JPH07155193A - Production of chitosan oligosaccharide derived from microorganism - Google Patents

Abstract

PURPOSE:To efficiently produce a chitosan oligosaccharide derived from a microorganism, especially the chitosan oligosaccharide having a high degree of polymerization of pentamer or a high polymer from a hypha of a microorganism capable of producing chitosan according to a simplified process. CONSTITUTION:The characteristic of this method for producing a chitosan oligosaccharide derived from a microorganism comprises treating a hyphal cell wall of a microorganism capable of producing chitosan such as Absidia coerulea IFO5301 strain or Mucor tuberculisporus IFO9256 strain with an enzyme capable of producing the chitosan oligosaccharide such as Chitosanase RD<(>R<)> derived from Bacillus sp. PI-7S or Cellulase onozuka R-10<(>R<)> derived from Trichoderma viride.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing chitosan oligosaccharides, and more particularly to a method for producing chitosan oligosaccharides by enzymatically treating the mycelium of a chitosan-producing microorganism.

[0002]

2. Description of the Related Art In recent years, chitosan has been attracting attention as a next-generation biomass that is abundantly present on the earth, and various application developments utilizing its biocompatibility and biodegradability have been studied. Since chitosan has a high molecular weight, when it is made into a solution, it is difficult to handle due to its high viscosity, and it is impossible to prepare a high-concentration solution. Further, when the solution is made neutral or alkaline, it becomes insoluble. These properties limit the application of chitosan. Therefore, as a way of expanding the application of chitosan, use of easily water-soluble chitosan oligosaccharide obtained by hydrolyzing high molecular weight chitosan is expected. In particular, chitosan oligosaccharides having a high degree of polymerization of pentamers or more have been found to have various physiological activities such as antibacterial properties, antitumor properties, plant elicitor activities, etc., and their added value is increasing.

Hitherto, as a method for producing chitosan oligosaccharides, chitosan obtained by chemically deacetylating chitin extracted from crustaceans such as crab and shrimp is hydrolyzed with an acid such as hydrochloric acid to reduce its molecular weight. How to do (J. Am.
Chem. Soc., 79, 5046-5049, (1957)) is known. However, this method has problems in safety due to the use of an acid and complicated post-treatment. A method of hydrolyzing chitosan obtained from crustaceans with chitosan-degrading enzymes such as chitosanase has been reported (Japanese Patent Publication No. 3-13).
No. 878), but this method is expected to have physiological activity.
Chitosan oligosaccharides having a high degree of polymerization equal to or higher than that of a monomer are not obtained in high yield.

On the other hand, it is reported that some microorganisms, such as zygomycetes, contain chitosan in the mycelium cell wall (Ann. Rev. Microbiol., 22, 87-108, (196
8)). As a method for producing a chitosan oligosaccharide derived from a microorganism,
A method of hydrolyzing chitosan extracted with an acid such as acetic acid by an enzyme after heat treatment of mycelium of a chitosan-producing microorganism to lower molecular weight has been reported (JP-A-2-
215393). However, when chitosan extracted from a microorganism is used, the production amount of chitosan per unit culture solution is low, so that there is a drawback that the price becomes extremely higher than that when chitosan produced from crustaceans is used.

[0005]

The object of the present invention is to efficiently produce chitosan oligosaccharides derived from microorganisms, especially chitosan oligosaccharides of pentamer or higher with a high degree of polymerization, in a simplified process from the mycelium of chitosan-producing microorganisms. The present invention is to provide a method of manufacturing the same.

[0006]

[Means for Solving the Problems] The present inventors have focused on the fact that cell wall lytic enzymes such as chitosanase decompose the cell wall of microorganisms, and chitosan having a high degree of polymerization can be obtained by enzymatic treatment of mycelium of chitosan-producing microorganisms. The inventors have found that oligosaccharides can be produced and completed the present invention.

That is, the present invention is a method for producing a chitosan oligosaccharide, which comprises treating the mycelium of a chitosan-producing microorganism with an enzyme that decomposes the mycelial cell wall of the chitosan-producing microorganism and produces a chitosan oligosaccharide. .

As the microorganism used in the present invention, any microorganism can be used as long as it can produce chitosan. For example, Absidia, Actinomucor, Choanephora
Examples of the genus, Cunninghamella genus, Mortierella genus, Mucor genus, Phycomyces genus, Rhizopus genus and the like. Specific examples thereof include Absidia coerulea IFO5301 strain, Mucor tuberculisporus IFO9256 strain, and the like. These microorganisms are found in the Fermentation Research Institute (I
FO).

