked cysteine protease-treated collagen. Can be done.

In the case of thermal crosslinking, the cysteine protease-treated collagen according to the present invention is heated under reduced pressure, preferably at a temperature of about 110 to 160 ° C., for about 3 to 48 hours, so that the cysteine protease-treated collagen is treated. Collagen can be obtained.

Such cross-linked cysteine protease-treated collagen is resistant to collagenogen. Zinc properties and strength can be improved.

As described above, the collagen treated with cysteine protease according to the present invention has high solubility in alcohol such as ethanol. For example, it is not necessary to perform another chemical modification to improve the solubility. If necessary, they may be chemically modified as long as they do not interfere with the purpose of the application. Examples of the type of chemical modification include, for example, Asinorei-Dani, Myristi / Rai-Dani, and polyethylene glycol modification.

Among them, for example, succinylated cystine protease-treated collagen, which is an acylation modification, reacts the cystine protease-treated collagen of the present invention with anhydrous succinic acid in a neutral pH solvent such as a phosphate buffer. You can get it. By saccharifying, the solubility of more neutral pH in a solvent can be improved, and the feel when used can be improved.

The cysteine protease-treated collagen modified with polyethylene glycol can be obtained by reacting with polyethylene dalicol activated with cyanuric chloride.

Use

The cysteine protease-treated collagen according to the present invention can be suitably used for food additives, medical materials, cosmetic materials, cell or embryo culture materials, and the like. For example, the cysteine protease-treated collagen of the present invention can be used in cosmetics because it has excellent moisture uptake performance, a high moisturizing effect, and excellent biocompatibility. Among them, for example, it can be suitably used by adding to basic cosmetics and the like. In addition, by applying a cosmetic containing the cysteine protease-treated collagen of the present invention to inflamed skin, symptoms can be reduced.

Further, for example, the cysteine protease-treated collagen of the present invention has a high uptake performance of red blood cells and platelets, has a high hemostatic effect, and is excellent in biocompatibility, and thus can be suitably used as a medical material.

Furthermore, the cysteine protease-treated collagen of the present invention is excellent in biocompatibility, has no problem for food use, and can be suitably used as a food additive. In addition, the cysteine protease-treated collagen of the present invention is expected to have an excellent effect on culturing various cells, tissues or embryos as an extracellular matrix, can be used safely, and can be used as a cell culture stabilizer. It is suitable.

The term “cell” used herein refers to a living organism that is surrounded by a membrane structure that isolates the outside world, has transit information with self-renewal capability inside, and has an expression mechanism.In a multicellular organism, it is a structural unit of tissue The term “tissue” refers to a cell population that differentiates in a specific direction and has the same function and morphology, and the term “embryo” refers to a multicellular organism at an early stage of ontogeny. Industrial applicability

The cysteine protease-treated collagen according to the present invention has a network structure, is inhibited from fibrosis, has excellent ability to take in water molecules or erythrocytes, and is excellent in biocompatibility. Also, it has excellent moisturizing effect, hemostatic power and cell retention power. Example

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

[Preparation Example 1]

Extraction of atelocollagen from the skin of yellowfin mag

To a Warinda blender containing 110 OmL of 50 Ommol / L citrate buffer (pH 2.5) was added 534 g of yellowfin tuna skin and ground. After stirring at 4 ° C for 3 days, the mixture was centrifuged at lOOOOXg for 30 minutes to obtain a primary supernatant. To the precipitate was added 60 OmL of 500 mmol / L citrate buffer (pH 2.5), and the mixture was stirred for 3 hours and centrifuged at 10 OO X g for 30 minutes to obtain a secondary supernatant.

Primary and secondary supernatants were collected, and pig'pepsin (Sigma) was added to a final concentration of 10 OU / mL, followed by stirring at 4 ° C for 3 days.

To this, sodium salt was added and dissolved at a concentration of 2.5 mol / L, left at 4 for 16 hours, and centrifuged at lOXOX g for 30 minutes to collect the precipitate. .

2 Ommol / L acetate buffer (pH 4.0) is dissolved in the collected precipitate. Was added. The amount of obtained atelocollagen was 139 _g (yield 26%).

