JP2763112B2 - Water-soluble low molecular weight chitosan and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a water-soluble low-molecular-weight chitosan (mainly containing low-molecular-weight chitosan having a maximum molecular weight of 4,000 to 12,000) used in various industrial applications, and its use. Related to manufacturing method.

[Conventional technology]

Chitosan is a basic polysaccharide in which 2-amino-2-deoxy-D-glucose is β-1,4 linked, and is usually obtained by deacetylating chitin. This chitosan does not dissolve in water but dissolves in a dilute acid aqueous solution. However, the use of chitosan is naturally limited because it is a high molecular compound and has a high viscosity, and if it is made neutral or alkaline, it becomes insoluble. Accordingly, there is a strong demand for establishing a technique for obtaining water-soluble low-molecular-weight chitosan. Therefore, various attempts have been made to reduce the molecular weight of chitosan.
000-12,000 which cannot be dissolved in an acidic or alkaline aqueous solution can be obtained.

Conventionally, as a method for obtaining low molecular weight chitosan which can be dissolved in neutral water, a method of bringing chitosan into contact with chlorine gas to reduce the molecular weight (JP-A-60-186504) or a method of nitrite treatment ( JP-A-60-184002). However, the water-soluble low-molecular-weight chitosan produced by these methods has a maximum molecular weight of less than 3,000, and these oxidative decomposition methods require pure low-molecular-weight chitosan for deamination by oxidation. Is difficult.

Other methods for reducing the molecular weight of chitosan include a decomposition method using aqueous hydrogen peroxide (Japanese Patent Application Laid-Open No. 54-148890) or a method of treating with an aqueous sodium perborate solution. Even if its molecular weight is low, it is about 12,000, so that it cannot be dissolved in neutral or alkaline water.

On the other hand, there is a method of reducing the molecular weight of chitosan by an enzymatic method. Chitosanase has been mainly reported as an enzyme that degrades chitosan. As a microorganism producing chitosanase, Myxobacter AL-1 [Hedge & Wolf: Journal of Bacteriology (A.
Hedges & RSWolfe: Journal of Bacteriology) 120
844-853 (1974)], and Bacillus (Bacillus s.
p.) R-4 [Tominaga et al .: Biohimica et Biophysica acta (Y. Tominaga et al: Biochimica et Biophys
ica Acta] Vol. 410, pp. 145-155 (1975)], Bacillus sp. No. 99-5 [Horiuchi: JP-A-60-18058]
No.5; Proceedings of the Japanese Society of Agricultural Chemistry 1984 Annual Meeting, No. 550
Page (1984)], Bacillus sp. No. 7-M
[Daihora et al .: JP-A-61-280277], Alkaligenes (Al
caligenes) MHK-1 strain [Yabuki et al .: JP-A-62-201571]
No.], Bacillus circulan
s) LCC-1 strain (Yabuki et al .: JP-A-63-94971), Bacillus pumilus BN-262 [Takebe et al .:
Japanese Patent Laid-Open No. 63-63382] and Alcaligenes faecalis IK-5 [Ichikawa et al .: Proceedings of the Japanese Society of Agricultural Chemistry 1988 Annual Meeting, p. 536] are known. In addition, Streptomyces (Streptomyces sp.) No. 6 [Price et al .; Journal of Bacteriology (JSPrice et al: Journal)
of Bacteriology) Vol. 124, pp. 1574-1584 (1975)
Year)], Streptomyces griseus (Streptomyces
griseus) HUT 6037 [Daihora et al .: Chitin, Chitosan and Related Enzyme (Chitin, Chitosan and
Related Enzymes) pp. 147-160 (1985) Academic Press (Academic Press), Streptomyces sp. WAK-861 [Terada et al .: Proceedings of the 62nd Annual Meeting of the Japanese Society of Agricultural Chemistry 1987, p. 651] And Penicillium islandicum
QM7571 [Fenton et al .: Journal of General Microbiology (DMFenton et al: Journal of
General Microbiology) 126, 151-165 (1981)
)] Is known to produce chitosanase.

Of these, chitosanase of Bacillus No. 99-5 and chitosanase of Penicillium isorandicum have been studied for the relationship between the degree of deacetylation of chitosan and enzymatic degradation, but a report on a product having a relatively large molecular weight has been made. Can't be found.

[Problems to be solved by the invention]

The present invention has been made in view of the above-mentioned circumstances regarding chitosan,
It is recognized that establishing a water-soluble low molecular weight chitosan having a maximum molecular weight of mainly 4,000-12,000 and soluble in an acidic to alkaline aqueous solution and its production technology is an important technical problem in the industry. Reached.

