JPH0313878B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention does not contain monosaccharides of D-glucosamine, and mainly has a degree of polymerization of D-glucosamine of 2 to 8.
This invention relates to a method for producing chitosan oligosaccharides that can obtain chitosan oligosaccharides containing oligosaccharides in high yield, and the chitosan oligosaccharides obtained by the present invention can be used as food additives, cosmetic ingredients, pharmaceuticals or medical treatments. It can be used for a wide range of applications such as materials. [Technical Background and Description of Prior Art] Chitin is a polysaccharide obtained from the shells of crustaceans such as shrimp and crabs, and has a chemical structure very similar to cellulose. 2-acetamido-2-deoxy-D in which the hydroxyl group at position 2 of is substituted with an acetamido group
-Glucose (N-acetylglucosamine) is β
-It is a linear polysaccharide with 1,4 bonds. On the one hand, chitosan oligosaccharides, which are degradation products of chitosan, such as chitobiose (GlcN) ₂ ,
Since chitotriose (GlcN) ₃ , chitotetraose (GlcN) ₄ , chitopentaose (GlcN) ₅ , and chitohexaose (GlcN) ₆ are amino sugars (basic sugars), these chitosan oligosaccharides It is thought that chitosan oligosaccharides can be used for a wide range of applications such as food additives (fillers), cosmetic ingredients, and pharmaceuticals, and there is a need for technology and development to economically produce these chitosan oligosaccharides. Recently, chitohexaose (GlcN) ₆ and chitoheptaose (GlcN) ₇ , both chitosan oligosaccharides, have been found to have antifungal properties.
Kendra and Lee A. Hadwiger: Experimental Mycology (DFKendra
and Lee A. Hadwiger: Experimental
Mycology) Volume 8, pp. 276-281 (1984)],
There is a need for the development of a technology for producing chitosan oligosaccharides with a relatively high degree of polymerization. Up to now, a hydrolysis method using hydrochloric acid [S.T. Horowitz et al.: Journal of American Chemical Society (ST.
Horowitz et al: JACS) Volume 79, No. 5046-
5049 pages (1957)], oxidative decomposition method using nitrous acid [F. Yaku et al.: Cellulose Chemistry &
Technology (F. Yaku et al: Cellulose
Chemistry and Technology) Volume 11, No. 421-
430 pages (1977)] and the oxidative decomposition method using chlorine (Shigehiro Hirano et al.: Japanese Society of Agricultural Chemistry, 1988 Annual Meeting,
Abstracts, page 330) is a known method for chemically decomposing chitosan. Although the hydrolysis method using hydrochloric acid can produce chitosan oligosaccharides, it requires a high concentration of hydrochloric acid and a long reaction time, and also produces a large amount of D-glucosamine monosaccharides, which makes it difficult to remove chitosan oligosaccharides from the reaction solution. It requires an isolation process, which makes the operation complicated,
The disadvantage is that the cost is also high. Furthermore, the oxidative decomposition method using nitrous acid or chlorine has the disadvantage that it is difficult to obtain pure chitosan oligosaccharides due to deamination caused by oxidation. On the other hand, when decomposing chitosan using an enzymatic method, the specificity of the enzyme can be utilized.
By reducing the production of monosaccharides such as D-glucosamine, it is possible to produce a significant amount of the desired chitosan oligosaccharide. Enzymes that degrade chitosan that have been reported so far include Bacillus
sp.) Chitosanase produced by R-4 [Tominaga and Y.Tsujisaka: Biohimica e.
Biochimica et Biophysica Acta) Volume 410, No.
145-155 (1975)], chitosanase produced by Penicillium islandicum [DMFenton et al: Journal of General
Microbiology), Vol. 126, pp. 151-165 (1981
Chitosanase produced by Bacillus sp. 99-5 (Horiuchi: Japanese Society of Agricultural Chemistry, 1982)
annual conference, lecture abstracts page 550), Streptomyces sp. No. 6 [G.S.
JSPrice et al: Journal of Bacteriology
124, pp. 1574-1585 (1975)] and Streptomyces griseus
chitosanase [Otakara: chitin, chitosan and related
Enzymes (A.Ohtakara: Chitin, Chitosan
and Related Enzymes) pp. 147-160 (1985
), Academic Press] is known.
There are no reports that chitosan oligosaccharides are produced in significant amounts. With the aim of developing a technology to economically produce chitosan oligosaccharides, the present inventors conducted a search for bacteria that produce chitosanase, and discovered a mutant strain of a microorganism belonging to the genus Bacillus.
We discovered that sp.) No. 7-M can produce chitosanase and that when this chitosanase is applied to chitosan, only chitosan oligosaccharides can be produced without producing D-glucosamine monosaccharides. We have arrived at the present invention based on the findings. [Objective of the Invention and Summary of the Invention] The object of the present invention is to provide an economically viable method for producing chitosan oligosaccharides that can produce a significant amount of chitosan oligosaccharides without producing D-glucosamine monosaccharides. It is about providing. The present invention uses chitosan as an enzyme produced by a microorganism belonging to the genus Bacillus, which has an action of PH5 to
7. Stable pH range 5-11 and molecular weight 30,000 (SDS)
