JPH08322554A - Aspergillus-fumigatus mutant bacteria and its production enzyme, and manufacture of chitosan-oligosaccharide using said bacteria or production enzyme - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a chitosan-oligosaccharide mutant and its producing enzyme and produce a chitosan-oligosaccharide useful for medicines, foods, cosmetics, etc., utilizing the mutant or its producing enzyme.

SOLUTION: An Aspergillus fumigatus mutant having properties of degrading chitosan into a chitosan-oligosaccharide is cultured to harvest a mycelium, which is then reacted with the chitosan. The reaction of the mycelium with the chitosan is preferably carried out at 50-60°C temperature and at pH 4.0-5.0 for 10-60 min reactional time. The chitosan concentration is preferably 1-5 wt.%.

COPYRIGHT: (C)1996,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an Aspergillus fumigatus mutant bacterium and a method for producing chitosan-oligosaccharide using the mutant bacterium, and more specifically, it is produced by an Aspergillus fumigatus mutant bacterium. A chitosan-oligosaccharide degrading enzyme is used to effectively utilize chitosan, which is contained in a large amount in crustaceans such as crabs and shrimps, as a natural resource. From chitosan to chitosan-oligosaccharide (Chitosa)
n-Oligosaccharide).

[0002]

Chitin, namely β-1,4-poly-N
-Acetyl-D-glucosamine (β-1,4-poly)
-N-acetyl-D-glucosamine)
Is a major polysaccharide such as arthropods and fungi, and naturally exists in the form of glycoprotein by binding to a protein. Chitin can be obtained by removing the protein from this glycoprotein with an alkali. If chitin is hydrolyzed with hydrochloric acid, it becomes D-glucosamine hydrochloride which is a monosaccharide, but it is heated in a concentrated alkaline solution,
Chitosan can be obtained by deacetylation.

Chitosan is composed of D-glucosamine, that is, 2-amino-2-deoxy-D-glucose is β-1,
It is a basic polysaccharide that is polycondensed with 4 bonds. Since chitin contained in crustaceans such as crab and shrimp is obtained through heating and deacetylation in a concentrated alkaline solution, the molecular weight of chitosan is slightly smaller than the molecular weight of chitin.

Chitosan has a reactive free amino group in the molecule and is used as a natural polymer flocculant in wastewater treatment.
Also, as a polymer material, it is of interest chemically, and its use research is actively conducted.

Here, "chitosan-oligosaccharide" means D
-It means an oligosaccharide derived from chitosan in which 2 to 10 glucosamines are linked by β-1,4 bond. Chitosan obtained by de-N-acetylating oligosaccharide derived from chitin can be obtained by partial hydrolysis.

Chitosan-oligosaccharides are known to have antitumor properties and physiologically active functions in the fields of medicine, food, cosmetics, agriculture and the like. Chitosan itself is insoluble in water, but chitosan-oligosaccharide is soluble in water, has no bitterness and astringency peculiar to chitosan, and has an excellent physiological activity function.

For example, N-acetyl-chitohexaose (Glc) in which six N-acetyl-D-glucosamines are bonded.
It has been reported that chitohexaose (GlcN6) having six NAc6) and D-glucosamine bound thereto has a tumor metastasis-suppressing effect, an immunopotentiating effect and an infection-preventing effect on a transplanted tumor of mice (Carbo-hyd, Res.,
151, 403 (1986)).

[0008] In addition, the plant can be utilized in the agricultural field by inducing a revival action of self-defense ability against pest damage and activation of plant cells. Further, it has an effect of inhibiting the growth of plant pathogens and is expected to be developed as a soil conditioner or a natural pesticide (Nippon Kagaku Kogyo 44.10.30-63. (1
991)).

Furthermore, food preservatives such as pancake syrup, yogurt, beverages, pickled foods and low calories,
It is expected to be developed as a novel sugar material having a low degree of sweetness (Nippon Kagaku Kogyo 44.10.30-63.
1)).