[Example 1]

Manufacture of collagen treated with cysteine protease

The mouth-collagen obtained in Preparation Example 1 was dialyzed three times with 4 against 100 volumes of 20 mmol / L citrate buffer (pH 3.0). After dialysis, a cysteine protease (actinidyne) was added to the atelocollagen so that the weight was ^1.0 / ₀ . The mixture was stirred at a temperature of 20 ° C. for 3 days to obtain a crude product.

The resulting crude product was purified by cation exchange chromatography. For details, a TSKgel SP-Toyopearl 650M column (trade name: manufactured by Tosoh Corporation) obtained by equilibrating the crude product with 2 Ommol / L citrate buffer (ρΗ3.5) (hereinafter referred to as “Αsolution”) ), And the components that did not adsorb to the gel were washed by flowing 30 A mL of solution A. Next, 2 Ommol / L citrate buffer containing 1.5 mol / L sodium chloride (pH 3.5)

It is called "B liquid." ) 30 OmL inflow, cysteine protease treated collagen

The fraction was analyzed by 7.5% SDS-polyacrylamide gel electrophoresis, and it was confirmed that the fraction consisted of only components with a molecular weight of about 100,000. (Fig. 5) 63 g of the purified collagen treated with cysteine protease was dialyzed three times against distilled water and freeze-dried Yield 11.8% Cation chromatography of collagen treated with cysteine protease Analysis

The α1, a2 components fractionated from the atelocollagen prepared in Preparation Example 1 and the purified cysteine protease-treated collagen were measured by cation chromatography. As shown in FIG. 6, the _α1 component and _α2 component contained in the atelocollagen usually give six peaks by cation chromatography or the like, whereas the cysteine proteinase-treated collagen according to the present invention usually has four peaks. Only one peak was given, and the elution times of atelocollagen and the α component of cysteine protease-treated collagen were all different.

-Electron microscopy of collagen treated with zeolite Atelocollagen and cysteine protease-treated collagen were separately dissolved in 5 O mmol / L acetate buffer (pH 4.0) and immobilized on a cell disk using dartartaldehyde according to a conventional method. The immobilized atelocollagen and collagen treated with cysteine protease were photographed with an electron microscope (S-900, 10 kV, manufactured by Hitachi, Ltd.) at a magnification of 60,000 (FIGS. 1 and 2).

As a result, the atelocollagen (FIG. 1) formed a typical fibrous aggregate structure in which the triple helical structure was regularly arranged, whereas the cysteine protease-treated collagen according to the present invention (FIG. 2). ) Was confirmed to have an irregular, mesh-like aggregate structure with many gaps.

It is presumed that this difference in the structure of the aggregate is due to the fact that the terminal structure of the polypeptide main chain of the collagen treated with cysteine protein is different from that of the atelocollagen. Air drying test of cysteine protease-treated collagen

Atelocollagen and cysteine protease-treated collagen were separately dissolved in 5 O mmol / L acetate buffer (pH 4.0), and 1 O / z L was dropped on the slide glass. Leave to dry for 6 hours. Each dried sample was analyzed with an optical biological microscope (manufactured by Shimadzu Corporation). As a result, it was confirmed that the collagen treated with cystine protease (FIG. 7) exhibited a finer pattern than the atelocollagen (FIG. 8). In addition, when the viscosity of each was analyzed by surface tension (droplet weight method), it was confirmed that collagen treated with cysteine protease had higher viscosity than atelocollagen.

As a result, it was confirmed that the collagen treated with cysteine protease was more effective in permeating and protecting human skin than in atherocollagen. Evaluation of hemostatic performance of cysteine protein treated collagen

A 10-week-old Os Wistar rat was anesthetized with ether, anesthetized by intraperitoneal administration of chloral hydrate, the tip of the spleen was partially removed, and bleeding was performed with a 18-G needle. Was. Cysteine protease-treated collagen and atelocora at bleeding sites Each gen was placed and the bleeding stopped. The average time required for hemostasis in each test group was 86 seconds for atelocollagen, 39 seconds for cysteine protease-treated collagen, and 91 seconds for collagen hemostat (commercially available).

