JPH057496A - Production of oligosaccharide - Google Patents

Abstract

PURPOSE:To efficiently produce a homo-oligosaccharide and a heterooligosaccharide both comprising high mol.wt. glucosamine and N- acetylglucosamine and having high physiological activities by hydrolyzing uniformly and partially deacetylated chitin with chitinase. CONSTITUTION:The powder of chitin is dissolved in an alkali solution and uniformly and partially deacetylated. The reaction product is dissolved in lactic acid, etc., and the solution is dropped in acetone to produce the precipitates of the partially deacetylated chitin lactic acid salt. The precipitates are filtered out, dried, dissolved in water, hydrolyzed with chitinase at 20-50 deg.C for 24hrs or longer, and subsequently heated to stop the hydrolytic reaction. The chitinase is removed by an ultrafiltration method, and the remaining reaction solution is treated with a cation exchange resin to make the partially deacetylated chitin adsorbed and removed the lactic acid. The resin is treated with diluted hydrochloric acid to elute a hetero-oligosaccharide comprising glucosamine and N-acetylglucosamine having high mol.wts. and glucosamine homo- oligosaccharide.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing chitin oligosaccharides and chitosan oligosaccharides having a high degree of polymerization and having a high physiological activity, which are obtained by degrading chitin and chitosan.

[0002]

2. Description of the Related Art Recently, oligosaccharides having high physiological activity have been found by degrading chitin and chitosan. An oligosaccharide having a high physiological activity is an oligosaccharide having a relatively high degree of polymerization. For example, chitin oligosaccharide hexaN-acetylchitohexaose (N-acetylglucosamine with a degree of polymerization of 6)
Have a remarkable effect on enhancing immune function (Suzuki Shigeo et al., 2nd chitin, chitosan symposium abstracts, 3-6, 198)
Five). In addition, the chitosan oligosaccharide heptachitoheptaose (glucosamine with a degree of polymerization of 7) inhibits the growth of Fusarium solani (DFKendra et.
al., Exp. Mycology, 8, 276-281 , 1984), useful for synthesizing tomato leaf protease inhibitors (M. Walke
r-Simmons et.al., Plant physiol., 76, 787-790,198
Four).

On the other hand, acid hydrolysis or enzymatic decomposition has been tried as a method for obtaining an oligosaccharide having a high physiological activity, but an efficient method has not been found so far. For example, chitin can be hydrolyzed with hydrochloric acid to obtain oligosaccharides of N-acetylglucosamine, but the ratio of oligosaccharides having a high degree of polymerization in the decomposed product is greatly affected by the concentration of hydrochloric acid, temperature, reaction time, and the like. In this case, if hydrolysis is limitedly carried out, the proportion of oligosaccharides having a high degree of polymerization in the degradation product increases, but the yield of the entire degradation product is low. On the other hand, if the hydrolysis is almost completely performed, the yield of the entire hydrolyzate increases, but the proportion of the oligosaccharides having a high degree of polymerization in the hydrolyzate significantly decreases. The same applies to the production of oligosaccharides of glucosamine by acid hydrolysis of chitosan, and a method for efficiently obtaining oligosaccharides with a high degree of polymerization has not been established (Kazuo Sakai, development and application of chitin and chitosan, industrial technology Kai, 111-134, 198
7).

In the enzymatic decomposition method, when chitin was decomposed with chitinase to obtain oligosaccharides of N-acetylglucosamine, most of the decomposition products were disaccharides (Daihomei, Showa 6).
1st Annual Meeting of the Japanese Society of Agricultural Chemistry, West Japan Chapter, 12, 1986
). Further, when degrading chitosan with chitosanase to obtain oligosaccharides of glucosamine, most of the degradation products are disaccharides to pentasaccharides, and among them oligosaccharides having a degree of polymerization of pentasaccharides or higher are small (M.Izumi et.al., Agric.Biol.Chem., 51, 1189-1191,1
987).

