JP3062893B2 - Enzymatic degradation of chitin-containing materials - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for enzymatically decomposing a chitin-containing material, and more particularly to a method for efficiently decomposing a chitin-containing material by an enzymatic reaction.

[0002]

2. Description of the Related Art Chitin is a polysaccharide that is abundant in nature and is contained in shells of crustaceans such as crabs and shrimps, exoskeletons of insects, and cell walls of certain molds.

[0003] Chitin is obtained by converting N-acetylglucosamine into β.
Long molecules connected by -1,4 glycosidic bonds,
When this is deacetylated in a high concentration hot alkaline solution, chitosan is obtained. This chitosan has a high ability to adsorb metal ions and proteins, and is used as a coagulant or a carrier for a bioreactor.

[0004] In addition, chitin and chitosan molded articles have good biocompatibility and are used for surgical sutures and artificial skin.

[0005] Furthermore, it is known that polymers or oligomers which are decomposed products of chitin and chitosan have antibacterial activity and antitumor activity.

[0006]

The substance obtained by using chitin as a material in this way has high industrial value. However, it is difficult to control the degree of decomposition in the acid hydrolysis conventionally performed for decomposing chitin and chitosan, so that the resulting decomposed products are often of low molecular weight.

On the other hand, the decomposition method using a degrading enzyme such as chitinase has the advantage that the reaction conditions are mild and the degree of decomposition is easy to adjust because the reaction specificity varies depending on the type of enzyme. However, there is a problem that conventionally known enzymes cannot be efficiently decomposed.

[0008] Therefore, there is a demand for a method capable of efficiently hydrolyzing chitin-containing materials and chitosan-containing materials and controlling the degree of decomposition.

[0009]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventor has found that chitin-containing materials are heat-treated in an organic solvent system or ultrasonically treated in an organic solvent system. As a result, the inventors have found that the reactivity to a decomposing enzyme is increased, and have reached the present invention.

That is, the present invention provides a method for decomposing a chitin-containing material by causing an enzyme reaction in a solution containing the chitin-containing material and performing an enzymatic reaction, wherein the chitin-containing material is an organic solvent or a solvent obtained by adding water thereto. Enzymatically decomposing a chitin-containing material, wherein the enzyme is decomposed in a solution containing the chitin-containing material. A method of decomposing a chitin-containing material by sonicating the chitin-containing material in an organic solvent or a solvent obtained by adding water thereto, and using a β-1,4 glycoside degrading enzyme. It is a method of enzymatic decomposition of materials.

Hereinafter, the present invention will be described in detail.

<1>. Chitin-Containing Material of the Present Invention The chitin-containing material used in the present invention can be obtained mainly from the crustacean, shrimp, krill and other crustacean exoskeletons, the fungus cell wall including fungi, yeast, and mushrooms.

These can be used as a material of the present invention, for example, by decalcifying them with a dilute acid, removing proteins with a concentrated alkali, and washing with water.

<2>. The organic solvent chitin used in the present invention has a strong crystal structure due to intramolecular or intermolecular hydrogen bonds or hydrophobic bonds. Therefore, it is presumed that heat treatment of chitin in a hydrophobic organic solvent or a solvent obtained by adding water thereto weakens hydrogen bonds or hydrophobic bonds and facilitates the action of enzymes.

As the organic solvent used in the present invention, any hydrophobic organic solvent that is incompatible with water can be used, and heating at 70 ° C. or more is possible with the organic solvent alone or in a mixed system with water. Are preferred.

Specifically, linear hydrocarbons such as hexane, heptane, octane, 2-methylheptane, 2,2,4-trimethylheptane, nonane, decane, undecane and dodecane, methylcyclobutane, cyclohexane, methylcyclohexane Cyclic saturated hydrocarbons such as benzene,
Toluene, o -xylene, m -xylene, p -xylene, ethylbenzene, aromatic hydrocarbons such as cumene, solvents obtained as petroleum fractions such as ligroin, carbon tetrachloride, 1,2-dichloroethane, 1,1, Halogenated hydrocarbons such as 2,2-tetrachloroethane, trichloroethylene, chlorobenzene, and 2,6-dichlorobenzene can be used, and these can be used alone or in a mixture or in a mixture of these and water. it can.