[Means for solving the problem]

Thus, the present inventors have conducted intensive studies on water-soluble low molecular weight chitosan, and as a result, using chitosan having various degrees of deacetylation as a raw material, acted on the chitonase, and obtained a low molecular weight chitosan by membrane treatment. As a result, it was found that there was a relationship between the degree of deacetylation of the raw material chitosan and the molecular weight of the obtained water-soluble depolymerized chitosan. That is, when chitosan having a relatively low degree of deacetylation is used, a water-soluble depolymerized chitosan having a maximum molecular weight of about 10,000 can be obtained. The molecular weight of chitosan tends to decrease, and when chitosan with a degree of deacetylation of about 100% is used, low molecular weight chitosan with a maximum molecular weight of about 1,000 becomes water-soluble, but low molecular weight with a maximum molecular weight of 2,000 The chitosan chloride was insoluble in water. In addition, in the depolymerization of chitosan by a conventional chemical treatment method, deamination of the produced low-molecular-weight chitosan was inevitable. However, by using an enzyme (chitosanase) having a high reaction specificity, the deamination of It has become possible to obtain water-soluble depolymerized chitosan having an extremely low degree under mild conditions. To define the extent of this deamination, the phenol-sulfuric acid method was employed.
That is, when amino sugars such as glucosamine and N-acetylglucosamine constituting chitosan are colored by the phenol-sulfuric acid method, absorption around 490 nm is not recognized, but the sugar generated by deamination is near 490 nm by the phenol-sulfuric acid method. This is based on the fact that a peak is observed. The present inventors have completed the present invention based on these findings.

An object of the present invention is to provide a water-soluble depolymerized chitosan having a maximum molecular weight of mainly 4,000 to 12,000 and soluble in an acidic to alkaline aqueous solution, and a method for producing the same.

The present invention is constituted by the following technical items (1) to (8), and all of the inventions (1) to (8) are included in the scope of the present invention.

(1) Chitosanase having a degree of deacetylation of 60-90% is reacted with chitosanase, and alkali is added simultaneously with or after the reaction to increase the pH of the reaction solution to 7-10. Water soluble low molecular weight chitosan which is -12,000 and is soluble in acidic to alkaline aqueous solutions.

(2) The above (1) obtained by increasing the pH of the reaction solution to 7-10 and then separating the enzyme reaction product by filtration or membrane treatment.
Water-soluble low molecular weight chitosan.

(3) The sugar equivalent in glucose by the phenol-sulfuric acid method is
(1) or (2) above, wherein the content is 10% or less.
Water-soluble low molecular weight chitosan.

(4) Chitosanase having a degree of deacetylation of 60-90% is allowed to act on chitosanase, and alkali is added simultaneously with or after the reaction to increase the pH of the reaction solution to 7-10. A method for producing water-soluble, low-molecular-weight chitosan, wherein the low-molecular-weight chitosan is 12,000 and is soluble in an acidic to alkaline aqueous solution.

(5) After raising the pH of the reaction solution to 7-10, the enzyme reaction product is separated by filtration or membrane treatment to obtain a low molecular weight chitosan, wherein the low molecular weight chitosan is reduced. Manufacturing method of chitosan.

(6) Chitosanase is Bacillus sp.
The method for producing a water-soluble depolymerized chitosan according to the above (4) or (5), which is produced by No. K-881 (Microtechnological Laboratory No. 10257).

(7) The method for producing a water-soluble depolymerized chitosan according to (5), wherein the membrane is a microfiltration membrane having a pore size of 0.05 μm to 5 μm or an ultrafiltration membrane having a molecular weight cut off of 10,000 or more.

(8) The method for producing water-soluble depolymerized chitosan according to (5), wherein the membrane is a ceramic filter having a pore size of 0.05 μm to 5 μm.

 Next, the configuration of the present invention will be described in more detail.

