JPS6230103A - Production of chitosan oligosaccharide - Google Patents

Abstract

PURPOSE:To produce a chitosan oligosaccharide containing no monosaccharide of D-glucosamine in high yield inexpensively, by decomposing chitosan with chitosanase produced by a microorganism. CONSTITUTION:A chitosan (e.g., colloidal chitosan, etc.,) is decomposed with chitosanase, an enzyme produced from a bacterium [e.g., Bacillus No7-M(FERM P-8139), etc.,], stable at 5-11pH, to give a chitosan oligosaccharide containing mainly an oligosaccharide with 2-8 polymerization degree without a D- glucosamine monomer. USE:Useful as a food additive, a cosmetic component, a drug or a medical material.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention provides a high-yield method for producing chitosan oligosaccharides that do not contain D-glucosamine monosaccharides and mainly contain D-glucosamine oligosaccharides with a degree of polymerization of 2 to 8. The present invention relates to a method for producing chitosan oligosaccharide that can be obtained at a high rate, and the chitosan oligosaccharide obtained by the present invention can be used for a wide range of applications such as food additives, cosmetic ingredients, pharmaceuticals, and medical materials. be.

[Technical background and explanation of conventional technology]

Chitin is a multitubular substance obtained from the shells of crustaceans such as shrimp and crabs, and has a chemical structure very similar to cellulose. It is a linear polysaccharide in which substituted 2-acetamido-2-deoxy-D-glucose (N-acetylglucosamine) is linked with β-1,4 bonds.

On the one hand, chitosan oligosaccharides which are degradation products of chitosan, such as chitobiose (c Ic N )2,
Chitotriose (GICN) 3, Chitotetraose (
G 1 c N) 4 , chitopentaose (GICN
) 5, and chitohexaose (G lc N )e
These chitosan oligo rings can be used in a wide range of applications such as food additives (filling agents), cosmetic ingredients, and pharmaceuticals, since these chitosan oligo rings are amino sugars (basic substances). There is a need for the development of techniques for economically manufacturing rings.

Recently, chitohexaose (GlcN) and chitoheptaose (C1cN) of chitosan oligo rings
7, a mold was found in [D.F.
Mycology (D, F.

Kendra and Lee A+Hadwiger
: ExperimentalMycolOg)'
) Volume 8, No. 276-281 m (1984)],
There is a need for the development of a technology for producing chitosan oligomers having a relatively high degree of polymerization.

So far, a hydrolysis method using hydrochloric acid [Niss T. Horowitz et al.: Journal of the American Chemical Society (S, T.

Horowitz et al: J.A.C.S.
) Volume 79, No. 5046-50490 (1957)]
, oxidative decomposition method using nitrous acid [F. Yaku et al.: Cellulose Chemistry and Technology (F. Yaku et al.
et aL: Ce1lulose Chemist
ry and Technology), Vol. 11, pp. 421-430 (1977)], and the oxidative decomposition method using chlorine (Shigehiro Hirano et al., Japanese Society of Agricultural Chemistry, 1980 Conference, Abstracts, p. 330) have been used to This is known as a chemical decomposition method. The hydrolysis method using hydrochloric acid is
Although it is possible to produce chitosan oligo rings, it requires a high concentration of hydrochloric acid and a long reaction time, and also requires a step to isolate the chitosan oligo rings from the reaction solution due to the production of a large amount of D-glucosamine (Ha). However, there are disadvantages in that the operations required for this purpose are complicated and the costs are high.

Furthermore, the oxidative decomposition method using nitrous acid or chlorine has the disadvantage that it is difficult to obtain pure chitosan oligo rings due to deamination by oxidation.