In addition, as a method for obtaining an oligosaccharide having a high degree of polymerization, a chitinase is allowed to act on the tetrasaccharide of N-acetylglucosamine to utilize the transglycosylation reaction to obtain the hexasaccharide of N-acetylglucosamine. N- of the raw material tetrasaccharide
Acetylglucosamine itself is difficult to obtain (F. Nanjo e
t.al., Agric. Biol. Chem., 53, 2189-2195 , 1989). Also,
A partially deacetylated chitin in which about 20 to 35% of N-acetylglucosamine residues are unevenly distributed in the molecule can be decomposed with chitinase to obtain an oligosaccharide, but the decomposition products are disaccharide, trisaccharide and Limited to tetrasaccharides (A. Ohtakara et.a
L., Agric. Biol. Chem., 52 , 3181-3182, 1988).

[0006]

The present invention has been made to solve the above problems, and an object thereof is to provide a method for efficiently producing glucosamine oligosaccharides and N-acetylglucosamine oligosaccharides having high physiological activity. And

[0007]

Means for Solving the Problems The method for producing an oligosaccharide of the present invention, which has been made to achieve the above-mentioned object, is a method of uniformly hydrolyzing partially deacetylated chitin with chitinase to give glucosamine having a high degree of polymerization. Hetero-oligosaccharides consisting of and N-acetylglucosamine and homo-oligosaccharides of glucosamine are obtained. Hydrolysis of chitin by chitinase is preferably performed inside the dialysis membrane, and at the same time, cation exchange resin is arranged outside the dialysis membrane to perform dialysis. The chitin used is preferably one obtained from crustaceans or filamentous fungi.

Chitin, which is uniformly partially deacetylated, is
It is sufficient that the N-acetylglucosamine residues are evenly distributed in the molecule, and the deacetylation ratio is 70 to 95.
%, Especially 80 to 90% is preferable.

As a method for uniformly partially deacetylating chitin, in the case of shellfish chitin, chitin powder is dissolved in a high-concentration alkaline solution to carry out partial deacetylation as alkaline chitin (T. Sannan et. al., Makromal. Chem., 1
77, 3589-3600 , 1976). For filamentous fungus chitin, deacetylated chitin can be prepared by subjecting the bacterial cells to alkali treatment and then extracting with acetic acid (Kobayashioka et al., Journal of the Japan Society for Agricultural Chemistry, 6
2 , 10 , 1988). Filamentous fungus chitin is easier to prepare than crustacean chitin. It is cumbersome to make chitin of crustacean into alkaline chitin at the time of partial deacetylation, and it is troublesome to treat a high-concentration alkaline waste solution (chitin, chitosan study group edition, chitin, chitosan experimental manual, Gihodo Publishing, 16-21, 1991).

The chitinase may be an endo-type chitinase that recognizes and decomposes N-acetylglucosamine residues in a partially deacetylated chitin molecule, and if possible, a chitobiase (β-N-acetylglucosaminase) activity-removed purified enzyme. Products are preferred. Examples of the chitinase include Trichoderma sp.AF6-T8 (Trichoderma sp.AF) manufactured by Takara Shuzo.
6-T8), SIGMA CHEMICAL COMPAN
Y) Streptomyces
griseus).

The dialysis membrane has a molecular weight cutoff of 1000 to 1500.
About 0, especially 2000 to 8000 is preferable. With commercial products, for example, SPECTRA / PORTR
A / POR) dialysis membrane.

The cation exchange resin may be a strong acid type which has been converted into H ⁺ ions in advance. Examples of commercially available products include DOW manufactured by DOW CHEMICAL COMPANY.
EX50W and DOWEX HGR are mentioned.

[0013]

[Function] In partially deacetylated chitin, N-acetylglucosamine residues are uniformly distributed in the molecule, and chitinase is N-.
Since the portion of the acetylglucosamine residue is preferentially cleaved, an oligosaccharide having a high degree of polymerization can be efficiently obtained.