41,000) to obtain a chitosan oligosaccharide containing mainly oligosaccharides having a degree of polymerization of 2 to 8 without containing monosaccharides of D-glucosamine. It is. In the production of chitosan oligosaccharide of the present invention, chitosanase is Bacillus No. 7-M
Chitosanase produced by culturing No. 8139) can be used, and colloidal chitosan can be used in addition to a chitosan solution, and the hydrolysis of chitosan by chitosanase can be carried out in a reaction solution around pH 6. It is preferable to carry out the [Specific description of the invention] The chitosan oligosaccharide produced by the present invention is
It is a chitosan oligosaccharide that mainly contains oligosaccharides with a degree of polymerization of D-glucosamine of 2 to 8 and does not contain monosaccharides of D-glucosamine. Since this chitosan oligosaccharide acts as a cation and has hygroscopic properties, it is useful as a skin moisturizer when used in cosmetics. In addition, among chitosan oligosaccharides, those with a relatively high degree of polymerization are
Since it is highly safe and has antifungal and antibacterial properties, it is suitable for use as a preservative for cosmetics, foods, etc., and as an antifungal and antibacterial agent in soil conditioners, seed coating agents, and the like. In the production of chitosan oligosaccharides of the present invention,
First, a chitosan solution is prepared. Since chitosan is soluble in acid, an aqueous acid solution is added to chitosan to obtain a chitosan solution. Separately, a chitosanase solution is prepared and pre-incubated together with the previously obtained chitosan solution at a reaction temperature, then the chitosanase solution is added to the chitosan solution, and the chitosan is decomposed by the chitosanase at the reaction temperature. The reaction temperature is 30 to 80 degrees above the action temperature of chitosanase.
℃, preferably around 40℃. Further, the PH of the reaction solution can be set to 3 to 9, which is the operating PH range of chitosanase, but it is preferably set to around 6, which is the optimum PH. Any chitosan can be used as long as it is decomposed by chitosanase, but the degree of deacetylation is 50 to 100%.
It is preferred to use chitosan. When chitosanase is added to these chitosan to degrade it,
In addition to the chitosan solution described above, colloidal chitosan can also be suspended in water. The acid used to prepare the chitosan solution can be any organic or inorganic acid as long as it can dissolve chitosan, but dilute solutions of hydrochloric acid or nitric acid, formic acid, ,
Preference is given to using dilute solutions of acetic acid, lactic acid, glutamic acid or ascorbic acid. Since the degree of polymerization distribution of chitosan oligosaccharide changes depending on the reaction time, the relationship between the reaction time and the degree of polymerization distribution of the chitosan oligosaccharide product is experimentally determined in a preliminary experiment, and based on this, the desired value can be determined. The reaction time can also be set as the reaction time required to obtain chitosan oligosaccharide having a polymerization degree distribution. After a predetermined reaction time, the chitosanase in the reaction solution is inactivated to stop the reaction, and the supernatant is collected by centrifugation or filtration of the reaction solution, which is then chromatographed using a conventional ion exchange resin. Graphie,
After impurities are removed by gel filtration or removal of colored substances using activated carbon, the mixture is dried to obtain the desired chitosan oligosaccharide powder. The chitosanase used in the production of the chitosan oligosaccharide of the present invention is stable in the PH range of 5 to 11, and does not produce D-glucosamine monosaccharides when chitosan is decomposed, and mainly has a polymerization degree of 2 to 1. Chitosanase that produces oligosaccharides of No. 8, and such chitosanase is Bacillus sp. No. 7-M (Feikoken Bacterial Serial No. 8139)
produced in the medium when cultured. Bacillus sp. No. 7-M is a bacterium that can grow in a medium containing chitin or chitosan as the sole carbon source, and was isolated from the soil of a virgin swamp in Unzen, Obama-cho, Minamitakagi-gun, Nagasaki Prefecture. This parent strain was treated with N-methyl-N'-nitroso-N-nitrosoguanidine (NTG) to induce mutations, and highly active chitosanase was selected from among the streptomycin-resistant mutants obtained. This is a mutant strain isolated as a product that can be produced, and has been deposited with the National Institute of Microbial Technology, Ministry of International Trade and Industry as FERM P-8139. The mycological properties of Bacillus No. 7-M are shown below. A Cell morphology (1) Cell shape and size: Short bacilli, (broth and broth agar slant culture, 37℃, 24
~72 hours of culture) (2) Presence or absence of cell pleomorphism: None, (3) Presence or absence of motility: Yes, (Meat juice agar semi-fluid high-layer puncture culture) (4) Presence or absence of spores: Yes, endospores and naked spores, spherical, [Dorner's staining method and modified Witz method] (5) Gram staining: positive, [broth agar slant culture,