On the other hand, in the decomposition of chitosan using an enzymatic method, the specificity of the enzyme can be utilized, so the production of 8Ms such as D-glucosamine can be reduced, and the target chitosan oligo ring can be produced in significant amounts. can be produced. Enzymes that degrade chitosan that have been reported so far include chitosanase produced by Bacillus (Bacillus Sp) R-4 [Tominaga and Tsujisaka: Biohimica
Engineering, Biofiguration, Acta (Y, Tominaga &
Y, Tsujisaka: Biochimica
et Biophysica Acta) No. 410
Vol., pp. 145-155 (1975)], Penicillium 1s
Chitosanase [D.
M. Puenton et al.: Journal Op General.
Microbiology (D, M, Fenton et
al: Journal of General M
iCrObiOIOgy) l Volume 126, 151
-165 pages (1981)], Bacillus
us, sp, ') chitosanase produced by 99-5 (inner layer: Japanese Society of Agricultural Chemistry, 1981 Conference Abstracts 4, p. 550), Streptomyces (Strep
tomyces sp-) No. 6 (G. Nis. Price et al.: Journal of Bacteriology)
(J, S, Pr1ceet al: Journal
of Bacteriology) Volume 124, No. 1
574-1585 (1975)] and Streptomyces griseus (Streptomyces g
chitosanase produced by riseuS) [Otakara:
Chitin, chitosan and related enzymes (A, 0htakara: Chitin+
Chitosan and RelatedEnzym
es), pp. 147-160 (1985), Academic Press], but there have been no reports of the production of chitosan oligo rings in significant amounts.

With the aim of developing a technology to economically produce chitosan oligo rings, the present inventors conducted a search for bacteria that produce chitosanase, and obtained a mutant strain of a microorganism belonging to the genus Bacillus (Bacillus sp. ) No. 7- We discovered that scales in which M produces chitosanase and that when this chitosanase acts on chitosan, scales that produce only chitosan oligo rings without producing the m-saccharides of D-glucosamine, and based on these findings Based on this, we have arrived at the present invention.

[Object of the invention and summary of the invention]

An 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.

The present invention decomposes chitosan with chitosanase, an enzyme produced by a microorganism belonging to the genus Bacillus, which is stable in the pH range of 5 to 11, and mainly polymerizes it without containing D-glucosamine. degree is 2-8
This is a method for producing chitosan oligosaccharide, which is characterized by obtaining a chitosan oligosaccharide containing an oligo moat.

In the production of the chitosan oligosaccharide of the present invention, chitosanase produced by culturing Bacillus No. 7-M (Feikoken Bacteria No. 8139) can be used; In addition to the solution, colloidal chitosan can be used, and the hydrolysis of chitosan by chitosanase is preferably carried out in a reaction solution around pH 6.

[Detailed description of the invention]

The chitosan oligotang produced by the present invention mainly contains oligosaccharides with a degree of polymerization of D-glucosamine of 2 to 8,
It is a chitosan oligosaccharide that does not contain D-glucosamine monosaccharides.

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, chitosan oligosaccharides with a relatively high degree of polymerization are highly safe and have fungal and fungal properties, so they can be used as preservatives in cosmetics, foods, etc.
It is also suitable for use as an antifungal and antibacterial agent in soil conditioners, plate coating agents, and the like.

In producing the chitosan oligosaccharide 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 after incubation with the previously obtained chitosan solution at the reaction temperature, the chitosanase solution is added to the chitosan solution, and the chitosan is decomposed by the chitosanase at the reaction temperature.

The reaction temperature can be set at 30-80'C, which is the action temperature of chitosanase, but is preferably around 40'C. 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 for producing the chitosan oligosaccharide of the present invention as long as it is decomposed by chitosanase.
Preferably, chitosan with a degree of deacetylation of 50 to 100% is used. When chitosanase is added to these chitosan for decomposition, colloidal chitosan can also be suspended in water in addition to the chitosan solution described above.

The acid used to prepare the chitosan solution may be any nurturing acid or inorganic acid as long as it can dissolve chitosan, but dilute solutions of hydrochloric acid or nitric acid, Preferably, a dilute solution of formic acid is used.

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 product chitosan oligosaccharide is experimentally determined in a preliminary experiment, and based on this, the desired degree of polymerization can be determined. It can also be the reaction time required to obtain a distribution of chitosan oligomers.

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 subjected to conventional chromatography using an ion exchange resin. After removing impurities by gel filtration or removing colored substances with activated carbon,
Dry to obtain the desired chitosan oligotang powder.

The chitosanase used to produce the chitosan oligosaccharide of the present invention is stable in the pH range of 5 to 11, and when chitosan is decomposed, it does not produce HLNs of D-glucosamine and mainly has a polymerization degree of 2 to 2. (Bacillus sp.) with chitosanase that produces 8 oligosaccharides.
No. 7-M is produced in the medium when culturing No. 7-M (Feikoken Bacteria No. 8139).