When cation exchange resin is placed outside the dialysis membrane while performing hydrolysis while hydrolyzing inside the dialysis membrane,
The hydrolysis can be carried out more efficiently, and the treatment after completion of the hydrolysis can be omitted.

[0015]

EXAMPLES Examples of the present invention will be described below, but the invention is not limited thereto.

After partially deacetylating the chitin uniformly,
This is dissolved in lactic acid and dissolved uniformly. As for lactic acid, 20 to 200 ml, preferably 60 to 100 ml of lactic acid of 2 to 20% by volume, preferably 5 to 10% by volume is added to 1 g of partially deacetylated chitin. When the solution is dropped into acetone, partially deacetylated chitin lactate is produced as a gel-like precipitate. Acetone is preferably used in an amount of 900 ml or more per 100 ml of the solution. The produced gel-like precipitate is filtered, washed with acetone several times to completely remove lactic acid, and then dried under reduced pressure to obtain partially deacetylated chitin lactate.

The partially deacetylated chitin lactate obtained is dissolved in water to give an aqueous solution of 0.1 to 1% by weight. Add chitinase to the aqueous lactate solution and add 24 at 20-50 ° C.
Hydrolyze for more than an hour. The chitinase is adjusted to a concentration containing 1 to 10 units (units) of chitinase in 10 ml of the aqueous lactate solution, and 1/500 to 1/10 times the volume of the aqueous lactate solution is added to the chitinase aqueous solution having the above concentration. After the hydrolysis is performed for 24 hours or more, the hydrolysis is stopped by heat treatment, and the chitinase is removed from the reaction solution by ultrafiltration.

The reaction solution from which chitinase has been removed is adsorbed on a cation exchange resin, and then the cation exchange resin is washed with distilled water to remove free lactic acid. The partially deacetylated chitin hydrolyzate adsorbed on the resin is eluted with dilute hydrochloric acid and evaporated to dryness. The products are glucosamine and N-acetylglucosamine heterooligosaccharides, and glucosamine homooligosaccharide hydrochlorides.

When hydrolysis is carried out inside the dialysis membrane, a partially deacetylated chitin lactate aqueous solution containing chitinase is put inside the dialysis membrane, and distilled water in which a cation exchange resin is dispersed from the outside of the dialysis membrane. Hydrolysis is carried out for 24 hours or more at 20 to 50 ° C in the same manner as described above while dialyzing with. The cation exchange resin dispersed in distilled water is 10 to 10 parts of the cation exchange resin per 100 mg of partially deacetylated chitin lactate.
It is a ratio of 50 ml. The volume of distilled water in which the cation exchange resin used for dialysis is dispersed is preferably 10 to 100 times the volume of the aqueous lactate solution containing chitinase. After hydrolysis for 24 hours or more, the cation exchange resin on the outside of the dialysis membrane is collected and washed with distilled water, and then the decomposed product is eluted with dilute hydrochloric acid and evaporated to dryness. Glucosamine and N-acetylglucosamine heterooligosaccharides and glucosamine homooligosaccharide hydrochlorides are produced in the same manner as described above.

Example 1 A commercially available reagent for chitin of crustacean (manufactured by Wako Pure Chemical Industries, Ltd.) was used. San
nan et. al. , Makromol. Che
m. , 177, 3589-3600 , 1976 for partial deacetylation. 2.0 g of the partially deacetylated chitin having a deacetylation degree of 87% thus obtained is added to 160 ml of 5% by volume lactic acid and uniformly dissolved. The solution was added dropwise to 1800 ml of acetone to form a partially deacetylated chitin lactate precipitate, which was filtered through a 500 micron sieve. The recovered partially deacetylated chitin lactate was dried at room temperature under reduced pressure. The yield was 2.02g.