Controls were untreated. Table 1 shows the results of measuring the time (seconds) until hemostasis. As a result, it was shown that cysteine protease-treated collagen can significantly reduce hemostasis in less than half the time (second) of atelocollagen or a commercially available collagen hemostatic agent, and a remarkable improvement in hemostatic performance was confirmed. Subcutaneous implantation test

10-week-old Os Wistar rats were anesthetized with ether, anesthetized by intraperitoneal administration of chloral hydrate, a skin incision was made in the back, and 20 mg of cysteine-lipase-treated collagen and atelocollagen were subcutaneously injected. Each was lumped and embedded. When the implanted site was incised 4 weeks later, the cystine protease-treated collagen and atelocollagen were absorbed and could not be visually confirmed. In addition, foreign body reactions such as tissue inflammation and adhesions could not be confirmed with the naked eye.

Table 1 Hemostatic effect

> 300 indicates that the bleeding is not stopped after 300 seconds. ND = not done Evaluation of cysteine protease-treated collagen and atelocollagen prepared in neutral pH buffer

The morphological characteristics of the supramolecular structure at neutral pH of the atelocollagen produced in Preparation Example 1 and the cysteine protease-treated collagen were evaluated as follows.

A solution having a buffering capacity at neutral pH was prepared for scanning electron microscope observation. Solution conditions: 50 mmol / L phosphate buffer (pH 7.4)

For scanning electron microscopy, 1.Omg / mL of cysteine protease-treated collagen and atelocollagen were each dialyzed against the above solution. Place a cell disk in each well of the 24-well plate, add each sample solution after dialysis to the wells, and leave it at 4 ° C. Then immobilize the collagen self-association product on the cell disk with daltonaldehyde. did.

Each collagen dialysis sample was photographed with a scanning electron microscope (Hitachi S-900) at a magnification of 60,000. FIGS. 9 and 10 show photographs of each dialysis solution obtained by treating cysteine protease-treated collagen and atelocollagen self-aggregate with different pH buffer solutions. From the electron micrographs of FIGS. 1, 2, 9, and 10, it was confirmed that the atelocollagen self-aggregate forms a fibrous structure even when the pH (4.0 to 7.4) is different. It was confirmed that the collagen treated with cysteine protease did not form a fibrous structure in this pH range but formed a three-dimensional network structure.

It is presumed that the difference in the supramolecular structure of the collagen self-association is influenced by the amino acid sequences involved in the self-association of the cysteine protease-treated collagen and the atelocollagen molecule. This is because collagen treated with cysteine protease has undergone limited hydrolysis by cysteine protease.

From these results, it was confirmed that the collagen treated with cysteine protease had a three-dimensional network structure within the range of pH 4.0 to 7.4, and was a supermolecular structure different from that of atelocollagen. Solubility of cysteine protease-treated collagen in ethanol

The atelocollagen produced in Preparation Example 1 and the cysteine protease-treated The solubility of Lagen in ethanol was evaluated.

These collagens were dissolved in a 2 O mmol / L acetate buffer (pH 4.0) to prepare a 1.0% cysteine proteinase-treated collagen solution and an atelocollagen solution.

Ethanol was added to each of the cysteine protease-treated collagen solution and the atelocollagen solution to a final concentration of 0, 10, 15, 20, 40, 60, 80% (VV) and mixed. After leaving at 4 for 1 hour, the supernatant was separated by centrifugation at 10,000 g for 30 minutes. The concentration of collagen contained in the supernatant was determined by measuring the absorbance at 21 O nm.

The relative ratio (%) of the absorbance at each ethanol concentration was calculated with the absorbance at 210 nm of the cysteine protease-treated collagen supernatant solution and the atherocollagen solution at 0% ethanol concentration as 100%. That is, here, the relative ratio is expressed as being directly proportional to the solubility. Figure 11 shows the results.

As is clear from Fig. 11, the relative ratio of atelocollagen solution was 15% at 15% ethanol concentration, but the relative ratio of cysteine protease treated at the same ethanol concentration was 100%. It was confirmed that the solubility of collagen at 0% and 15% ethanol concentration did not change. In addition, the relative ratio at 20% ethanol concentration was only 6% for atelocollagen but 80% for cysteine protease-treated collagen. The ethanol concentration giving 50% solubility showed a difference of about 10% between atelocollagen and cysteine protease-treated collagen.