Regarding enzymes, in the food field, it is expected that the market development of new enzymes will be accelerated along with the importance of functional foods. In fact, several tens of billion won of various oligosaccharides have already been commercialized in Japan alone. In other fields as well, for example, intake suppression research using cholesterol reductase, which is under study in the United States, may create a new market of more than 100 million dollars per year by commercialization (Nippon Kagaku Kogyo 44.10.30). ~ 63. (1991)).

On the other hand, as methods for producing chitosan-oligosaccharides having such excellent physiological activity, (1) acid hydrolysis method, (2) enzymatic decomposition method, (3) glycosyl transfer reaction utilization method, etc. are known. There is.

(1) Acid Hydrolysis Method In 1964, Rupley decomposed chitin by acid hydrolysis and isolated the product using an activated carbon-zeolite column. -Acetylchitopentaose is obtained. Also, recently H
Chitin-oligosaccharides have been produced in combination with a column for PLC (Biochem. Biophys Acta.
83, 245.1964)). Furthermore, JP-A-61-2
Japanese Patent Laid-Open No. 1102 is an improved method of acid hydrolysis, wherein chitosan is added with concentrated hydrochloric acid of 10 normal or more, and chitosan is added in an amount of 5% by weight.
It is disclosed that an oligosaccharide having a high degree of polymerization was obtained by adding an amount of 30 to 30 times and partial hydrolysis at a temperature of 60 to 100 ° C. for 1 to 4 hours. However, the acid hydrolysis method is problematic in that it requires a large amount of acid and heating, the manufacturing cost rises, waste liquid treatment is required, and secondary environmental pollution may occur.

(2) Enzymatic Degradation Method Vibrio anguillarum E-383, a mesophilic bacterium isolated in seawater
a is 3 in liquid medium containing chitin (medium A in Table 1)
After shaking culture at 5 ° C. for 16 days, N, N′-acetylchitobiose (GlcNAc2) has been obtained (Agri).
c. Biol. Chem. 53.1537-1541,
(1989)). However, the method using Vibrio anguillarum E-383a has a problem that the hydrolysis rate is as low as 53% and that a long culture time of 16 days is required.

Utilizing Bacillus licheniformis X-7u, which is a thermophilic bacterium isolated in a hot spring, by rotary shaking culture at 50 ° C. for 3.5 days in a liquid medium (medium B in Table 1),
N, N'-acetylchitotriose (GlcNAc3)
Has been obtained (Journal of the Japanese Society of Agricultural Chemistry 63, 7, 1199)
~ 1205,1989). However, the method utilizing Bacillus licheniformis X-7u has a hydrolysis rate of 72.
%, Which is relatively high, and the obtained chitin-oligosaccharide is also trisaccharide, but there is a problem in that it must be shaken by rotation for 3.5 days.

In addition, Bacillus No. Utilizing chitosanase, which is a chitosan-degrading enzyme derived from 7-M, chitosan-oligosaccharides from disaccharides to pentasaccharides have been obtained (Ag
ric. Biol. Chem. 51. (1989)).
However, most of them are monosaccharides.

Also, a chitosan-degrading bacterium isolated from soil, Pseudomonas spp.
In a medium (medium C in Table 1) at a temperature of 25 ° C. for 3 to 1
After culturing for 0 days, inoculating seed bacteria and culturing at 26 to 28 ° C., it was reported that two days after that, chitosanase was secreted into the medium, and the peak of the amount of enzyme in the medium was on the 4th day (Agric). Biol Chem. 54.1
2, 3341-3433, 1990). However, the Pseudomonas genus (Pseudomon) isolated in soil
as spp. ), The hydrolysis rate and product are unknown.

(3) Utilization of glycosyl transfer reaction Nocardia orientalis (Nocarda or)
ientalis) or Trichodoma lactis (Tri
A white precipitate of hexasaccharide was obtained by a transglycosylation reaction using a chitinase derived from choderma reesi) in a nitrate buffer with N-acetylchitotetraose (tetrasaccharide) (5 to 10% by weight) as a substrate, while Using the sugar as a substrate, the same procedure as above was carried out to obtain a hepta sugar. However, although the method is strongly influenced by the substrate concentration, reaction temperature and pH, the hydrolysis rate is low and is 34%. For this reason, the existing chitin-oligosaccharide tetrasaccharide and disaccharide are required, and the hydrolysis rate is low.