The partially deacetylated chitin lactate obtained 1
Dissolve 00.0 mg in 14.6 ml distilled water and add 20 units.
s / ml chitinase (Taboo Shuzo, Trichoderma sp.AF6-T8,
400 μl of an aqueous solution having an activity of 10 units / mg) was added, and a hydrolysis reaction was carried out at 37 ° C. After hydrolysis for 48 hours, 90 ℃
Then, heat treatment is performed for 5 minutes to stop the reaction. After removing the chitinase from the reaction solution by ultrafiltration with a molecular weight of 20000, a cation exchange resin (Bio-Rad) was used.
(Manufactured by AG50W-X8, H ⁺ type). The resin was washed with distilled water and eluted with 5.5 N hydrochloric acid, and the eluate was evaporated to dryness at 40 ° C with an evaporator.
63.0 mg of the hydrolyzate of the decomposition product was obtained, and the yield was 78%.

FIG. 1 shows the results of high performance liquid chromatography (hereinafter referred to as HPLC) analysis of the hydrochloride of the obtained decomposition product. The position of each glucosamine oligosaccharide hydrochloride (GlcN.HCl _n : n is an integer of 2 to 6, the same applies to Example 2 and the following) in the figure was determined by the following commercially available reagents. Chitobiase Hydrochloride, Chitotriose Hydrochloride, Chitotetolase Hydrochloride, Chitopentaose Hydrochloride, Chitohexaose Hydrochloride
oride, Chitotriose Hydrochloride, Chitotetraose Hy
drochloride, Chitopentaose Hydrochloride, Chitohexa
ose Hydrochloride, manufactured by Seikagaku Corporation).

Among the degradation products, the heterooligosaccharides of glucosamine and N-acetylglucosamine cannot be directly analyzed. Therefore, the entire hydrochloride of the degradation product was N-acetylated, and the degree of polymerization was examined as N-acetylglucosamine oligosaccharides. The reaction for N-acetylating the hydrochloride of the decomposition product was carried out by reacting the hydrochloride of the decomposition product with acetic anhydride in a mixed solvent of distilled water-methanol containing triethylamine. Obtained N
-The analysis result of the acetylated product by HPLC is shown in FIG. In the figure, GlcNAc _n (n is an integer of 1 to 8) represents each N-acetylglucosamine oligosaccharide having a degree of polymerization of 1 to 8 (the same applies to Example 2 and the following).

Further, the analysis results of FIG. 2 were analyzed by a calibration curve method of N-acetylglucosamine oligosaccharide standard reagent to determine the weight ratio of each oligosaccharide in the decomposed product. The results are shown in Figure 3. As shown in FIG. 3, the ratio of oligosaccharides having a degree of polymerization of 4 or more in the decomposed product was 65%, and the ratio of oligosaccharides having a degree of polymerization of 5 or more was 36%, indicating that oligosaccharides having a high degree of polymerization could be efficiently produced. I can say.

Example 2 Partially deacetylated chitin lactate 100 prepared in Example 1.
07 mg was dissolved in 14.6 ml of distilled water, and 400 μl of the same chitinase as in Example 1 and a concentration of 20 units / ml was added.
A dialysis tube with a molecular weight cut off of 8000 (SPEC
TRA / POR manufactured by NO.132116), and hydrolyzed at 37 ° C. while dialyzing from outside the dialysis tube with 200 ml of distilled water in which 20 ml of the same cation exchange resin as in Example 1 was dispersed. After hydrolyzing for 48 hours, the cation exchange resin outside the dialysis tube is collected and the resin is washed with distilled water. The decomposition product adsorbed on the resin is eluted with 5.5N hydrochloric acid, and the eluate is evaporated to dryness at 40 ° C with an evaporator. 49.3 mg of decomposition product hydrochloride was obtained, and the yield was 61%.

The results of HPLC analysis of the obtained decomposition product hydrochloride are shown in FIG. The position of the glucosamine oligosaccharide hydrochloride in the figure was determined using the same reagents as in Example 1.