Bacillus sp. No. 7-M is a bacillus (Bacillus sp.) No. 7 that was isolated from the primeval soil of Unzen, Obama Town, Minamitakagi District, Nagasaki Prefecture, as a bacterium that can grow in a medium containing chitin or chitosan as the sole carbon source. strain as a parent strain, and this parent strain as N-methyl-N'-nitroso-N
- A mutant strain capable of producing highly active chitosanase from among the streptomycin-resistant mutant strains obtained by treatment with nitrosoguanidine (NTG) to induce mutations; It has been deposited with the Microbial Technology Research Institute of the Ministry of International Trade and Industry as FERMP No. 8139 (FERM P-8139).

The mycological properties of Bacillus No. 7-H are shown below.

A. Cell morphology (1) Cell shape and size: Brachybacillus, (broth and broth agar slant culture, 37°C, 24-72 hours culture) (2) No pleomorphism of cells; (3) Presence or absence of motility, (Meat juice agar semi-liquid Takafu perforation @ culture) (4) Presence or absence of spores: present, endospores and naked spores, spherical, [Dorner's staining method and Witz] (Witz) Modified method j (5) Durham staining: positive, [broth agar slant culture, 37°C, 18 hours, Hützker
(Stained by a modified method of Hucker) B. Growth status in each medium (1) Broth agar plate culture (37°C, 24-168 hours)
: Forms a round, raised, milky-white colony with a filamentous periphery. The surface of the colony is uneven, slightly shiny, and translucent. It will increase as time passes. Does not produce pigment.

(2) Meat juice agar slant culture (37°C for 124 to 168 hours) A milky white colony is formed in the form of a cloth.

Colonies have convex circular ridges and are shiny. It grows well and will spread over time. Does not produce pigment.

(3) Succulent liquid culture (37°C, 24-168 hours): No film is formed on the surface. The whole thing becomes cloudy over time. Precipitates of vx <m granularity) 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) Lidmus 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.

CO Physiological properties (1) Reduction of nitrate (malt salt broth medium, 37°C, 24゛~120 hours) (2
) Denitrification reaction knee 24-120 hours) (3) MR test: 10 (37°0124-168 hours) (4) VP test (acetyl methyl carbinol production test: + (37℃, 24-168 hours) (5 ) Formation of indole (37°C, 24-168 hours) (6) Formation of hydrogen sulfide (TSI agar method, 37°C, 24-168 hours) (7) Hydrolysis of starch: 10 (37°C, 24-168 hours) (168 hours) (8) Utilization of citric acid (Koser's medium, 37'C, 24-168 hours)
- (Christensen's medium, 37' (:, 24-168 hours): 10 (9) Utilization of inorganic nitrogen sources (37'' (:, 24-168 hours); diatonic: apical! 2; ammonium salts: undetermined; (10) Pigment production (mannitol/yeast extract agar slant t@ground): [King A agar slant medium Kony (11) No fluorescence residue: None 12) Urease: + (Christensen-urea agar Medium, 37'(:.

24-168 hours) (13) Oxidase: + (Meat juice agar medium, 37' (:, 24-48 hours) (14
) Catalase: + (Meat juice agar medium, 37°C, 24-48 hours) (15) Range of growth = (Meat juice agar medium) Temperature: undetermined, pH: 5-10° Added salt concentration: undetermined, (16) Resistance to oxygen Attitude: Aerobic (1% glucose broth high-rise agar medium, 37℃, 24-7
2 hours) (+7) O-F test [Hugh Lifeson (llug
h-Leifson) method, 37°C, D-glucose]: Acid is produced fermentatively.

(fermentative) (18) Presence or absence of acid and gas generation from tongs (37℃
, 24-168 hours): Sugar Acid Gas D-glucose + - D-mannose - D-galactose - D-fructose - L-arabinose - D-xylose - D-sorbitol - D-mannite −− Inosit −− Maltose + − Saccharose − Lactose −− Starch + − Cellulose −− Glycerin −− The above mycological properties are described in the Virgies Manual of Determinative Pacteriology (Be
rgey's Manual of Determi
Native Bacteriology) 8th edition (
1974), it was found that the lh7-M strain most likely belongs to Bacillus I2I.

The chemical properties of chitosanase produced by Bacillus No. 7-H are as shown below.

(1) Action: Acts on chitosan and optionally removes β-1,4 from the internal chain of the molecule.
By decomposing the bonds, mainly chitosan oligo M (Glc
N) (n=2-8) (2ffi body ~ 8!! body)
generate. Chitosan oligosaccharides can be separated from the chitosan decomposition solution using high performance liquid chromatography. The decomposition degree of chitosan in this decomposition liquid is about 45%. It also acts on carboxymethyl cellulose (CMC) and decomposes it to some extent, but it does not work on chitin. The optimum working temperature is 50°C.