[0018] In order to solve the above problems, it has been earnestly desired to develop a method for producing a chitosan-oligosaccharide having a high efficiency and a desired degree of polymerization.

[0019]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems of the prior art, the present inventors have found that an enzyme that decomposes chitosan produced by an Aspergillus fumigatus mutant or the above Aspergillus fumigatus. The inventors have found that the use of the mutated bacterium itself can produce chitosan-oligosaccharide with a high hydrolysis rate, economically and without causing environmental pollution, and completed the present invention.

That is, in the present invention, chitosan is
The Aspergillus fumigatus mutant strain deposited under the deposit number KCTC0139BP at the Institute of Genetic Engineering, Korea Institute of Science and Technology, which has the property of degrading into oligosaccharides, is provided. The present invention also provides an enzyme produced by the Aspergillus fumigatus mutant bacterium that decomposes chitosan into chitosan-oligosaccharide. In addition, the Aspergillus fumigatus mutant bacterium is cultured to collect mycelium,
The present invention provides a method for producing a chitosan-oligosaccharide, which comprises reacting the obtained mycelium with chitosan. Hereinafter, the present invention will be described in detail.

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention comprises culturing an Aspergillus fumigatus mutant bacterium, collecting the mycelium produced by the culturing, and collecting the mycelium with chitosan at 50 to 60 ° C.
The chitosan-oligosaccharide is produced by reacting at 60 ° C. for 10 to 60 minutes, particularly preferably 30 minutes. Therefore, unlike the acid hydrolysis method, the use of hydrochloric acid or heating is unnecessary. Further, since the mycelium thus cultivated is used, an operation such as salting out is unnecessary in the process of using the enzyme, and the chitosan-oligosaccharide can be easily produced at a high hydrolysis rate in a short time.

In the method for producing chitosan-oligosaccharide of the present invention, the medium for culturing the Aspergillus fumigatus mutant strain is 5 to 15 g of chitosan and 0.25 to 1 of yeast extract per 1 liter of distilled water. 0.0 g, ammonium nitrate 0.5 to 2.0 g, sodium chloride 0.
25 to 1.5 g, dibasic potassium phosphate 0.5 to 2.
0 g, dibasic sodium phosphate 2.0 to 5 g, magnesium sulfate 0.25 to 1.0 g, calcium chloride 0.0
2 to 0.1 g, 10 to 20 g of agar, and 10 ml of nitric acid are preferable, and the Aspergillus fumigatus mutant is cultured in a liquid medium at a temperature of 20 to 30.
The shaking culture is preferably carried out at 70 ° C. and 70 to 150 rpm for 3 to 6 days.

The reaction between the mycelium and chitosan is carried out at a temperature range of 50 to 60 ° C., a pH of 4.0 to 5.0, and a reaction time of 1
It takes 0 to 60 minutes, and the concentration of chitosan is 1 to 5
It is preferably in the weight%.

For culturing the Aspergillus fumigatus mutant of the present invention, it is preferable to use the medium D shown in Table 1.

(Separation of Enzyme-Producing Bacteria) With respect to the enzyme-producing bacterium according to the present invention, a chitosan-degrading function was examined in a medium containing chitosan (medium D in Table 1), and a strain having the highest hydrolysis rate of chitosan was selected. The characteristics of the selected bacteria are shown in Table-2.

(Mycological properties) The selected bacteria were slide-cultured and observed for their morphological properties with an optical microscope. The hypha has a thickness of 4 to 6 μm, and a part of it thickens. The thickness of the conidia spores is 6 to 9 μm, the length is 300 to 500 μm, and they branch vertically from the hyphae. The conidium surface is smooth. The color of the colony on the Czapek agar plate medium was initially yellowish brown, but gradually changed to brown over the course of the culture period. The Fungi (Ai-nsworth
h, G. C. Sprrw, F.F. K. and Su
ssman A. S. : The Fungi, Vol
4A. Academic Press. New
York, pp 45-68 (1973)) and Th.
e Genus Aspergillus (Pape
r, K. B. and Fennell, D.M. I. :
The Genus Aspergillus, Ro
bert E. Kriege Pub. Co. Hunt
ingt-on. New York, pp 13-
577 (1973)), etc., and the results of the ubiquinone system, confirming that Aspergillus fumigatus,
It was named Aspergillus fumigatus KB-1.