37°C, 18 hours, stained by Hucker's modified method] B Growth status in each medium (1) Broth agar plate culture (37°C, 24-168 hours): Round, raised, milky white with a filamentous periphery. form a colony. The surface of the colony is uneven, slightly shiny, and translucent. It will get better as time passes. Does not produce pigment. (2) Broth agar slant culture (37°C, 24-168 hours): Forms a milky white colony that rises in a spread shape. Colonies have convex circular ridges and are shiny. It grows well and will spread over time. Does not produce pigment. (3) Meat juice liquid culture (37℃, 24-168 hours): No film is formed on the surface. The whole thing becomes cloudy over time. Granular sediments are formed at the bottom and gradually increase in number. (4) Meat juice gelatin puncture culture (25°C, 24-168 hours): Grows along the puncture line and liquefies. The surface and interior grow funnel-shaped and liquefy. The liquefied part becomes cloudy. (5) Litmus milk (37°C, 24-168 hours): After two days, the upper part gradually liquefied, and on the fourth day, the color completely changed and became acidic. It does not coagulate. As time passed, liquefaction progressed and it became translucent. C Physiological properties (1) Nitrate reduction: - (Nitrate broth medium, 37℃, 24-120 hours) (2) Denitrification reaction: - (Komagata et al.'s method, using fermentation tube, 37℃, 24 hours)
~120 hours) (3) MR test: + (37℃, 24-168 hours) (4) VP test (acetylmethylcarbinol production test: + (37℃, 24-168 hours) (5) Indole production: - (37℃, 24-168 hours) (6) Hydrogen sulfide generation: - (TSI agar method, 37℃, 24-168 hours) (7) Starch hydrolysis: + (37℃, 24-168 hours) ) (8) Utilization of citric acid (Koser's medium, 37℃, 24-168 hours):
− (Christensen's medium, 37°C, 24–168
time): + (9) Utilization of inorganic nitrogen source (37℃, 24-168 hours) Nitrate: undetermined, ammonium salt: undetermined, (10) Pigment production (mannitrate/yeast extract agar slant): - [King ( King) A agar slant medium]: - (11) Presence of fluorescence: None (12) Urease: + (Christensen-Urea agar medium, 37
℃, 24-168 hours) (13) Oxidase: + (broth agar, 37℃, 24-48 hours) (14) Catalase: + (broth agar, 37℃, 24-48 hours) (15) Growth Range: (gravy agar medium) Temperature: undetermined, PH: 5-10, Added salt concentration: undetermined, (16) Attitude towards oxygen: aerobic (1% glucose gravy high-rise agar medium, 37
(°C, 24-72 hours) (17) 0-F test (Hugh-Leifson method, 37°C, D-glucose): Acid is produced fermentatively.
(fermentative) (18) Presence or absence of acid and gas production from sugars (37℃, 24-168 hours): Sugar Acid Gas D-glucose + - D-mannose - - D-galactose - - D-fructose + - L-arabinose − − D-xylose − − D-sorbit − − D-mannite − − Inosyte − − Maltose + − Satucarose + − Lactose − − Starch + − Cellulose − − Glycerin − − Regarding the above mycological properties, Bergey's Manual of Determinative Bacteriology
When searching the 8th edition (1974) of Bacteriology, it was found that strain No. 7-M most likely belongs to the genus Bacillus. The enzymatic chemical properties of chitosanase produced by Bacillus No. 7-M are as shown below. (1) Action: Acts on chitosan, arbitrarily decomposes β-1,4 bonds from the internal chains of the molecule, and mainly forms chitosan oligosaccharides (GlcN ₎ (n=2 to 8) (dimers to 8
(quantity). Chitosan oligosaccharides can be separated from the chitosan decomposition solution using high performance liquid chromatography. The decomposition degree of chitosan in this decomposition solution is about 45%. It also acts on carboxymethyl cellulose (CMC), degrading it to some extent, but has no effect on chitin. (2) Action temperature range and optimal action temperature: When soluble chitosan is used as a substrate, it works up to 80°C, and the optimal action temperature is 50°C. Figure 1 shows the relationship between temperature and specific activity when reacting for 10 minutes at pH 6.0. (3) Action PH range and optimum PH: It works in the range of PH3 to 9, and the optimum PH is PH
It is 6. 1 ml of 1% soluble chitosan and 2 ml of each pH buffer
FIG. 2 shows the relationship between pH and specific activity of the enzyme when a reaction solution containing 1 ml of the enzyme solution was reacted at 37° C. for 10 minutes. (4) Thermostability: Almost stable until kept at 50°C for 15 minutes; heating at 60°C for 15 minutes deactivates approximately 40% of the enzyme; heating at 70°C for 15 minutes completely deactivates the enzyme. Deactivated. Figure 3 shows the relationship between temperature and specific activity. (5) PH stability: The remaining enzyme activity was measured after being left in a 0.1M buffer at 30°C for 2 hours.
It was stable within the range of 11. One of the major characteristics of chitosanase produced by Bacillus No. 7-M is that it is stable at pH 10 to 11. Figure 4 shows the relationship between PH and specific activity. (6) Inhibitor: Chitosanase produced by Bacillus No. 7-M was treated with HgCl ₂ , PbCl ₂ , at a final concentration of 1×10 ⁻³ M;
AgNO ₃ , and nearly 100 due to the presence of PCMB
% was inhibited. (7) Substrate specificity: Various substrates were used, and the final concentration of the substrate was 0.25%.
1 enzyme protein per 4 ml of enzyme reaction solution
The amount of total reducing sugars and hexamine released after 1 hour (mg/mg protein/hour) was measured.
The results are shown in Table 1.

