JP2009247824A - Degradable composite material containing chitin or chitosan - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a degradable composite material which contains at least one polysaccharide of chitin or chitosan and has an autolytic ability. <P>SOLUTION: A molded article which contains at least one polysaccharide of chitin or chitosan is made to carry a degrading enzyme which can hydrolyze the polysaccharides, whereby the polysaccharide can be gradually decomposed in the presence of water e.g. in vivo. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a degradable composite material containing at least one polysaccharide of chitin and chitosan. Specifically, the present invention relates to a degradable composite material that contains at least one polysaccharide of chitin and chitosan and that is gradually degraded in a water-existing environment such as a living body.

  Chitin and chitosan are used as various medical materials because they do not adversely affect the ecology (see, for example, Patent Document 1). For example, a sheet manufactured using chitin or chitosan as a raw material is used as a wound dressing, and further, a fiber manufactured using chitin or chitosan as a raw material is used as a medical prosthetic material. Chitin and chitosan are known to be soluble carbohydrates that are absorbed in vivo when decomposed, and it is known that the biocompatibility of the decomposed products is high. However, since chitin and chitosan cannot be decomposed by enzymes in the human body, there is a problem that medical materials produced using these as raw materials are difficult to use in a form of use left in the living body. Medical materials that are used to stay in living tissue for a long period of time can be removed from the unnecessary medical materials if they are absorbed and extinguished in the body at the stage where damage to the living tissue has been healed to some extent after surgery. There are advantages in terms of safety and ease of use.

Therefore, if chitin and chitosan can also be provided with self-degradability, it is possible to provide a medical material with higher utility. However, to date, a technology for providing self-resolution to chitin and chitosan has not yet been established.
JP 2003-265591 A

  An object of the present invention is to provide a degradable composite material having at least one polysaccharide of chitin and chitosan and having self-resolution.

  As a result of intensive studies to solve the above problems, the present inventors have made a molded product containing at least one kind of chitin and chitosan by supporting a degrading enzyme capable of hydrolyzing the polysaccharide. The present inventors have found that the polysaccharide can be gradually decomposed in a water-existing environment such as a living body, thereby providing a degradable composite material having self-resolution. In particular, it has been found that by using a thermostable enzyme as the decomposing enzyme, the degrading activity on the polysaccharide can be stably maintained for a long period of time, and the composite material can be continuously and stably decomposed in a water-existing environment. It was. The present invention has been completed by making further improvements based on these findings.

That is, the present invention provides the following degradable composite materials:
Item 1. A degradable composite material, wherein a decomposing enzyme capable of hydrolyzing a polysaccharide is supported on a molded article containing at least one polysaccharide selected from the group consisting of chitin and chitosan.
Item 2. Item 2. The degradable composite material according to Item 1, wherein the molded product containing the polysaccharide is in the form of a sheet or fiber.
Item 3. Item 3. The degradable composite material according to Item 1 or 2, wherein the degrading enzyme is a heat-resistant chitinase that exhibits activity at 4 ° C or higher and an optimum temperature is 95 ° C or higher.
Item 4. Item 3. The decomposable composite material according to Item 1 or 2, wherein the decomposing enzyme is a thermostable chitinase derived from Pyrococcus furiosus.
Item 5. Item 5. The degradable composite material according to any one of Items 1 to 4, which is a medical material.

  The degradable composite material of the present invention can self-decompose chitin and / or chitosan into soluble carbohydrates having high biocompatibility in the presence of water. Therefore, when the degradable composite material of the present invention is applied to a damaged tissue as a medical material, it disappears after staying in the damaged tissue for a desired period of time, so there is no need to remove the medical material after the operation. The burden can be reduced.

  In the degradable composite material of the present invention, various temperature conditions can be obtained by using a degrading enzyme that exhibits activity in a wide temperature range of about 4 to 100 ° C. as a degrading enzyme capable of hydrolyzing chitin and / or chitosan. Can induce self-degradation in the presence of water and can be applied to a wide range of uses.