The degradation product was N-acetylated in the same manner as in Example 1, and the N-acetylglucosamine oligosaccharide was analyzed by HPLC.
The result of the analysis is shown in FIG. The results of FIG. 5 were analyzed by the calibration curve method of the N-acetylglucosamine oligosaccharide standard reagent to determine the weight ratio of each oligosaccharide in the decomposed product. The results are shown in Figure 6.

Compared with Example 1, the yield of hydrolyzate as a degradation product was reduced to 61%, but the proportion of oligosaccharides having a degree of polymerization of 4 or more in the degradation product was 67%, and that of oligosaccharides having a degree of polymerization of 5 or more was 67%. The proportion was 47%, and the proportion of high-polymerization degree oligosaccharides was high. Therefore, it can be said that by using the dialysis membrane, the highly polymerized oligosaccharide could be produced more efficiently.

Example 3 Filamentous fungus (Absidia coerule
a): IFO NO.5301) was used to obtain partially deacetylated chitin with a degree of deacetylation of 86% by the method described by Kobayashioka et al., Journal of Japan Society of Agricultural Chemistry, 62, 10 , 1988. 0.81 g of the partially deacetylated chitin was added to 80 ml of 5% by volume lactic acid and uniformly dissolved, and the solution was added dropwise to 720 ml of acetone to form a precipitate of partially deacetylated chitin lactate of filamentous fungi. . Filter the precipitate through a 500 micron sieve,
It was dried at room temperature under reduced pressure. 1.01 g of partially deacetylated chitin lactate was obtained.

Partial deacetylated chitin lactate of filamentous fungi 1
Dissolve 0.09 mg in 14.6 ml distilled water and add 20un
400 μl of its / ml chitinase solution in Example 1
1 was added and hydrolysis was carried out at 37 ° C. After the hydrolysis for 48 hours, the hydrolysis was stopped by heat treatment at 90 ° C. for 5 minutes, and the chitinase was removed by ultrafiltration with a molecular weight of 20000. The solution from which chitinase had been removed was adsorbed on 30 ml of the same cation exchange resin as in Example 1 and then washed with distilled water. The decomposition product adsorbed on the resin was eluted with 5.5 N hydrochloric acid, and the eluate was evaporated to dryness at 40 ° C. with an evaporator. The hydrolyzate of the decomposition product was 43.5 mg, and the yield was 5
It was 4%.

The hydrochloride of the obtained decomposition product was analyzed by HPLC, and FIG. 7 shows the result. The position of the hydrochloride of glucosamine oligosaccharide in the figure was determined by the same reagent as in Example 1.

The degradation product was N-acetylated in the same manner as in Example 1, and the N-acetylglucosamine oligosaccharide was analyzed by HPLC.
The result of the analysis is shown in FIG. The results of FIG. 8 were analyzed by the calibration curve method of the N-acetylglucosamine oligosaccharide standard reagent to determine the weight ratio of each oligosaccharide in the decomposed product. The results are shown in Figure 9. As shown in FIG. 9, the ratio of oligosaccharides having a degree of polymerization of 4 or more in the decomposed product was 67%, the ratio of oligosaccharides having a degree of polymerization of 5 or more was 40%, and this ratio was in Example 1 or more.

Example 4 100.04 mg of the partially deacetylated chitin lactate of the filamentous fungus used in Example 3 was dissolved in 14.6 ml of distilled water to obtain the chitinase used in Example 1 at a concentration of 20 units / ml. 400 μl of the aqueous solution was added. The solution was cut to a molecular weight of 8
000 dialysis tubing (SPECTRA / POR, NO.13211
The solution was placed in 6) and hydrolyzed at 37 ° C. while dialyzing from the outside of the dialysis tube with 200 ml of distilled water in which 20 ml of the same cation exchange resin as in Example 1 was dispersed. After hydrolysis for 48 hours, the cation exchange resin outside the dialysis membrane was collected and washed with distilled water. The decomposition product adsorbed on the resin was eluted with 5.5 N hydrochloric acid, and the eluate was evaporated to dryness at 40 ° C. with an evaporator. 24.8 mg of hydrolyzate hydrochloride were obtained,
The yield was 31%.