As the raw material chitosan used in the present invention, those having a degree of deacetylation of 60 to 90% among conventionally known chitosans can be used. For example, commercially available chitin or naturally occurring chitin is deacetylated by a conventional method. Examples include chitosan obtained and chitosan obtained by acetylating chitosan having a degree of deacetylation of 90% or more. As an example of the former, for example, chitin obtained by decalcifying and deproteinizing a crab shell is immersed in an aqueous solution of sodium hydroxide having a concentration of 30 to 50%, and
After the reaction is performed at a reaction time set at 130 ° C. so that the desired degree of deacetylation is achieved, the alkali is removed, and then the dried flakes or further pulverized dry substances obtained by washing and drying are dried. No. Deacetylation degree 60%
When prepared according to the production method of the present invention using less than the starting chitosan, a higher molecular weight, water-soluble, lower molecular weight chitosan can be obtained. It is not impossible to obtain a water-soluble depolymerized chitosan having a maximum molecular weight of 4,000 to 12,000 by increasing the enzyme treatment amount or increasing the reaction time, but the production efficiency is 60 to 90. % Less than chitosan. In addition, the low molecular weight chitosan obtained in this case has a low amino group content per unit weight and is inferior in polycationic effect, and is not suitable for the practice of the present invention.

In the present invention, a chitosan solution must be prepared in order for chitosanase to act on the raw material chitosan. However, since chitosan is soluble in acid, an aqueous acid solution is added to chitosan to obtain a chitosan concentration of 1-30%, preferably 5-10%. % Solution. In this case, the amount of the acid to be added is adjusted so that the pH of the solution is near the optimum pH of the action of chitosanase. For example, in the case of chitosanase derived from Bacillus No. K-881, since the optimum pH is 6, the amount of acid added is adjusted so that the pH of the chitosan solution becomes 5-6.

As the acid used for preparing the chitosan solution, any organic or inorganic acid can be used as long as it can dissolve chitosan, but hydrochloric acid, formic acid, acetic acid, lactic acid, glutamic acid, etc. Is preferred. A chitosanase powder or solution is added to the chitosan solution to decompose chitosan at the working temperature of chitosanase. The action temperature of chitosanase is, for example, 30-60 ° C in the case of Bacillus-derived chitosanase, but is preferably around 40 ° C.

In the present invention, the molecular weight distribution of low molecular weight chitosan generated by the enzymatic reaction varies depending on the reaction time or the amount of enzyme, etc., so that the reaction time (or amount of enzyme) is required to obtain a high yield of water-soluble chitosan having the desired molecular weight. It is necessary to strictly determine the reaction conditions based on the relationship between and the product molecular weight.

The chitosanase used in the present invention decomposes chitosan having a degree of deacetylation of 60 to 90%, and has a maximum molecular weight of 4,000.
Any enzyme capable of mainly producing -12,000 low molecular weight chitosan can be used. For example, commercially available chitosanase (sold by Wako Pure Chemical Industries, derived from Bacillus pumilus BN-262) can be used in the present invention, but is very expensive.
Water-soluble depolymerized chitosan produced by enzymatic treatment is also very expensive.

Thus, the present inventors conducted a search for microorganisms capable of producing chitosanase at a lower cost than in nature and found that Bacillus sp. No. K-881 isolated from soil.
(No. 10257) has been found to produce chitosanase efficiently.

The mycological properties of the bacterium (Bacillus sp. No. K-881) are as follows.

(A) Cell morphology Cultured at 37 ° C for 24 to 72 hours in broth or broth agar medium,
Observation.

Cell system and size: short rod, 0.5-1.0 × 1.0-3.
0μm Cell polymorphism: Not determined Motility: None Spore presence: Spherical to elliptic endospores, spores are sub-original or edged Gram stain: Positive Negative (b) Growth state in each medium Gravy agar plate culture (37 ° C., 24-72 hours): forms opaque, thick, milky white colonies.

The surface of the colony is uneven and glossy and does not produce any pigment.

Gravy agar slant culture (37 ° C, 24-72 hours): Growth is good and spreads over time, as described under.

Broth liquid culture (37 ° C, 24-72 hours): forms a milky white pellicle on the medium surface, but the liquid is not turbid.

Broth gelatin stab culture (25 ° C., 24-72 hours): Growing on stab line, gelatin is liquefied.

Litmus milk (37 ° C., 24-72 hours): Liquefaction and discoloration are observed from about the second day and become acidic. No coagulation is observed. Liquefaction proceeds with the passage of time and becomes translucent.

(C) Physiological properties Nitrate reduction: positive (Succinate nitrate medium with yeast extract, 37 ° C, 24-
120 hours) Denitrification: Negative (Komagata et al., 37 ° C, 24-72 hours) MR test: Negative (37 ° C, 24-72 hours) VP test: Positive (37 ° C, 24-72 hours) Indole Production: Negative (37 ° C, 24-72 hours) Production of hydrogen sulfide: Negative (TSI agar method, 37 ° C, 24-72 hours) Starch hydrolysis: Negative (37 ° C, 24-72 hours) Use of citric acid : (Coser medium, 37 ° C, 24-72 hours): Not used (Christensen medium, 37 ° C, 24-72 hours): Used Inorganic nitrogen source Nitrate: Not used Ammonium salt: Ammonium salt is the only nitrogen Can grow as a source.