As the enzyme used in the present invention, any enzyme can be used as long as it decomposes the mycelial cell wall of the above chitosan-producing microorganism to produce chitosan oligosaccharide. For example, the genus Bacillus, Streptomyces (Str
eptomyces) genus, chitosanase derived from the penicillium (Penicillium) genus, Aspergillus genus,
Cellulase derived from the genus Trichoderma and the like can be mentioned. More specifically, Bacillus
Examples include p.PI-7S-derived chitosanase-RD (manufactured by Pierce) and Trichoderma viride-derived cellulase Onozuka R-10 (manufactured by Yakult). These enzymes are first prepared in, for example, 0.1 M acetate buffer (pH 5.5) at about 0.1-5 U /
Add ml to make an enzyme preparation. The pH of the enzyme preparation is 4.0-7.0
Is preferred.

The enzyme activity is measured by the following method. pH
A reaction solution containing a 1% chitosan solution dissolved in 6.0 acetate buffer and the enzyme solution is reacted at 37 ° C for 10 minutes. About 100 reactions
After heat treatment at 5 ° C for 5 minutes to complete the enzyme treatment, the modified Schales method (Agric.Biol.Chem., 35, 1154-1156 (1
971)) to measure the chitosanase activity. 1 unit is
1 μ of soluble chitosan per minute at pH 6.0 and 37 ℃
It is the amount of enzyme that releases a reducing sugar equivalent to mol of glucosamine.

Step 1: Preparation of mycelium The mycelium of the chitosan-producing microorganism used in the present invention is obtained by culturing the microorganism subcultured in a slant medium or the like in a liquid medium for producing chitosan and growing it. Examples of the liquid medium for producing chitosan include yeast extract 0.1 to 2.5%, polypeptone 0.5 to 5%, glucose 1 to 10%, ammonium sulfate 0.5 to 1%, potassium dihydrogen phosphate 0.1 to 1%.
%, Sodium chloride 0.1-1%, magnesium sulfate (7
Hydrate) 0.05 to 0.1% and calcium chloride (dihydrate) 0.01 to 0.1%, and a liquid medium having a pH of 4.5 is exemplified. The culture conditions are about 25 in liquid medium for chitosan production.
Incubate with shaking at ℃. The culture period is preferably 4 to 5 days. Spore concentration is preferably 10 ⁶ to 10 ⁷ cells / ml. Step 2: Crushing of mycelium The cultured mycelium is immersed in an aqueous solution such as an acetate buffer, crushed by a homogenizer or the like, and then subjected to enzyme treatment. Step 3: Enzyme treatment For example, the mycelium crushed in the enzyme preparation to which the enzyme is added is immersed in an aqueous solution such as an acetate buffer solution and shaken, and the enzyme treatment is performed at 30 to 60 ° C for 1 hour or more By carrying out, chitosan oligosaccharide is produced in the reaction solution. The enzyme treatment is performed by gently shaking. Step 4: Reaction completion Generally, heat treatment is performed at about 100 ° C. for about 5 minutes to complete the enzyme treatment. Step 5: Purification The produced chitosan oligosaccharide can be separated according to the degree of polymerization by using ion chromatography, thin layer chromatography or the like.

[0012]

The present invention will be described in detail below with reference to examples. Example 1 Potato dextrose agar (manufactured by Nissui Pharmaceutical Co., Ltd.) Absidia coelrea (Abs) subcultured at 25 ° C. on a slant medium
Spores of idia coerulea) IFO5301 strain were suspended in sterile water.
The spore suspension was prepared by adding yeast extract 0.1%, polypeptone 1%, glucose 2%, ammonium sulfate 0.5%, potassium dihydrogen phosphate 0.1%, sodium chloride 0.1%, magnesium sulfate (heptahydrate) 0.05% and calcium chloride (2%). Hydrate) 0.01% to pH 4.5 liquid medium 100ml, 10 ⁶ ~
Inoculate to a spore concentration of 10 ⁷ cells / ml and spin at 12
The mixture was stirred at 0 rpm and 25 ° C and cultured for 4 days. The grown mycelium was collected by suction filtration, washed, and then subjected to enzyme treatment.

4 g of the obtained mycelium was added to 0.1 M acetate buffer (pH 5.
It was immersed in 5) and crushed with a homogenizer. After removing the buffer solution, the crushed mycelium was treated with 0.1 M acetate buffer (pH 5.5).
Bacillus sp. PI- added at a concentration of 0.1 U / ml to
The mycelium was treated with enzyme by immersing it in 120 ml of an enzyme preparation of 7S-derived chitosanase-RD (manufactured by Pierce) and gently shaking it at 37 ° C for 1 hour or more. After the enzyme treatment, the reaction solution was heated at 100 ° C. for 5 minutes to stop the reaction, and then the enzymatic decomposition product in the enzyme treatment reaction solution was analyzed by thin layer chromatography (TLC). A TLC plate of silica gel (Kieselgel 60F manufactured by Merck & Co., Inc.) was coated with the enzyme-treated reaction solution and a standard solution of chitosan degradation product derived from crab shell, developed with 1-butanol-acetic acid-water = 2: 1: 2, and developed with a ninhydrin solution. Then, spots were detected. As a result, spots corresponding to the spots of chitosan oligosaccharide derived from crab shell were found in the enzyme-treated reaction solution. FIG. 1 shows the result.