FIG. 1 shows the relationship between temperature and specific activity when reacting for 10 minutes at pH 6.0.

(3) Working pH range and optimum pH: It acts in the pH range of 3 to 9, and the optimum pH is pH 6.

1% soluble chitosan 177ZA! 2 m of each pH buffer
J and enzyme solution 1 rnI! Figure 2 shows the relationship between the pH and the specific activity of the enzyme when the reaction solution to which the enzyme was added was reacted for 10 minutes at 37°C.

(4) Thermal stability: Stable for up to 15 minutes at 50°C,
Approximately 40% of the enzyme was inactivated by heating at 6C for 15 minutes, and completely inactivated by heating at 706C for 15 minutes.

Figure 3 shows the relationship between temperature and specific activity.

(5) pH stability: 0.1M mild I11? After standing in the solution for 2 hours at 30°C, the remaining enzyme activity was measured.
It was stable in the range of 1. One of the major characteristics of chitosanase produced by Bacillus No. 7-H is that it is stable at pH 10 to 1. p
The relationship between H and specific activity is shown in Figure 4.

(6) Inhibitor: Chitosana-PbC produced by Bacillus Na7-H
1, AgN0, and the presence of PCMB
00% inhibited.

(7) Substrate specificity: When various substrates were used and the final concentration of the substrate was 0.25%, the f of total reducing sugars and hexosamine released after 1 hour by 1 ml of enzyme protein per 4 ml of enzyme reaction solution !
k (~/k protein/hour) was measured.

The results are shown in Table 1.

(Margin below) Table 1 Substrate specificity Note) *: Based on the Reisig (Re1551g) method.

Chitosanase produced by Bacillus No. 7-H degraded colloidal chitosan, soluble chitosan, and glycol chitosan well, and also slightly degraded carboxymethylcellulose (CMC), but had no effect on powdered chitosan. Furthermore, colloidal chitin, glycol chitin, powdered chitin, and methylcellulose were not decomposed at all.

(8) Molecular weight: Figure 5 shows the results of measuring the molecular weight by 5OS-polyacrylamide electrophoresis. In FIG. 5, (◯) indicates the molecular weight of chitosanase produced by Bacillus No. 7-H, which is approximately 41,000.

The results of molecular weight measurement by gel filtration using Sephadex G-100 are shown in FIG. In Figure 6 (
0) is the molecular weight of chitosanase produced by Bacillus No. 7-M, which is approximately 30,000.

(9) Enzyme titer determination method: Dissolve in 0.1M acetic acid aqueous solution, adjust the pH to 6.0 with O.1M sodium acetate aqueous solution, and then add 0.1M acetate buffer (pH: 6.0). Prepare a 1% soluble chitosan solution of the substrate by adjusting the total volume to +00ml.

Pre-incubate for 5 minutes at 376C 1ml of 1% soluble chitosan solution of the substrate! Add 1 rnl of the similarly incubated enzyme solution to the solution and allow the enzyme reaction to occur at 37°C for exactly 10 minutes. The reaction solution is boiled for 3 minutes to stop the enzyme reaction, and the reducing sugar produced in the reaction solution is quantified.

Under these conditions, the amount of enzyme that releases reducing sugar equivalent to 1 μmol of glucosamine is defined as 1 unit of chitosanase activity.

The present invention will be explained in more detail below using reference examples and examples.

Reference example (preparation of seed culture) 250m/! In a Erlenmeyer flask, yeast extract 0.8%,
Peptone 0.4%, Meat Extract 0.2%, Colloidal Ki 1
-4+-non, 5Q<K-7) tp Aki/Ni Oh (
After adding nLI ° 7.91Rnm, 5 and sterilizing it by a conventional method, add Bacillus (Bacillus) cultured in advance to this.
llus sp-) No, 7-M (FERMP
-8139) and cultured with shaking at 30°C for 1 day.

(Preparation of culture solution for enzyme production) Fill two 511-volume Erlenmeyer flasks with 1 liter of a liquid medium having the same composition as above, and sterilize the flasks using a conventional method.
This was inoculated with 40TrLl of the seed culture solution obtained above,
The cells were cultured with shaking at 30°C for 4 days. 6+ culture solution
Cells were removed by centrifugation at OOOr, p, m, and the chitosanase activity of the obtained supernatant was measured by the enzyme titer measurement method described in h''J.0 per 1rrLl of supernatant. It was .99 units.