Pigment generation (King A agar slant medium): Negative urease: Positive (Christensen-urea agar medium, 37 ° C, 24-72)
Time) Oxidase: Positive (meat agar medium, 37 ° C, 24-48 hours) Catalase: Positive (meat agar medium, 37 ° C, 24-48 hours) Growth range Optimum temperature: 25-35 ° C pH: 5-9 Growth in the presence of 7% NaCl: grows Attitude to oxygen: aerobic (1% glucose broth high agar medium, 37 ° C, 24-72
Time) OF test: oxidation (Hugh-Rifson method, 28 ° C, D-glucose) Presence or absence of acid and gas generation from saccharides (28 ° C, 24-72 hours) (D) Other properties Egg yoke reaction (37 ° C, 24-48 hours): positive Based on the above mycological properties, Bergey's Manual of Determinative Bacteriology
A search of the 8th edition (1974) revealed that strain No. K-881 belongs to the genus Bacillus.

The enzymatic properties of chitosanase produced by Bacillus sp. No. K-881 are as follows.

(1) Action Acts on chitosan to hydrolyze β-1,4 bonds. The disassembly type is considered to be the end type.

(2) Substrate specificity The present chitosanase acts on chitosan and colloidal chitosan, but does not act on powdered chitin, colloidal chitin, glycol chitin, carboxymethylcellulose (CMC) and α-1,4-polygalactosamine. The chitosanase degrades chitosan with a degree of deacetylation of about 80% best, and when the degree of deacetylation further decreases, the rate of degradation sharply decreases.

FIG. 1 shows the relationship between the degree of deacetylation of chitosan and the relative activity when reacted at pH 6.0 and 40 ° C. for 10 minutes.

(3) Optimum pH and stable pH It works in the range of pH 4-8, and the optimum pH is about 6.0. FIG. 2 shows the relationship between the reaction solution pH and the relative activity when reacted at 40 ° C. for 10 minutes.

Further, when heat treatment is performed at 40 ° C. for 30 minutes, the stable pH range is 5 to 8 as shown in FIG.

(4) Method of measuring enzyme titer 0.5 g of chitosan (degree of deacetylation: 75 to 90%, 16 mesh or less) is dissolved in 90 ml of 0.075N acetic acid, adjusted to pH 6.0 of sodium hydroxide solution, and then diluted with water. A chitosan solution of the substrate is prepared with a total volume of 100 ml.

0.5 ml of this enzyme solution is added to 0.5 ml of this chitosan solution (about 0.025uni).
t of chitosanase), and the enzyme reaction is carried out at 40 ° C. for 10 minutes. Then, add acetylacetone solution to the reaction solution.
1 ml is added, and the amount of reducing sugar generated in the reaction solution is measured by the Londre-Morgan method.

Under the above conditions, the enzyme titer to release reducing sugar corresponding to 1 μmol of glucosamine per minute was determined as 1 unit (uni).
t).

(5) Range of suitable operating temperature The operating temperature is up to 60 ° C, and the optimum temperature is about 55 ° C.

FIG. 4 shows the relationship between the temperature and the relative activity when the reaction was carried out at pH 6.0 for 10 minutes.

(6) Thermal stability and stabilization Although heat treatment at pH 6, 15 minutes is stable at 50 ° C or less,
It is completely inactivated at ℃.

 FIG. 5 shows the relationship between the temperature and the residual activity.

The thermal stability of the present chitosanase is affected by the ionic strength, and is completely inactivated by dialysis by heating at pH 6, 60 ° C. for 15 minutes.

The chitosanase is stabilized by 0.1 M or more sodium chloride.

FIG. 6 shows the influence of the concentration of sodium chloride on the residual activity when heated at pH 6, 50 ° C. for 15 minutes.

In addition, stabilization is recognized by alkali metal salts such as potassium chloride, ammonium chloride, manganese chloride, ammonium sulfate, and magnesium sulfate.

(7) Inhibition and Activation Almost 100% of the present chitosanase is inhibited by 0.05 M of NiSO ₄ , ZnSO ₄ and CuSO ₄ .