  Furthermore, in the degradable composite material of the present invention, by using a thermostable enzyme as the degrading enzyme, the self-decomposition in the degradable composite material can be stably maintained based on the high stability of the enzyme. The composite material can be continuously and stably decomposed in a water-existing environment, and can also have long-term storage stability.

  The degradable composite material of the present invention includes a polysaccharide containing at least one polysaccharide selected from the group consisting of chitin and chitosan (hereinafter, sometimes simply referred to as “chitinous molded article”). It is characterized in that a degrading enzyme capable of hydrolyzing is supported. Hereinafter, embodiments of the present invention will be described in detail.

  In general, chitin is a polysaccharide in which N-acetylglucosamine, which is a constituent monosaccharide, is β-1,4 glycoside-linked, but chitin used in the present invention has less than 50% acetyl group deacetylated. Also good. Chitosan is a deacetylated polysaccharide, but chitosan used in the present invention only needs to have 50% or more of the acetyl group of chitin deacetylated, and a portion not deacetylated is mixed. You may do it.

  The chitinous molded article used for the degradable composite material of the present invention may contain only one of chitin and chitosan as a polysaccharide, or may contain them in combination.

  Moreover, the chitinous molded product used for the degradable composite material of the present invention may be composed of only the polysaccharide, but contains other biodegradable resins and additives as necessary. Also good.

  In the chitin molded article used for the degradable composite material of the present invention, the blending ratio of the polysaccharide is not particularly limited, but from the viewpoint of improving the self-resolution of the degradable composite material, Examples are such that the total amount of the polysaccharide is 30 to 100% by weight, preferably 50 to 100% by weight, more preferably 70 to 100% by weight, based on the total weight.

  In the decomposable composite material of the present invention, the shape of the chitinous molded article may be appropriately set according to the form of the target decomposable composite material, and is not particularly limited. For example, a sheet shape, fiber (yarn) , Non-woven fabric, cotton, sponge and the like. Among these, the sheet form and the fiber form are suitable because they are easy to use as medical materials.

  The chitinous molded product can be produced according to a known production method, and can also be obtained as a commercial product.

  The degrading enzyme used in the degradable composite material of the present invention is not particularly limited as long as it can hydrolyze the polysaccharide and is pharmaceutically acceptable. Specific examples of such degrading enzymes include chitinase and chitosanase. For example, chitinase is used when the polysaccharide is chitin, and chitosanase is used when the polysaccharide is chitosan. As the degrading enzyme used in the degradable composite material of the present invention, one kind of enzyme may be used alone, or two or more kinds of enzymes may be used in combination.

  The working temperature of the decomposing enzyme used in the present invention is not particularly limited as long as it exhibits activity in the temperature environment in which the decomposing composite material is used, but the decomposing enzyme exhibiting activity in a wide temperature range of about 4 to 100 ° C. preferable. By using an enzyme that exhibits activity in such a high temperature range, self-degradation of the decomposable composite material can be induced in a water-existing environment of various temperature conditions, and can be used in a wide range of applications. Become.

  Moreover, a thermostable enzyme is mentioned as a suitable example of the decomposing enzyme used by this invention. The use of such a thermostable enzyme can improve the storage stability of the decomposable composite material of the present invention in addition to allowing the composite material of the present invention to have self-resolution at high temperatures. Examples of such thermostable enzymes include those having an optimum temperature of 60 ° C. or higher, more preferably around 90 ° C. As a suitable example of the thermostable enzyme used in the present invention, a thermostable chitinase having an activity at 4 ° C. or higher and an optimum temperature of 95 ° C. or higher is exemplified. Specific examples of such thermostable chitinases include those derived from Pyrococcus furiosus. A thermostable chitinase derived from Pyrococcus furiosus is known and can be produced, for example, according to the method described in JP-A-2004-344160.

  As a preferred embodiment of the degradable composite material of the present invention, a chitinase is supported on a molded product containing chitin; more preferably, a chitinase is supported on a sheet-like or fibrous molded product containing chitin. Particularly preferably, there are exemplified those in which a heat-resistant chitinase is supported on a sheet-like or fibrous molded article containing chitin.