(Purification of enzyme solution) The supernatant liquid obtained above was mixed, and the resulting mixture was 1.8
1,015 g of solid ammonium sulfate (corresponding to 80% saturation of ammonium sulfate) was added to No. 11 and filtered, and the resulting precipitate was dissolved in distilled water to make 177 ml. This enzyme solution was dialyzed against distilled water and then against 0.02 M phosphate buffer (pH: 6.0), and then the obtained enzyme solution was
CM-Sephadex C- equilibrated with M phosphate buffer
Column (2.6 cm (diameter) x 45 cm (
length)] to adsorb chitosanase. Most of the impure proteins were concentrated in the pass-through section. After washing this column with 350 mJ of 0.02M phosphate buffer solution,
The enzyme protein was eluted with a linear gradient with ~0.5M sodium chloride.

Next, the 218th to 240th fractions that showed chitosanase activity were combined and concentrated 17 times using an ultrafiltration device using a diaflow membrane filter PH-10 (manufactured by Amicon). The liquid was subjected to gel filtration using Sephadex G-100.

Nos. 50 to 63, which showed chitosanase activity of this gel filtration.
Combine the fractions and add again CM-Sephadex C-
Column chromatography using 50 was performed. The enzyme was adsorbed under the same conditions as the previous time, and the enzyme protein was eluted using a linear concentration gradient of 0 to 0.5 M sodium chloride.

In this column chromatography, 2 to 42 un
A fraction showing chitosanase activity of it/rILl was obtained.

Example 1 15 g of chitosan (degree of deacetylation: 99%) was placed in a 500 rrLl beaker, and 150 g of deionized water was added to it.
mJ and 75 mJ of IN acetic acid were added and thoroughly stirred.
Add deionized water and the total volume is 300m7! Closed. The pH of this chitosan acetic acid solution was 5.68.

Take this chitosan acetic acid solution IQmJ in a test tube and hold it at 37°C.
The mixture was incubated for 10 minutes in a constant temperature bath.

Apart from this, reference example CM-Sephadex C-50
The chitosanase solution obtained by column chromatography was diluted with water to give 10.5 units/cure, part 1.
1 was placed in a test tube, incubated in the same manner as above, added to the above chitosan acetic acid solution, and incubated in a thermostat at 379C.
; River formula nubuga 2- Soak the test tube in a boiling bath,
The reaction solution was heated to stop the reaction, and the formation of reduced adhesion m<trty glucosamine/m7 in each reaction solution and the ratio of the amount of chitosan oligosaccharide produced depending on the degree of polymerization by high performance liquid chromatography were investigated.

The results were as shown in FIGS. 7 and 8.

Figure 7 shows the relationship between the reaction time and the amount of reducing sugar produced, and the vertical axis is the production of reducing sugar ffi (1 glucosamine/a
l), and the horizontal axis is the reaction time (hours).

Figure 8 shows the proportions of chitosan oligosaccharides by degree of polymerization, the horizontal axis is the degree of polymerization of the chitosan oligosaccharides, and the vertical axis is the content ratio of chitosan oligosaccharides of each degree of polymerization in the produced chitosan oligosaccharides. (%). Second
The content ratio of chitosan oligosaccharide of each degree of polymerization in the figure is shown by the area ratio of the bar graph.

Example 2 Take 10ml of the chitosan acetic acid solution of Example I into a test tube,
Pre-incubation was performed for IO minutes in a constant temperature bath at 37°C.

Apart from this, reference example CM-Sephadex C-50
The chitosanase solution obtained by column chromatography is diluted with water. unit/rrLI! and 3.5 units/ml of chitosanase solution each! 1IllllL-z, respectively I Rj?
were placed in each test tube and pre-incubated in a constant temperature bath at 37°C as before.These were added to each of the above chitosan acetic acid solutions and allowed to react in a constant temperature bath at 37°C. At the time shown in Figure 9, the test tube was immersed in a boiling bath, the reaction solution was heated to stop the reaction, and the amount of reducing sugar produced (glucosamine/TrLl) in each reaction solution was measured. did.

The results were as shown in FIG.