The result of HPLC analysis of the obtained hydrolyzate of the decomposition product is shown in FIG. The position of the hydrochloride of glucosamine oligosaccharide in the figure was determined by the same reagent as in Example 1.

The degradation product was N-acetylated in the same manner as in Example 1, and the N-acetylglucosamine oligosaccharide was analyzed by HPLC.
The result of analysis by is shown in FIG. The result of FIG.
-Acetylglucosamine oligosaccharide standard analysis was performed by a calibration curve method to determine the weight ratio of each oligosaccharide in the degradation product. Results are shown in FIG.

Compared with Example 3, the yield of the hydrolyzate of the degradation product was reduced to 31%, but as shown in FIG. 12, the ratio of oligosaccharides having a degree of polymerization of 4 or more in the degradation product was 68% and the degree of polymerization was 5%. The ratio of the above oligosaccharides is 45%. Therefore, by using the dialysis membrane, oligosaccharides having a high degree of polymerization could be produced more efficiently.

[0037]

As described above in detail, if chitin is decomposed by the method for producing an oligosaccharide of the present invention, an oligosaccharide having a high degree of polymerization and a high physiological activity can be reliably obtained. Further, if a dialysis membrane is used during hydrolysis, an oligosaccharide having a high degree of polymerization can be obtained more easily and efficiently. Therefore, oligosaccharides having high physiological activity can be obtained from easily obtainable chitin, which is also very useful for medical research.

[Brief description of drawings]

FIG. 1 is a diagram showing the analysis results of glucosamine oligosaccharide hydrochloride of Example 1.

FIG. 2 is a view showing the analysis result of N-acetylated oligosaccharides of Example 1.

FIG. 3 is a view showing the weight ratio of each oligosaccharide of the degradation product of chitin of Example 1.

FIG. 4 is a view showing the analysis result of glucosamine oligosaccharide hydrochloride of Example 2.

FIG. 5 shows the analysis results of N-acetylated oligosaccharides of Example 2.

FIG. 6 is a view showing the weight ratio of each oligosaccharide of the degradation product of chitin of Example 2.

FIG. 7 is a diagram showing the analysis result of glucosamine oligosaccharide hydrochloride of Example 3.

FIG. 8 shows the analysis results of N-acetylated oligosaccharides of Example 3.

FIG. 9 is a view showing the weight ratio of each oligosaccharide of the degradation product of chitin of Example 3.

FIG. 10 shows the analysis results of glucosamine oligosaccharide hydrochloride of Example 4.

FIG. 11 shows the analysis results of N-acetylated oligosaccharides of Example 4.

FIG. 12 is a view showing the weight ratio of each oligosaccharide of the degradation product of chitin of Example 4.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Toru Chiba             Shin-Etsu 100-1 Sakado, Takatsu-ku, Kawasaki, Kanagawa Prefecture             Chemical Industry Co., Ltd. Corporate Research Center             In the center (72) Inventor Watanabe Kotsuna             3-11-17-308, Ebisu Minami, Shibuya-ku, Tokyo

Claims (3)

[Claims] 1. An oligosaccharide characterized by obtaining a heterooligosaccharide composed of glucosamine and N-acetylglucosamine having a high degree of polymerization and a homooligosaccharide of glucosamine by hydrolyzing uniformly partially deacetylated chitin with chitinase. Manufacturing method. 2. The hydrolysis is performed inside a dialysis membrane, and at the same time, a cation exchange resin is placed outside the dialysis membrane to perform dialysis, and a heterooligosaccharide composed of glucosamine and N-acetylglucosamine having a high degree of polymerization, and The method for producing an oligosaccharide according to claim 1, wherein a homooligosaccharide of glucosamine is obtained. 3. The method for producing an oligosaccharide according to claim 1, wherein the chitin is obtained from crustaceans or filamentous fungi.