  In the degradable composite material of the present invention, it is sufficient that the decomposing enzyme is supported on the molded article containing the polysaccharide by physical adsorption or chemical bonding. The degradable composite material of the present invention is produced, for example, by applying or impregnating a molded article containing the polysaccharide with a solution containing the decomposing enzyme. Furthermore, it can also be produced by kneading the decomposing enzyme into a molded product containing the polysaccharide.

In the degradable composite material of the present invention, the amount of the degrading enzyme supported on the molded product containing the polysaccharide is not particularly limited, and is appropriately set depending on the type of the polysaccharide, the form and use of the degradable composite material do it. As an example of the carrying amount of the degrading enzyme, the degrading enzyme is 100-100,000 U, preferably 1000-10000 U, more preferably 2000-5000 U per 1 g of the polysaccharide. Here, the activity unit of the degrading enzyme is defined as 1 U of enzyme activity that liberates 1 μmol of N-acetylglucosamine or glucosamine per minute under the following conditions:
Substrate solution : Chitin powder or chitosan powder is added to 200 mM acetate buffer (pH 5.6) to a concentration of 0.5% by weight and suspended.
Degradation enzyme solution : Dissolve the degradation enzyme to be the target of activity measurement in distilled water to 0.8 mg / ml.
Measurement protocol :
(1) Preincubate 1 ml of substrate solution in a test tube at 37 ° C. for 5 to 10 minutes.
(2) Next, 0.2 ml of the degrading enzyme solution is added to the test tube, and the degrading enzyme is incubated at 37 ° C. for 1 hour under gentle shaking conditions.
(3) After cooling this with ice, 1000 μl of DMAB (para-dimethylaminobenzaldehyde) reagent was added, heated at 37 ° C. for 20 minutes, and then the absorbance at 585 nm was measured to measure the increase in reducing end. Then, the amount of N-acetylglucosamine or glucosamine released is quantified. The DMAB reagent is a solution obtained by adding 10 g of DMAB to 100 ml of acetic acid containing 12.5% by weight of 10N hydrochloric acid, and diluted 10-fold with acetic acid immediately before use.

  The use of the decomposable composite material of the present invention is not particularly limited and can be used in various fields. As a suitable example of the use of the degradable composite material of the present invention, medical materials such as wound dressings and medical prosthetic materials are exemplified. When the degradable composite material of the present invention is applied to an affected area, it contacts the moisture present in the affected area, whereby the supported degrading enzyme is activated and starts self-degradation. As described above, since the degradable composite material of the present invention is self-degraded and absorbed in the living body, the medical material used so as to remain in the living tissue for a long time (for example, a wound dressing material, a medical prosthetic material) Useful as. Here, when the degradable composite material of the present invention is used as a medical material, it may be used for human medical purposes or may be used for mammalian (non-human) medical purposes.

  For example, when the decomposable composite material of the present invention is a sheet, it can be suitably used as a wound dressing. When the degradable composite material of the present invention is used as a wound dressing, the degradable composite material applied to the wounded area is decomposed and absorbed in the living body when the wound is healed to some extent. There is an advantage that there is no need. Furthermore, when used as a wound dressing, the therapeutic effect of the wound can be enhanced by gradually decomposing in the process of wound healing.

  For example, when the decomposable composite material of the present invention is fibrous, it can be suitably used as a medical prosthetic material. When the decomposable composite material of the present invention is used as a medical prosthetic material, it is absorbed into the living body along with the healing of the affected affected area, so that there is no need to remove the thread after the operation and the burden on the patient can be reduced.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited thereto.
Example 1
1. Preparation of chitinase
An Escherichia coli holding a plasmid in which a DNA encoding a chitinase derived from Pyrococcus furiosus (amino acid sequence represented by SEQ ID NO: 2 described in JP-A-2004-344160) was incorporated was treated with LB medium (10 g of polypeptone in 1 L). , Containing 5 g of yeast extract and 10 g of sodium chloride at 37 ° C. with shaking, and isopropyl βthiogalactopyranoside (IPTG) during the logarithmic growth phase (optical density 0.2 to 0.4 at 600 nm) Was added to a final concentration of 0.2 mM in the medium. Cultivation was continued overnight, and then E. coli cells were recovered by centrifugation (6000 × g for 7 minutes).