Figure 9 shows the relationship between the reaction time and the amount of reducing sugar produced, and the vertical axis is the amount of reducing ring produced: 01 glucosamine/1 rLJ
), and its horizontal axis is the reaction time (hours). And (-〇-) is the result of chitosanase of 7 units/pane, and (-Δ-) is the result of chitosanase of 3.5 unit/ml.

Example 3 15 g of chitosan (degree of deacetylation = 99%) was placed in a 50 om, g-volume beaker, and deionized water was added to it.
After adding 82.5 TLl of IN lactic acid and stirring thoroughly, deionized water was added to bring the total volume to 300 ml. The pH of this chitosan lactic acid solution was 5.90.

Take 1077LJ of this chitosan lactic acid solution into a test tube and
It was incubated for 10 minutes in a constant temperature bath at 7°C.

Apart from this, reference example CM-Sephadex C-50
The chitosanase solution obtained by column chromatography was diluted to 10.5 units/me, the ITJ was taken into a test tube, pre-incubated in the same manner as above, and this was added to the above chitosan lactic acid solution. ℃
The reaction was carried out in a constant temperature bath. After the time shown in Figure 1O has elapsed, the test tube is immersed in a boiling bath, the reaction solution is heated to stop the reaction, and reducing sugar production fI in each reaction solution is performed.
k (7 ng glucosamine/7 nl>) was measured.

The results were as shown in Figure 1O.

Figure 10 shows the relationship between the reaction time and the amount of reducing sugar produced, and the vertical axis is the amount of reducing ring produced (rrLgglucosamine/r
rLI, and its horizontal axis is the reaction time (hours).

Example 4 500tyu! Chitosan ■ [degree of deacetylation: 97%, viscosity (viscosity when 0.5 g of chitosan is dissolved in 0.5% acetic acid IQQmI!): 270 c
ps) Take 3y and add 100rrLJ of deionized water to it.
i! and IM acetic acid ismz were added, and after thorough stirring, deionized water was added to bring the total volume to 300ml. The pH of this chitosan/acetic acid solution was 5.30.

This chitosan■acetic acid solution 10TL! ! Take it in a test tube,
It was incubated for 10 minutes in a constant temperature bath at 37°C.

Apart from this, reference example CM-Sephadex C-50
The lnI! was obtained by column chromatography using
Place it in a test tube and incubate it in the same way as above.
This was added to the above chitosan/acetic acid solution and reacted in a constant temperature bath at 37°C.

After the time shown in Figure 1f had elapsed, the test tube was immersed in a boiling bath, the reaction solution was heated to stop the reaction, and the production of reducing sugar 1it (71 g glucosamine/ml) in each reaction solution was measured. .

Chitosan ■ [Degree of deacetylation = 99%, viscosity (0.5%
Viscosity (viscosity when 0.5 g of chitosan is dissolved in 100 mJ of acetic acid): 100 cps) and chitosan ■ [degree of deacetylation: 99%, viscosity (viscosity when 0.5 g of chitosan is dissolved in 100 iIl of 0.5% acetic acid)] :20cps
) was used to measure the amount of reducing sugar produced in each reaction solution in the same manner as described above.

The results were as shown in FIG.

Figure 11 shows the relationship between the reaction time and the amount of reduced rings produced, and the vertical axis is the reduced ring produced m (77L9 glucosamine/m
e), the horizontal axis is the reaction time (hours), and (-〇-) is the result of chitosan ■, (-X-)
is the result of chitosan ■, and (-he-) is the result of chitosan ■.

Example 5 Chitosan 51/(m acetylation degree: 99%) was placed in a 25+) mJ Erlenmeyer flask, deionized water 50 mg and IN acetic acid 27.5 m/! After adding and stirring thoroughly,
Add deionized water and the total volume is 100ml! Closed. The pH of this chitosan acetic acid solution was 5.74. This chitosan acetic acid solution was incubated in a thermostat at 37°C for 15 minutes.

Separately, the chitosanase bath solution obtained by column chromatography using CM-Sephadex C-50 in the reference example was diluted to 10.5 units/ml, and +omg of the solution was taken into a test tube and pre-injected in the same manner as above. Add this to the above chitosan acetic acid solution, 37
The reaction was carried out in a constant temperature bath at °C.

After 1 hour and 40 minutes, the Erlenmeyer flask was placed in a boiling bath for 6 minutes to heat the reaction solution and stop the reaction. The amount of reduced layer produced in the reaction solution was measured using glucosamine hydrochloride as a standard sample, and a result of 22.5 mli/ml was obtained.