Also, 0.05M NaCl, KCl, NH ₄ Cl, (NH ₄ ) ₂ SO ₄ , MgSO ₄ , Mn
Activation by Cl ₂ is observed. However, the degree of activation differs depending on the chitosan substrate.

This chitosanase is not activated by EDTA.

(8) Purification method The chitosanase is purified, for example, as follows. An SP-Toyopearl 650S column (manufactured by Tosoh Corporation) was prepared by fractionating the water-soluble fraction of the culture solution by ammonium sulfate precipitation or organic solvent precipitation, dialyzing, and equilibrating with a 0.005 M acetate buffer (pH 4.5). Is used for ion exchange chromatography. Next, purification is performed by gel filtration chromatography using Toyopearl HW-50S.

(9) Molecular weight Using a 0.1 M phosphate buffer (containing 0.2 M sodium sulfate) and gel filtration chromatography using a TSK gel G3000SW glass column (manufactured by Tosoh), a molecular weight of about 31,000 was obtained.
It is.

(10) Isoelectric point The isoelectric point was measured to be 8.4 by acrylamide focal electrophoresis.

In the present invention, after the chitosanase treatment or during the enzymatic reaction, an alkali is added to adjust the pH of the reaction solution to 7-10.
To break down the ammonium salt formation of amino groups in chitosan and precipitate water-insoluble chitosan. The alkali used in the present invention may be any substance that exhibits alkalinity in an aqueous solution. For example, a hydroxide of sodium or potassium can be used, and an aqueous solution of sodium hydroxide is preferably used. When the amount of water-insoluble chitosan is small, it can be used as it is depending on the intended use. In this case, desalting operation is performed if necessary, and the powder is dried and powdered by freeze-drying or spray-drying. On the other hand, when preparing water-soluble depolymerized chitosan from which water-insoluble chitosan has been removed, filtration or membrane separation is performed, and then dry powdering is performed depending on the intended use. If necessary, desalting operation is performed before or after the membrane separation step. In the present invention, the membrane that can be used for the membrane treatment of chitosan includes a microfiltration membrane having a pore size of 0.05 μm to 5 μm or a molecular weight cutoff of 1
There are more than 10,000 ultrafiltration membranes. As the microfiltration membrane mentioned here, for example, a ceramic membrane (manufactured by Toshiba Ceramics Co., Ltd.)
Membrane Rocks ceramic filter, NGK Insulators ceramic filter, Nippon Cement Co., Ltd. ceramic filter, Kubota Iron Works ceramic filter, etc.), Polyolefin membrane (Asahi Kasei Microza membrane, etc.), Polyvinyl alcohol membrane (Kuraray Co., Ltd.) SF filter, etc.), polypropylene-based membrane (NTTO Denko Corporation NT
M film, etc.), polycarbonate-based film (WO film, etc., manufactured by Sulzer), polysulfone-based film (PSE film, etc., manufactured by Fuji Film), polyvinylidene fluoride-based film (Durapore film, manufactured by Millipore Limited). Teflon-based membranes (Sartorius's Sartoflor membrane, Cuno's Teflon filter, etc.), cellulose-based membranes (Fujifilm's FM
Membrane, MF-Millipore membrane manufactured by Millipore Limited, Zarto Blanc manufactured by Sartorius, and the like). Examples of the ultrafiltration membrane mentioned here include a polysulfone-based membrane (a PT membrane manufactured by Millipore Limited, a NUT membrane manufactured by Nitto Denko Corporation), a cellulose-based membrane (a PT membrane manufactured by Millipore Limited), a polyacrylonitrile-based membrane, and the like. (ACV film manufactured by Asahi Kasei Corporation).

〔The invention's effect〕

As described above, according to the production method of the present invention, a water-soluble depolymerized chitosan having a relatively large molecular weight can be produced from chitosan having a degree of deacetylation of 60 to 90% with good yield under mild conditions. Moreover, the conventional method has a maximum molecular weight of 3,00.
The production method of the present invention was able to eliminate the drawbacks such that no more than 0 or more than 12,000 were obtained, and there was a disadvantage such as quality deterioration due to deamination. Originally, chitosan is a highly functional polymer with properties such as polycationicity and film-forming ability, and can be widely used in the fields of food, pharmaceuticals, cosmetics, etc. Has the disadvantage of being insoluble. However, the water-soluble depolymerized chitosan obtained by the production method of the present invention has a maximum molecular weight of mainly 4,4.
000-12,000 high, chitosan's original polycationic,
While having the property of film forming ability, it is soluble in acidic or alkaline aqueous solutions, and has no chemical modification, so it has high safety like chitosan as a raw material,
It can be widely used in medicine and cosmetics.