  Escherichia coli recovered from 1 L of the culture solution was suspended in 20 mL of buffer A (25 mM Tris-HCl [pH 7.5], 1 mM EDTA, 25 mM NaCl) and subjected to ultrasonic disruption. After crushing, an extract was obtained by high-speed centrifugation (13,000 × g for 15 minutes). The extract was heated at 85 ° C. for 30 minutes, and the supernatant was recovered again by high-speed centrifugation (13000 × g for 15 minutes).

  The collected supernatant was added to an anion exchange column HiTrapQ (Amersham, 5 ml). AKTA prime (Amersham) was used for addition and elution to the column. Elution was performed with a concentration gradient (25 mM to 1 M) of a salt (NaCl) contained in buffer A. The fraction from which the target chitinase was eluted was collected and precipitated overnight at 4 ° C. under conditions of 80% ammonium sulfate saturation. The precipitate was recovered by high-speed centrifugation (13000 × g for 15 minutes) and redissolved with buffer A. The sample after dissolution was added to a gel filtration column HiLoad 26/60 Superdex-200pg (Amersham) equilibrated with buffer A to remove contaminating low molecular weight protein and residual ammonium sulfate. The fraction eluted with chitinase was collected and concentrated with CentriPrep YM-10 (Amicon).

  The chitinase thus obtained had a chitinase activity of 18000 U at 37 ° C. per 1 g (in terms of dry weight).

2. Evaluation of Chitinase Degradability of Sheets Containing Chitin A sheet containing chitin (Unitika Ltd., Besquitin W) was cut into a size of 20 mm × 20 mm (about 0.0125 g), and each of the sheets was 50 mM with 50 mM pH 4.5. In 20 ml of acetate buffer or Tris buffer at pH 7.4. To each of these two types of solutions, 0.0006 g of the chitinase obtained above was added and kept at 37 ° C. and 60 ° C., respectively. After a certain time, the sheet was taken out and its collapse was confirmed. For comparison, the same experiment was performed without adding chitinase.

  The result of evaluating the disintegration property of the sheet in the acetate buffer under the temperature condition of 60 ° C. is shown in FIG. As shown in FIG. 1, it was revealed that the sheet containing chitin is decomposed when it coexists with chitinase. Similar results were obtained with acetate buffer at 37 ° C, Tris buffer at 37 ° C, and Tris buffer at 60 ° C. From the above results, the chitin-containing sheet was decomposed in the presence of chitinase even at a temperature of 37 ° C., so the chitin sheet carrying chitinase has self-degradability in vivo. It was confirmed to be effective for medical use.

3. Production of degradable composite material Sheet containing chitin (Unitika Ltd., Vesquitin W, 100 mm ×) in 10 mL of 50 mM Tris buffer (pH 7.5) containing the chitinase obtained above at a concentration of 0.011 g / mL 120 mm, 0.4 g) was impregnated at 4 ° C. for 30 minutes. Thereafter, the sheet was taken out and dried at 50 ° C. to obtain a chitin sheet (degradable composite material) carrying chitinase.

In an acetate buffer, it is a photograph which shows the result of having evaluated the disintegration property of the sheet | seat containing a chitin on 60 degreeC temperature conditions.

Claims (5)

  A degradable composite material, wherein a decomposing enzyme capable of hydrolyzing a polysaccharide is supported on a molded article containing at least one polysaccharide selected from the group consisting of chitin and chitosan.   The decomposable composite material according to claim 1, wherein the molded product containing the polysaccharide is in a sheet form or a fiber form.   The degradable composite material according to claim 1 or 2, wherein the degrading enzyme is a heat-resistant chitinase having an activity at 4 ° C or higher and an optimum temperature of 95 ° C or higher.   The degradable composite material according to claim 1 or 2, wherein the degrading enzyme is a thermostable chitinase derived from Pyrococcus furiosus.   The decomposable composite material according to any one of claims 1 to 4, which is a medical material.

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