The reaction solution was centrifuged, further filtered through a filter paper, and then freeze-dried to obtain 6.18 g of chitosan oligo-coated powder.

A reduced layer was produced Wk 25.8 m9/ml (D Reaction H) in the same manner as above except that the reaction time was 6 hours, and 6.38 g of chitosan oligosaccharide powder was also obtained.

A portion of each reaction solution was subjected to high performance liquid chromatography to examine the amount of chitosan oligomer produced depending on the degree of polymerization.

The results were as shown in FIG. - In Figure 12, the horizontal axis is the degree of polymerization of chitosan oligosaccharide;
The vertical axis represents the content ratio (%) of chitosan oligosaccharide of each degree of polymerization in the produced chitosan oligosaccharide. The content ratio of chitosan oligosaccharides of each degree of polymerization in FIG. 12 is shown by the area ratio of the bar graph. FIG. 12(a) shows the results for a reaction of 1 hour and 40 minutes, and FIG. 12(b) shows the results for a reaction of 6 hours.

Example 6 In the same manner as in Example 5, except that 31 m/l of IN lactic acid was used instead of 27-5 ml of 1 N acetic acid, pH 5,
A chitosan lactic acid solution of No. 74 was prepared, and the reduction [41] of the reaction solution was measured in the same manner as in Practical Example 5, and the amount of chitosan oligosaccharide produced depending on the degree of polymerization was investigated. Obtained.

The amount of reducing sugar produced in the reaction solution in the reaction for 1 hour and 40 minutes was 19.4 mli/rrLl, and the amount of chitosan oligotang powder taken was 6.53.9 mli/rrLl. The amount of reduced layer produced is 24,3
m! g/ml, and the yield of chitosan oligosaccharides was 6.92 g.

The results of measuring the amount of chitosan oligosaccharide produced depending on the degree of polymerization are as shown in FIG.

In Figure 13, the horizontal axis represents the degree of polymerization of the chitosan oligosaccharides, and the vertical axis represents the content ratio (%) of chitosan oligosaccharides at each degree of polymerization in the produced chitosan oligosaccharides
). Chitosanio IJ-fin of each degree of polymerization in FIG. The right side of 1 is indicated by the area ratio of the bar graph. FIG. 13(a) shows the results after a reaction of 1 hour and 40 minutes, and FIG. 13(b) shows the results after a reaction of 6 hours.

〔Effect of the invention〕

Chitosan oligosaccharides mainly containing oligosaccharides with a degree of polymerization of 2 to 8 can be efficiently produced without producing 111Ms of D-glucosamine.

[Brief explanation of drawings]

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 relationship between pH and specific activity in the action pH range of chitosanase.
The figure is a chart showing the relationship between temperature and specific activity in the thermostability of chitosanase, Figure 4 is a chart showing the relationship between pH and specific activity in the pH stability of chitosanase, and Figures 5 and 6 are charts showing the relationship between temperature and specific activity in the thermostability of chitosanase. Figure 7 is a diagram showing the results of molecular ffi measurement, Figure 7 is a diagram showing the relationship between the reaction time and the amount of reduced layer produced in Example I, and Figure N8 is a diagram showing the ratio of chitosan oligosaccharides according to the degree of polymerization in Example 1. 9 is a chart showing the relationship between the reaction time and the amount of reducing sugar produced in Example 2. FIG. 10 is a chart showing the relationship between the reaction time and the amount of reducing sugar produced in Example 3. 1+ figure is Example 4
A diagram showing the relationship between reaction time and amount of reducing sugar produced in
FIG. 12 is a chart showing the ratio of chitosan oligo shreds according to degree of polymerization in Example 5, and FIG. 13 is a chart showing the ratio of chitosan oligo shredded according to polymerization degree in Example 6. Applicant Katakura Chikkarin Co., Ltd. (and 2 others)

Claims (4)

[Claims] (1) Chitosan is decomposed by chitosanase, an enzyme produced by a microorganism belonging to the genus Bacillus, which is stable in the pH range of 5 to 11, and the degree of polymerization is mainly reduced without containing monosaccharides of D-glucosamine. A method for producing chitosan oligosaccharides, which comprises obtaining chitosan oligosaccharides containing 2 to 8 oligosaccharides. (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) Claim 1, characterized in that the decomposition of chitosan by chitosanase is carried out at around pH 6.
A method for producing chitosan oligosaccharide according to any one of Items 1 to 3.