From these results, it was confirmed that the collagen treated with cysteine protease had an extremely higher solubility in ethanol than that of atelocollagen. This result is considered to be due to the fact that collagen treated with cysteine protease has a surface structure different from that of atelocollagen.

Further, high solubility in ethanol has the following advantages. That is, for example, in cosmetics, ethanol is often added, and the higher the solubility in ethanol, the higher the concentration of cysteine protease-treated collagen can be added. Therefore, cysteine protease-treated collagen The effect of the addition can be further enhanced.

Further, the cysteine protease-treated collagen according to the present invention has high solubility in ethanol even without special chemical modification as described above, and there is no fear of deterioration in properties due to the chemical modification. In addition, for example, cysteine protease-treated collagen is modified by acylation to ethanol. It is also possible to increase the solvability, in which case it is presumed that the higher the concentration, the smoother the touch, the less powdery and the more effective cosmetics.

Furthermore, in the case of dissolving other water-insoluble compounds in cosmetics, medical materials, etc., the ratio of ethanol content as a solvent increases, but even in this case, a sufficient amount of cysteine protease-treated collagen can coexist. It is possible.

Since the cysteine protease-treated collagen has a high solubility, it is possible to prepare a sample by uniformly dispersing it even in a high-concentration aqueous ethanol solution. Therefore, it can be used in various fields. Furthermore, by adding cystine protease-treated collagen to ethanol and evaporating the solvent, a uniformly dispersed solid can be obtained. This can be used for forming a multilayer film. Amount of water evaporation of collagen

The water evaporation of the atelocollagen produced in Preparation Example 1 and the cysteine protease-treated collagen was evaluated.

First, the following three solutions were prepared.

Control: 20 mmol / L acetate buffer (pH 4.0),

Atelocollagen: 0.5% atelocollagen solution dissolved in 20 mmol / L acetate buffer (pH 4.0)

-Zeta-treated collagen: 20 mmolL acetate buffer (pH 4.

0.5% CP-collagen solution dissolved in (0)

For each solution, one 24-well plastic plate (Libro, manufactured by ICN) with a 16-mm diameter (2.0 mm ² ) was prepared, and the weight (tare) was measured. Add 200 ml of the same solution to each well of each plastic plate. μL was added and the weight was measured with an electronic balance. Specifically, the weight (mg) of the solution added to each plate was calculated from the difference from the tare weight.

Each plate was placed in an incubator at 22 ° C and a humidity of 50% to 60%, and was left as it was for 11 hours, 13 hours, 14 hours, 18 hours, 22 hours, 30 hours, and 37 hours. At each time, the weight of each plate was measured with an electronic balance, the tare weight and the protein concentration were measured, and the amount of water evaporation was determined. Specifically, the protein weight (0.5 mg ZmL) was subtracted from the weight of the solution of atelocollagen and cysteine protease-treated collagen (CP collagen) added at 0 hours, and the respective water amounts W (0). W (0) force of atelocollagen and cysteine protease-treated collagen Calculate coefficients to be equal to W (0) of S control, correct W (t) at each time with each correction coefficient, and calculate W ^£ (t). Calculated. Next, dW '(t) was calculated by subtracting W (0) from the solution weight W' (t) at each time. Relative ratio of weight change dW '(t) of solution at each time, assuming dW (37) of control weight change after standing for 37 hours as 100%

(%) (= dW * (t) x 100 ÷ dW (37) was calculated to evaluate the changes in the standing time (hour) and the relative ratio of water evaporation (%). All were expressed as relative values, with the amount as 100.

The results are shown in Table 2.

As is evident from ¾2, atelocollagen showed a gradual decrease in the effect of inhibiting water evaporation from the control, from 6% after 13 hours, and showed little difference from the control after 18 hours. . In other words, the effect of suppressing water evaporation was lost over time. On the other hand, cysteine protease-treated collagen showed a 10% reduction in water evaporation compared to the control after 13 hours. Even more surprisingly, this remarkable water evaporation suppression effect was 10.5% even after 22 hours, and it was confirmed for the first time that the water evaporation suppression effect lasted for a long time. From this experimental result, it was clarified that the water retention capacity of cysteine protease-treated collagen was significantly different from that of atelocollagen.