【table】

〔Effect of the invention〕

Chitosan oligosaccharides mainly containing oligosaccharides with a degree of polymerization of 2 to 8 can be efficiently produced without producing monosaccharides of D-glucosamine.

[Brief explanation of the drawing]

Figure 1 is a chart showing the relationship between temperature and specific activity in the action temperature range of chitosanase, Figure 2 is a chart showing the relationship between PH and specific activity in the action PH range of chitosanase, and Figure 3 is a chart showing the thermostability of chitosanase. Figure 4 is a diagram showing the relationship between temperature and specific activity in PH stability of chitosanase. Figures 5 and 6 are diagrams showing the results of molecular weight measurement of chitosanase. , FIG. 7 is a chart showing the relationship between the reaction time and the amount of reducing sugar produced in Example 1, and FIG. 8 is a chart showing the ratio of chitosan oligosaccharides according to the degree of polymerization in Example 1.
FIG. 9 is a chart showing the relationship between the reaction time and the amount of reducing sugar produced in Example 2, and FIG. 10 is a chart showing the relationship between the reaction time and the amount of reducing sugar produced in Example 3.
FIG. 11 is a chart showing the relationship between the reaction time and the amount of reducing sugar produced in Example 4, and FIG. 12 is a chart showing the relationship between the reaction time and the amount of reducing sugar produced in Example 4.
FIG. 13 is a chart showing the ratio of chitosan oligosaccharide according to degree of polymerization in Example 5, and FIG. 13 is a chart showing the ratio according to degree of polymerization of chitosan oligosaccharide in Example 6.

Claims (1)

[Scope of Claims] 1. Chitosan is an enzyme produced by a microorganism belonging to the genus Pacillus, which has an action of PH5 to 7 and is stable.
It is decomposed by chitosanase having a pH range of 5 to 11 and a molecular weight of 30,000 (41,000 in SDS) to produce chitosan oligos containing mainly oligosaccharides with a degree of polymerization of 2 to 8 without containing D-glucosamine monosaccharides. A method for producing chitosan oligosaccharide characterized by obtaining sugar. 2 The microorganism belonging to the genus Bacillus is Bacillus No.7.
-M (Feikoken Bibori No. 8139). 3. The method for producing chitosan oligosaccharide according to claim 1 or 2, wherein the chitosan is colloidal chitosan. 4. The method for producing chitosan oligosaccharide according to any one of claims 1 to 3, characterized in that the decomposition of chitosan by chitosanase is carried out before or after pH 6.