When used in a mixture with water, if the amount of water is too small, the enzyme is easily deactivated, and if the amount is too large, the effect of the solvent on the substrate is reduced.
It is preferably vol%.

Among the above-mentioned hydrophobic organic solvents, linear hydrocarbons are preferred, and hexane and dodecane are particularly preferred.

Further, in a preferred embodiment, 10 vol
% Dodecane.

<3>. Method of heat treatment of chitin-containing material In order to heat-treat the chitin-containing material, for example, it may be boiled in the above-mentioned solvent system. At this time, in order to efficiently destroy the crystal structure, it is desirable to heat while stirring.

The chitin-containing material may be suspended in a solvent and subjected to a heat treatment using an autoclave.

There is no particular limitation on the heating time, but if the temperature of the solvent itself has reached a temperature close to the boiling point, several minutes to several hours are sufficient.

<4>. Decomposition of Chitin by Enzyme Decomposition of a chitin-containing material can be performed by adding a decomposing enzyme to a suspension of the chitin-containing material that has been heat-treated in a solvent and causing an enzymatic reaction. The reaction is preferably performed with stirring in order to increase the area of the interface between the organic solvent and water and to increase the number of times of contact between the enzyme and the substrate. For example, the reaction can be carried out in a stationary state, or may be carried out while stirring in the presence of a surfactant.

As this surfactant, Triton
X-100 (polyoxyethylene (10) octyl phenyl ether), Tween 20 (polyoxyethylene sorbitan monolaurate) and the like.

As a degrading enzyme, β-constituting chitin is used.
A β-1,4 glycosidic bond degrading enzyme that cleaves 1,4 glycosidic bonds is used. As this enzyme, for example, lysozyme (Mucopeptide N-acetylmuramyhydrolase, EC 3.
2.1.17) and hydrolases such as chitinase (EC 3.2.1.14), which are commercially available.

Among them, lysozyme is easily available and inexpensive. The temperature at which the enzyme reaction is carried out may be within a temperature range in which the enzyme is not inactivated, but is preferably carried out within an optimum temperature range for the enzyme activity.

When lysozyme is used as a chitinolytic enzyme, preferable reaction conditions are a temperature of 30 to 60 ° C.
H is neutral, the ionic strength of the buffer is 0.1 to 0.6 M, the enzyme concentration is 2 to 8 (W / V)%, and the substrate (chitin) concentration is 2 to 8
(W / V)%.

When a heat-resistant enzyme is used, the enzyme can be added during the heat treatment of the chitin-containing material, and the enzyme reaction can be carried out simultaneously with the heat treatment.

The enzyme reaction can be carried out in a state in which the enzyme is dissolved in a solvent, but it is also possible to use the enzyme in which the enzyme is immobilized on an insoluble carrier. The enzyme can be immobilized by a method generally used for immobilizing an enzyme, such as a method of entrapping in a polyacrylamide gel or the like, or a method of adsorbing the enzyme on an ion exchange resin.

The decomposition product of the chitin-containing material produced by the decomposition reaction can be obtained as a concentrated liquid by leaving the reaction solution to stand after the reaction and concentrating the aqueous phase which naturally separates from the organic solvent phase.

The decomposition product can be purified, for example, by removing the residual solvent by treatment with an adsorbent such as activated carbon, and by liquid chromatography, gel filtration or the like.

<5>. Ultrasonic treatment of chitin-containing material When pretreatment of chitin-containing material is performed by ultrasonic treatment in an organic solvent instead of heat treatment in an organic solvent system, digestion of the chitin-containing material is easily performed.

It is presumed that the ultrasonic treatment in an organic solvent loosens the crystal structure of chitin, which makes it easier for the enzyme to work.

As the organic solvent, the same solvent as used in the heat treatment can be used. In this case, a solvent unsuitable for the heat treatment can be used.

Further, the heat treatment and the ultrasonic treatment may be used in combination.