How to calculate “6% of 13 hours” above>

Comparing the values in the table at 13 hours, the control is 37.4%, the atelocollagen is 35.3%, and the collagen treated with cysteine protease is 33.6%. Previous The above 6% indicates the ratio when the difference in evaporation change, 2 · 1 (= 37.4-35.3), is used as the numerator and 37.4 is used as the denominator. Table 2 Changes in water evaporation relative to storage time

[Preparation Example 2]

Extraction of atelocollagen from the ears and tail of a heron

5. Eg of the heron 5.45 g and 18.2 g of the tail were each finely chopped, and 10 OmL of an O.lmol / L acetic acid aqueous solution was added, followed by pulverization with a Perling blender. After stirring at 4 ° C. for 3 days, the mixture was centrifuged at 10, OX g for 30 minutes to obtain a primary supernatant of each sample. Further, 10 OmL of a 10 Ommol / L acetic acid aqueous solution was added to the precipitated portion, and the mixture was stirred and centrifuged in the same manner to obtain a secondary supernatant.

These primary and secondary supernatants were collected, and porcine pepsin (manufactured by Cygnet) was added to each sample solution to a final concentration of 3 OU / mL, followed by stirring at 4 ° C for 3 days. Add sodium chloride to the total supernatant after centrifugation under the same conditions to a final concentration of 2.5 mol / L, dissolve, leave at 4 ° C for 1 hour, and centrifuge at 10,000 Xg for 30 minutes The precipitate was collected with care. Add the appropriate amount of 2 Ommol / L acetate buffer (pH 4.0) to the precipitate. After dissolution, these were used as an ear-derived atelocollagen sample and a tail-derived atelocollagen sample.

[Example 2]

調製 Preparation of cysteine protease-treated collagen from heron ear and tail

A part of the atelocollagen of the egret ears and tail obtained in Preparation Example 2 was transferred to separate containers each having a lid.

Cysteine protease (acti-dyne) was activated at 25 ° C. for 1 hour in a 2 O mmol / L phosphate buffer (pH 6.5) containing 5 mmol / L dithiothreitol and 1 mmol / LEDTA. To each atelocollagen sample solution was added 3.0% (w / v) activated actinidyne. The mixture was stirred at 20 ° C for 7 days to obtain a crude product.

Purification of cysteine protease-treated collagen from heron ear and tail

The prepared herring-derived cysteine protease-treated collagen (ear-derived and tail-derived) and acti-dyne were separated and purified by a patch method using an anion exchange gel. Specifically, an appropriate amount of TSKgel DEAE-Toyopearl 650C gel (manufactured by Tosoh Corporation) equilibrated with 20 mmol / L acetate buffer (pH 4.0) was added to the crude enzyme reaction product,

After standing at 25 ° C for 1 hour, the mixture was centrifuged at 10, OOOX g for 30 minutes to obtain cystine protease-treated collagen in the supernatant.

The crude product was analyzed by 5% SDS polyacrylamide gel electrophoresis.As a result, as shown in FIG. 12, the obtained cystine protease-treated collagen derived from egrets had a molecular weight of only 100,000 It was confirmed that it consisted of only components. The purified cystine protease-treated collagen from the perforated ears and tail obtained was dialyzed three times against distilled water and then lyophilized.

[Embodiment 3]

Preparation of collagen treated with cysteine protease derived from citrate-soluble collagen (type I)

2 Ommol / L acetate buffer (pH 4.0) so that commercially available citrate-soluble collagen (Type I) (Nacalai Tester, Part No. # 093 50 34) has a concentration of 1.0 mg / mL. Was added and dissolved. A cysteine protease that had been activated in advance by leaving it in a 20 mmol / L phosphate buffer (pH 6.5) containing 5 mmol / L dithiothreitol and 1 mmol / LE DTA at 25 ° C for 1 hour (Acti-dyne) was added to the citrate-soluble collagen sample solution at a concentration of 1.0% (w / v). The mixture was stirred at 20 ° C for 7 days to obtain a crude product.