Next, TLC of the whole reaction solution was performed to collect a thin layer of a fraction corresponding to the chitosan oligosaccharide hexamer. The collected thin-layer fraction was suspended in 0.1 M acetic acid, insoluble matter was removed by filtration, and the eluate was lyophilized to give a high-polymerization degree chitosan oligosaccharide fraction mainly containing a hexamer. 118 mg was obtained.

Example 2 As in Example 1, Mucor tubercusporus (Mucor)
tuberculisporus) mycelium obtained by culturing IFO9256 strain
Trichoderma viride-derived cellulase Onozuka R-10 (manufactured by Yakult) prepared by adding 4 g of 0.1 M acetate buffer (pH 5.5) at a concentration of 0.1 U / ml 12
Treated in 0 ml. After that, the same operation as in Example 1 was carried out, and as a result, a spot corresponding to the spot of the crab shell-derived chitosan oligosaccharide was observed in the TLC of the enzyme-treated reaction solution.
Next, as a result of TLC of all the reaction solutions, a high degree of polymerization chitosan oligosaccharide fraction 39 mg was obtained.

Comparative Example 1 In the same manner as in Example 1, Absidia coelrea (Abs
idia coerulea) IFO5301 strain was cultivated to obtain 4 g of mycelium
300 ml of a 2% aqueous sodium hydroxide solution was added to and heated at 121 ° C. for 1 hour in an autoclave. The obtained alcal-insoluble substance was collected with a glass filter and washed with purified water until the washing liquid became neutral. This alkaline insoluble substance 7
10 mg was immersed in 300 ml of a 2% acetic acid aqueous solution, crushed with a homogenizer, and then stirred at 37 ° C. for 30 minutes. After stirring, it was centrifuged to separate into a supernatant and a precipitate. The supernatant of the two precipitations was filtered through a glass filter to remove insoluble matter. A 20% aqueous sodium hydroxide solution was added to this acetic acid extract to adjust the extract to pH 8.5 to 9.0 to precipitate chitosan.
After washing the precipitated chitosan with water and centrifugation a total of 3 times,
Vacuum dried. The total amount of chitosan obtained from mycelium is 2
It was 04 mg. Chitosan 204 mg from Absidia coerulea IFO5301 strain obtained by the above method was dissolved in 0.1 M acetate buffer to prepare 1% chitosan solution (pH 5.5) 0.1 U / m
Chitosanase from Bacillus sp.
The enzyme reaction was carried out at 0 ° C for 1 hour or more. After the reaction solution was heated at 100 ° C. for 5 minutes to stop the reaction, TLC was performed in the same manner as in Example 1. As a result, a spot corresponding to the spot of the chitosan oligosaccharide derived from crab shell was found in the enzyme reaction solution. Next, as a result of TLC of all reaction solutions,
Chitosan oligosaccharide fraction with a high degree of polymerization, which is mainly composed of hexamers 41
mg was obtained.

[0017]

According to the present invention, a chitosan oligosaccharide having a high degree of polymerization can be obtained by enzymatically treating the mycelium of a chitosan-producing microorganism. INDUSTRIAL APPLICABILITY According to the present invention, chitosan oligosaccharide can be obtained by a more simplified process without extracting chitosan as in the conventional method for producing chitosan oligosaccharide.

[Brief description of drawings]

FIG. 1 shows the results of TLC analysis of a reaction solution obtained by enzymatically treating a mycelium of Absidia coerulea IFO5301 strain with chitosanase derived from Bacillus sp. PI-7S.

Claims (5)

[Claims] 1. A method for producing a chitosan oligosaccharide derived from a microorganism, which comprises treating a mycelium of a chitosan-producing microorganism with an enzyme that decomposes a mycelial cell wall of the chitosan-producing microorganism and produces a chitosan oligosaccharide. 2. The method for producing a microorganism-derived chitosan oligosaccharide according to claim 1, wherein the chitosan-producing microorganism is Absidia coerulea or Mucor tuberculisporus. 3. The method for producing a chitosan oligosaccharide derived from a microorganism according to claim 1, wherein the enzyme that decomposes the mycelium cell wall of the chitosan-producing microorganism to produce chitosan oligosaccharide is chitosanase or cellulase. 4. The method for producing a microorganism-derived chitosan oligosaccharide according to claim 1, wherein the chitosanase is a Bacillus-derived chitosanase. 5. The method for producing a microorganism-derived chitosan oligosaccharide according to claim 1, wherein the cellulase is a cellulase derived from Trichoderma.