<6>. Applications of the Invention The method of the invention is equally applicable to enzymatic degradation of chitosan as well as chitin. In this case, chitosanase or the like may be used as the enzyme.

In addition, polysaccharides such as cellulose, xylan, other hemicellulose, hardly soluble starch, and the like, or hardly decomposable polymer compounds such as lignin, which have a strong three-dimensional structure and are difficult to efficiently decompose with enzymes, are also used. Similar applications are possible.

[0038]

EXAMPLES The present invention will be described in more detail with reference to Examples.

[0039]

Example 1 First, the improvement of the reactivity of a degrading enzyme by heating treatment of chitin in a solvent system containing a hydrophobic organic solvent was examined.

The reactivity of chitin (Wako Pure Chemical Industries, Ltd., for biochemistry) pretreated under the following five conditions with respect to lysozyme was examined. 0.2 in 10 ml of 50 mM acetate buffer (pH 5.0)
g of chitin added 2 ml of 50 mM acetate buffer (pH 5.0) and 8 ml of dodecane and 0.04 g of chitin added 0.2 ml to 10 ml of 50 mM acetate buffer (pH 5.0)
g of chitin, and boiled for 1 hour while stirring 2 ml of 50 mM acetate buffer (pH 5.0) and 8 ml of dodecane, 0.04 g of chitin was added, and the mixture was boiled for 1 hour with stirring 8 ml of dodecane Was added with 0.04 g of chitin, and the mixture was boiled while stirring for 1 hour.
5.0) Addition of 2 ml Of these substrate solutions, an aqueous system, that is, egg white lysozyme (Wako Pure Chemical Industries, Ltd., No. 122-02673) was added in an amount of 0.1 ml.
2 g of an organic solvent system, that is, 0.04 g of egg white lysozyme was added thereto, and the mixture was placed in a 30 ml reaction vessel.
The reaction was performed while stirring with a magnetic stirrer in a thermostat at 0 ° C.

The analysis of the reaction product was performed by Asahi Pak.
NH2P-50 column (made by Asahi Kasei Kogyo) or YMC-
This was performed by high performance liquid chromatography using a Pack Polyamine column (manufactured by Yamamura Chemical Laboratory Co., Ltd., purchased from YMC).

FIG. 1 shows the conversion rate (the ratio of the total amount of the produced N-acetylglucosamine monomer, dimer and trimer to the amount of chitin added) with respect to the reaction time of each reaction solution. Note that, for the above conditions (1) and (2),
□ (), ● (), ■ (), ▲ ().

From these results, it was found that lysozyme easily acts on chitin by heat treatment in dodecane, particularly in dodecane containing water. Furthermore, it was found that the heat treatment was not effective with the buffer alone, and that the heat treatment was not effective even with the use of dodecane.

[0044]

Example 2 Next, the chitin decomposition efficiency was examined by changing the enzyme concentration for a constant chitin concentration.

1 m of 50 mM acetate buffer (pH 5.0)
l, 9-8 ml of a mixture of dodecane, 2-8% of chitin was added, and the mixture was boiled while stirring for 1 hour, and then 0.5-4% of lysozyme was added. The object was analyzed. Table 1 shows the reaction conditions.

[0046]

[Table 1]

FIG. 2 shows the decomposition rate for each reaction time, and the decomposition rate (chitin concentration 2
%, Reaction time 120 hours) are shown in FIG.

From the results, it was found that the buffer and dodecane were 1: 9
It was found that in the case of the solvent system, the highest decomposition efficiency was obtained when both chitin and enzyme were 2 (W / V)%.

[0049]

Example 3 The relationship between the ratio of water contained in the solvent system and the efficiency of chitin decomposition was examined.

Chitin was added to a mixture of various ratios of dodecane and 50 mM acetate buffer (pH 5.0) to a concentration of 2 (W / V)%, and heat treatment was performed at 120 ° C. (1 atm in an autoclave). After 30 minutes, lysozyme is added to 2 (W
/ V)%, the reaction was carried out in the same manner as in Example 1, and the decomposition products after 90 hours were analyzed. The results are shown in FIG.