Purification of cystine-protease-treated collagen derived from citrate-soluble collagen

The collagen-treated cysteine-mouth-tease-treated collagen and actinidain prepared as described above were separated and purified by a column method using an anion exchange gel.

Specifically, TSKgel equilibrated with 20 mmol / L acetate buffer (pH 4.0)

The crude product of the enzymatic reaction was passed through a column filled with DEAE-Toyopearl 650C gel (manufactured by Tosoichi Co., Ltd.) to fractionate the CP-collagen derived from E.

The crude product was analyzed by 5% SDS polyacrylamide gel electrophoresis. As a result, it was confirmed that the crude product consisted of only the _α- chain component having a molecular weight of about 100,000 without the J3 and γ chains.

[Example 4]

Preparation of chicken-derived atelocollagen

Ninety-three grams of chicken cartilage was cut into small pieces, 100 mL of a 0.5 mol / L acetic acid aqueous solution was added, and the mixture was pulverized with a Warlinda blender. After stirring at 4 ° C for 2 days, the mixture was centrifuged at 100,000 X g for 30 minutes to collect the supernatant. Porcine pepsin (manufactured by Sigma) was added to the supernatant solution to a final concentration of 30 U / mL, and the mixture was stirred at 30 ° C for 3 days. Add sodium chloride to the total supernatant after centrifugation under the same conditions to a final concentration of 2.5 mol / L, dissolve, allow to stand for 1 hour, and add 100,000 X g The precipitate was collected by centrifugation for minutes. The precipitate was dissolved by adding an appropriate amount of a 20 mmol / L acetate buffer (pH 5.0), and these were used as a sample of atelocollagen derived from -Petria.

Preparation of Collagen Treated with Cysteine Protease from Chickens

The cysteine protease (acti-dyne) activated as described above was added to the chicken atelocollagen solution obtained as described above to a concentration of 3.0% (w / v). The mixture was stirred at 30 ° C for 3 days to obtain a crude product. The crude product was analyzed by 5% SDS-polyacrylamide gel electrophoresis. It has been shown that it has no molecular weight of about 90,000 to 130,000.

Claims

The scope of the claims

1. A method for producing cysteine protease-treated collagen, wherein collagen or atelocollagen is brought into contact with cysteine protease.

2. The method for producing cysteine protease-treated collagen according to claim 1, wherein the collagen or atelocollagen is fish, bird, or mammal-derived atelocollagen.

3. The method for producing collagen treated with cysteine protease according to claim 1 or 2, wherein the collagen or atelocollagen is fish-derived atelocollagen.

4. Collagen or collagen that has been treated with cysteine protease obtained by contacting cysteine protease with cysteine protease.

5. The method for producing cysteine protein-treated collagen according to claim 4, wherein the collagen or atelocollagen is fish, bird or mammal-derived atelocollagen.

6. The cysteine protease-treated collagen according to claim 4, wherein the collagen or atelocollagen is fish-derived atelocollagen.

7. The cysteine protease-treated collagen has a triple helical structure in which one cysteine protease-treated collagen molecule is composed of three polypeptide chains, and a network-like aggregate structure formed by a plurality of cysteine protease-treated collagen molecules. The cysteine protease-treated collagen according to any one of claims 4 to 6, wherein the collagen is formed.

8. The cysteine protease-treated collagen according to any one of claims 4 to 7, wherein the cysteine protease-treated collagen inhibits fibrosis into one fiber by a plurality of cysteine protease-treated collagen molecules. Collagen.

9. The cysteine according to any one of claims 4 to 8, wherein the temperature at which the cysteine protease-treated collagen is denatured by heat is 25 ° C or more. Protease-treated collagen.

10. A cosmetic containing the cysteine protease-treated collagen according to any one of claims 4 to 9.

11. A medical material containing the cysteine protease-treated collagen according to any one of claims 4 to 9.

12. A food additive containing the cysteine protease-treated collagen according to any one of claims 4 to 9.

13. A culture material for cells, tissues or embryos containing the cysteine protease-treated collagen according to any one of claims 4 to 9.