Hereinafter, the present invention will be described specifically with reference to Reference Examples, Examples, and Comparative Examples, but the present invention is not limited thereto.

Reference Example (1) (Seed culture) In a 500 ml Erlenmeyer flask, starch 3%, peptone 5
%, Monopotassium phosphate 0.1%, sodium nitrate 0.1%, and 100% in a liquid medium (pH 6.0) containing 0.05% magnesium sulfate, sterilized by a conventional method, and then subjected to Bacillus sp.
sp.) No. K-881 (Microtechnological Laboratory No. 10257)
C. for 1 day with shaking to obtain a seed culture solution.

(Culture for enzyme production) 5 Erlenmeyer flasks each containing 600 ml of a liquid medium having the same composition as above were sterilized by a conventional method.
The medium was inoculated with 10 ml of the above seed culture solution, and cultured with shaking at 28 ° C. for 5 days.

(Preparation of enzyme) The culture solution obtained above was centrifuged (7000 rpm) to remove bacterial cells. The chitosanase activity of the obtained supernatant is
It was 5.2 u / ml. 1515 g of solid ammonium sulfate in 2700 ml of this supernatant
(Equivalent to 80% saturation of ammonium sulfate), and the mixture was filtered. The resulting precipitate was dissolved in ion-exchanged water, dialyzed against ion-exchanged water for 1 day, lyophilized in vacuo, and crude enzyme chitosanase was added. 77.5 g of powder was obtained. Chitosanase activity of this product is 1
It was 05u / g.

Reference Example (2) 10 g of the crude enzyme chitosanase powder obtained in Reference Example (1)
Was dissolved in 150 ml of a 0.005 M acetate buffer (pH 4.5), and the insoluble matter was removed by centrifugation.
SP-Toyopearl 650S column (diameter 2.2 cm x
Ion exchange chromatography (flow rate: 3 ml / min) using a length of 20 cm, manufactured by Tosoh Corporation was performed. The enzyme was eluted with a linear concentration gradient of 0 to 0.5 M sodium chloride, the enzyme active fraction was collected, and then concentrated with a Diaflow membrane filter PM-10 (manufactured by Amicon). Add this solution to 0.
Gel filtration chromatography was performed using a Toyopearl HW50S column (2 cm in diameter × 100 cm in length, manufactured by Tosoh Corporation) equilibrated with a 1 M phosphate buffer (pH 6). The active fractions were collected to obtain a purified chitosanase solution (15 ml, total activity: 326 units). The specific activity was 39.4 units / mg protein.

Reference Example (3) Chitosan having a degree of deacetylation of 81% [Wako Pure Chemical Industries, Chitosan
80H; Viscosity (0.5W / V%, 20 ° C) 180cp] 7.5g ion-exchanged water
115 ml and 25 ml of 1N acetic acid aqueous solution were added to dissolve (pH 5.
9). Take 14 ml of this chitosan solution into L-shaped test tubes,
Chitosanase solution (chitosanase powder of Reference Example (1)
Dissolve 143 g in 1 ml of water: 15 u / ml) Add 1 ml and react for 15 minutes, 1 hour, 5 hours and 23 hours while shaking in a constant temperature water bath at 40 ° C. Then, place the L-shaped test tube in a boiling water bath. The reaction was stopped by immersion. The molecular weight of these reaction solutions was measured by high performance liquid chromatography using an Asahipak GS-320 column (manufactured by Asahi Kasei Corporation). The results were as shown in FIG.

When reacting under the above conditions, the reaction time was appropriately around 15 minutes.