From these results, it was found that the effect of the water ratio on the decomposition efficiency was very large, and that the water content was 30%.
The following cases were found to be effective.

[0052]

Example 4 Subsequently, the effect of pH on the reaction was examined.

200 mM acetate buffer (pH 4.0 to 4.0)
8.0) Alternatively, 2 (W / V)% of chitin was added to a mixture of 10% of buffer and 90% of dodecane, and the mixture was boiled with stirring for 1 hour, and 2 (W / V)% of lysozyme was added. The reaction was performed at 40 ° C.

After 90 hours, the total amount of the produced N-acetylglucosamine monomer, dimer and trimer was measured by high performance liquid chromatography, and the relative activity to the activity at pH 6 is shown in FIG.

As a result, it was found that a pH around neutrality was suitable.

[0056]

Example 5 The effect of ionic strength on the reaction was examined using acetate buffers of different concentrations. In an acetate buffer (pH 5.0) having a different concentration or a mixture of 10% of the same buffer and 90% of dodecane, 2 (W / V)% of chitin was heated and boiled for 1 hour to give 2 (W / V). Decomposition was performed at 40 ° C. with V)% lysozyme.

FIG. 6 shows the decomposition efficiency after 90 hours. From this result, the ionic concentration of the buffer solution was 100 to 100 in the aqueous system.
It has been found that 300 mM and 100-600 mM are suitable for the water-dodecane system.

[0058]

Example 6 Improvement in reactivity of a degrading enzyme was investigated by sonicating chitin in a solvent system containing a hydrophobic organic solvent.

Chitin was added to 50 mM acetate buffer (pH 5.0) or 10% of the same buffer-90% of dodecane with 2 (W / W).
V)%, a cell crusher sonicator (manufactured by Tommy Seiko Co., Ltd., Model UR-200P, oscillation frequency 20 kHz)
After sonication for 5 minutes using z), 2 (W / V)% lysozyme was added and reacted at 40 ° C.

The conversion rate after 44 hours was as follows:
The results were compared with those subjected to heat treatment at 00 ° C. for 30 minutes (FIG. 7).
In addition, each condition was shown in Table 2.

[0061]

[Table 2]

From these results, it was found that when ultrasonic treatment was applied to chitin in an organic solvent system, the chitin was easily decomposed by a hydrolytic enzyme as in the case of heat treatment.

[0063]

As described above, the chitin-containing material is heat-treated in an organic solvent or a solvent obtained by adding water thereto, and / or
Alternatively, chitin can be efficiently degraded by sonication and the action of β-1,4 glycoside degrading enzyme.

[Brief description of the drawings]

FIG. 1 is a graph comparing the degradation rates of chitin with different solvent systems.

FIG. 2 is a graph comparing chitin degradation rates depending on the difference between an enzyme concentration and a substrate concentration.

FIG. 3 is a graph showing a chitin decomposition rate with respect to an enzyme concentration.

FIG. 4 is a graph showing the relationship between the water content and the degradation of chitin.

FIG. 5 is a graph showing the relationship between pH and the degradation of chitin.

FIG. 6 is a graph showing the relationship between ionic strength and chitin degradation.

FIG. 7 is a graph showing the effect of sonication on the degradation of chitin.

Claims (2)

(57) [Claims] 1. A method for decomposing a chitin-containing material by causing an enzyme to be present in a solution containing the chitin-containing material and performing an enzymatic reaction, wherein the chitin-containing material is heated in an organic solvent or a solvent obtained by adding water thereto. And a step of coexisting the chitin-containing material and β-1,4 glycoside-degrading enzyme. 2. A method for decomposing a chitin-containing material by allowing an enzyme to be present in a solution containing the chitin-containing material and performing an enzymatic reaction, wherein the chitin-containing material is dissolved in an organic solvent or a solvent obtained by adding water thereto. A method for enzymatically decomposing a chitin-containing material, comprising a step of applying a sound wave and a step of coexisting a chitin-containing material and a β-1,4 glycoside-decomposing enzyme.