Example (1) 12.5 g of chitosan (Wako Pure Chemical Industries, Chitosan 70H) having a deacetylation degree of 72% was placed in a 500 ml beaker, 210 ml of ion-exchanged water and 39 ml of a 1N acetic acid aqueous solution were added, and the mixture was thoroughly stirred and uniformly mixed. Solution. The pH of this chitosan acetic acid solution is 5.
It was eight. 1 ml of a chitosanase solution (dissolve 38 mg of the chitosanase powder of Reference Example (1) in 1 ml of water: 4 u / ml) is added to this chitosan acetic acid solution, and the mixture is stirred in a 40 ° C. constant temperature water bath.
After reacting for 2 hours, the reaction solution was heated to stop the enzyme reaction. Then add 14 ml of 10% sodium hydroxide solution and adjust the pH.
Is adjusted to 8 to produce insolubles. After desalting the reaction solution by dialysis using a dialysis membrane, 345 ml of the desalted solution was subjected to 0.2-pore diameter.
Filtration was performed using a ceramic filter (manufactured by Nippon Cement Co., Ltd.) having a membrane size of 0.02 m ² and a 305 ml transparent filtrate was obtained. This liquid was dried by a freeze vacuum drying method to obtain 8.2 g of powdery water-soluble depolymerized chitosan. (Note that the residue after filtration with the ceramic filter was dried and found to be 3.0 g.) Example (2) Chitosan having a deacetylation degree of 81% (Wako Pure Chemical Industries, Chitosan
80H), and 207 ml of ion-exchanged water and 42 ml of a 1N acetic acid aqueous solution were added thereto to dissolve (pH 5.9). Then, enzyme treatment, pH adjustment, desalting, membrane treatment, and drying were performed under the same conditions as in Example (1) to obtain 8.8 g of water-soluble low molecular weight chitosan. (The ceramic filter residue was 2.7 g when dried.) Example 3 Chitosan having a degree of deacetylation of 87% (Wako Pure Chemical Industries, Chitosan
12.5 g of 90M) was dissolved by adding 199 ml of ion-exchanged water and 50 ml of 1N aqueous acetic acid solution (pH 5.8). Then, enzyme treatment, pH adjustment, desalting, membrane treatment, and drying were performed under the same conditions as in Example (1) to obtain 7.3 g of water-soluble, low-molecular-weight chitosan.
(Note that when the residue of the ceramic filter is dried, 3.
6 g. Comparative Example (1) 12.5 g of chitosan having a degree of deacetylation of 59% was taken, and dissolved in 223 ml of ion-exchanged water and 26 ml of 1N acetic acid aqueous solution (pH 5.3). Then, enzyme treatment, pH adjustment, desalting, membrane treatment, and drying were performed under the same conditions as in Example (1) to obtain 7.7 g of water-soluble, low-molecular-weight chitosan. (Furthermore, there was almost no filtration residue on the ceramic filter.) Comparative Example (2) Chitosan with a degree of deacetylation of 93% (Hokkaido Soda CS-90)
12.5 g was taken and dissolved by adding 166 ml of ion-exchanged water and 83 ml of 1N acetic acid aqueous solution (pH 5.3). Then, enzyme treatment, pH adjustment, desalting, membrane treatment, and drying were performed under the same conditions as in Example (1) to obtain 3.0 g of water-soluble, low-molecular-weight chitosan. (Note that the filtration residue of the ceramic filter was 5.4 g when dried.) Comparative Example (3) 12.5 g of chitosan (Wako Pure Chemical, 100 L of chitosan) having a deacetylation degree of 100% was taken, and 185 ml of ion-exchanged water and 1N
64 ml of an acetic acid aqueous solution was added to dissolve (pH 5.5). Then, enzyme treatment, pH adjustment, desalting, membrane treatment, and drying were performed under the same conditions as in Example (1) to obtain 1.2 g of water-soluble, low-molecular-weight chitosan. (The residue after filtration of the ceramic filter was 4.9 g when dried.) Next, the aqueous solutions obtained in Examples (1), (2) and (3) and Comparative Examples (1), (2) and (3) were obtained. Table 1 shows a comparison between the molecular weight of the functional low molecular weight chitosan and the low molecular weight chitosan and the increase in color tone during heating.

The maximum molecular weight is measured by Waters high-performance liquid chromatograph (M600 multi-solvent delivery system, 710B fully automatic sample processor, M410 differential refractometer) and
Using an Asahipak GS-320 column (manufactured by Asahi Kasei Kogyo), measurement was performed by the GPS method using pullulan as a standard substance (the molecular weight determined from the retention time at the peak apex is defined as the maximum molecular weight). The degree of deacetylation was measured by a colloid titration method in which an aqueous acetic acid solution of chitosan was titrated with an aqueous solution of potassium potassium sulfate using methylene blue as an indicator.

From the results shown in Table 1, a relationship was found between the degree of deacetylation of the raw material chitosan and the molecular weight of the obtained water-soluble depolymerized chitosan. The molecular weight of the resulting low molecular weight chitosan tends to decrease. When the molecular weight is reduced, the proportion of reducing groups increases, so that browning tends to occur in an aqueous solution due to an amino-carbonyl reaction (Maillard reaction) or the like. on the other hand,
The water-soluble low molecular weight chitosan obtained by the present invention has a low degree of browning and a relatively high molecular weight.

Example (4) Chitosan having a degree of deacetylation of 87% (Wako Pure Chemical Industries, Chitosan
Enzyme treatment and pH adjustment were carried out using 12.5 g of 90M) under the same conditions as in Example (3), and using a microfiltration membrane Microza XPW-11 (pore size 0.1 µ, membrane area 0.1 ² ) manufactured by Asahi Kasei Kogyo Co., Ltd. After filtration, the filtrate was desalted with an electrodialyzer MicroAcilyzer G3 (manufactured by Asahi Kasei Corporation) and then dried by freeze-vacuum drying to obtain 9.7 g of a powdery water-soluble depolymerized chitosan.

Comparative Example (4) Chitosan having a degree of deacetylation of 87% (Wako Pure Chemical Industries, Chitosan
90M) 65ml of aqueous solution containing 13.9g of sodium nitrite 0.95g
After immersion for 5 minutes at room temperature, 7.5 g of acetic acid was added and reacted at room temperature for 2 hours. After the reaction, 210 ml of water was added, the pH was adjusted to 9 with a 40% sodium hydroxide solution, filtration was performed in the same manner as in Example (4), dialyzed, and freeze-vacuum dried.
6.2 g of powder were obtained.

Next, Table 2 shows a comparison between the molecular weights of the powders obtained in Example (4) and Comparative Example (4) and the amount of glucose in terms of glucose by the phenol-sulfuric acid method.

According to the results in Table 2, the low molecular weight chitosan obtained by the conventional nitrite treatment had a maximum molecular weight of 3000
The value is low, near, and deamination occurs, so that the glucose-equivalent sugar value by the phenol-sulfuric acid method shows a high value.
On the other hand, the water-soluble low molecular weight chitosan obtained according to the present invention has a relatively large maximum molecular weight and a low degree of deamination, which is inferred from the glucose-equivalent sugar amount by the phenol-sulfuric acid method.

[Brief description of the drawings]

FIG. 1 shows the relationship between the degree of deacetylation of chitosan and the relative activity of chitosanase obtained according to the present invention.
Fig. 2 shows the optimum pH, Fig. 3 shows the stable pH range, Fig. 4 shows the optimum temperature range for operation, Fig. 5 shows the effect of sodium chloride concentration on thermal stability, and Fig. 6 shows the effect of sodium chloride concentration on thermal stability. . FIG.
It is a figure which shows the time of the enzyme reaction and the molecular weight distribution of chitosan in reference example (3).

Claims (8)

(57) [Claims] (1) A chitosanase having a degree of deacetylation of 60-90% is reacted with chitosanase, and an alkali is added at the same time as or after the reaction to raise the pH of the reaction solution to 7-10. Water-soluble, low-molecular-weight chitosan that is mainly 4,000 to 12,000 and is soluble in acidic to alkaline aqueous solutions. 2. The water-soluble depolymerized chitosan according to claim 1, which is obtained by raising the pH of the reaction solution to 7-10 and separating the enzymatic reaction product by filtration or membrane treatment. 3. The water-soluble, low-molecular-weight chitosan according to claim 1, wherein the sugar content in terms of glucose by the phenol-sulfuric acid method is 10% or less. 4. The chitosanase having a degree of deacetylation of 60-90% is reacted with chitosanase, and an alkali is added simultaneously with or after the reaction to raise the pH of the reaction solution to 7-10. A method for producing a water-soluble, low-molecular-weight chitosan, which mainly comprises 4,000 to 12,000 and is soluble in an acidic to alkaline aqueous solution. 5. The water-soluble low-molecular-weight compound according to claim 4, wherein after raising the pH of the reaction solution to 7-10, the enzymatic reaction product is separated by filtration or membrane treatment to obtain a low-molecular-weight chitosan. Production method of chitosan. 6. The method according to claim 6, wherein the chitosanase is a bacterium belonging to the genus Bacillus.
p.) The method for producing a water-soluble depolymerized chitosan according to claim 4 or 5, wherein the method is produced by No. K-881 (No. 10257). 7. A microfiltration membrane having a pore size of 0.05 μm to 5 μm or an ultrafiltration membrane having a molecular weight cut-off of 10,000 or more.
A method for producing the water-soluble depolymerized chitosan according to the above. 8. The method according to claim 5, wherein the membrane is a ceramic filter having a pore size of 0.05 μm to 5 μm.