(Deposit) The present inventor has deposited the Aspergillus fumigatus KB-1 of the present invention with the Institute of Genetic Engineering, Korea Institute of Science and Technology on December 15, 1994, and the deposit number is KCTC0139BP.

When Aspergillus fumigatus KB-1 is stored, potato dextrose liquid medium (pot) is used.
100 ml of ato dextrose broth was inoculated with Aspergillus fumigatus KB-1 at 25 ° C,
After culturing with shaking at 120 rpm for 5 days, the obtained bacterial suspension is aseptically filtered with 6 pieces of cheese cloth to remove mycelium, and then centrifuged at 1,500 rpm to obtain conidia and stored.

When the above-mentioned conidia of Aspergillus fumigatus KB-1 are used, they are washed with sterilized distilled water to prepare a conidial suspension, and the culture is carried out by using agar in the composition of medium D in Table-1. Liquid culture medium from which is removed at 25 ° C., 120 rpm,
What was shake-cultured for 5 days is used.

[0030]

EXAMPLES The present invention will now be described in detail with reference to examples, but the present invention is not limited thereto. In addition,% regarding a density | concentration shows weight%.

Example 1 Effect of Temperature on Enzyme Activity After adding mycelium of Aspergillus fumigatus KB-1 and chitosan, the enzyme activity at each temperature was increased from 30 to 80 ° C. at 10 ° C. intervals. Was measured. The result is shown in Figure 2.
Shown in The optimum temperature was 60 ° C., and the enzyme activity was about 87% at 50 ° C., but the enzyme activity decreased sharply at 40 ° C. or lower, or at 70 to 80 ° C.

(Example 2: Effect of pH on enzyme activity) The pH of the reaction solution was changed by 0.5, and the enzyme activity at each pH was examined. The results are shown in Fig. 3. The optimum pH of the enzyme produced by Aspergillus fumigatus KB-1 is 4.0 to 5.0, pH 3.5 or lower or pH
At 6.5 or more, there was almost no enzyme activity.

(Example 3: Influence of chitosan concentration on chitosan-oligosaccharide production) A chitosan solution in which the chitosan concentration was changed from 1 to 8% in 1% steps was prepared by dissolving chitosan in 1% nitric acid. If chitosan is 5-8%,
It was melted at a temperature of 60 ° C. 50 ml of each chitosan solution,
The pre-cultured mycelium of Aspergillus fumigatus KB-1 was mixed and allowed to react by gently shaking in a 60 ° C water bath for 30 minutes. Chitosan remaining without reacting with the enzyme is
The same amount of ethanol was added and the precipitate was removed by centrifugation. This supernatant was converted to chitosan-oligosaccharide. FIG. 4 shows the results. In the chitosan concentration range of 1 to 3%, 95% or more was decomposed into chitosan-oligosaccharide, and 74% was decomposed even when the chitosan concentration was 5%. However, at higher chitosan concentrations, chitosan-oligosaccharide production was greatly reduced.

The chitosan-oligosaccharide produced in Example 3 was analyzed by high performance liquid chromatography. Results are shown in FIG. When the concentration of chitosan is 1%, the produced chitosan-oligosaccharide has 43% disaccharide or less and 5% disaccharide or more.
It was 7% (FIG. 5B). When the chitosan concentration was 3% or higher, disaccharide was 22%, trisaccharide was 34%, and tetrasaccharide was 44% (FIG. 5C). The chitosan-oligosaccharide produced by using the mycelium of Aspergillus fumigatus KB-1 used in the present invention produces almost no monosaccharide as compared with acid hydrolysis, and at a chitosan concentration of 3%, 78 % Was chitosan-oligosaccharide with 3 to 6 sugars.

[0035]

[Table 1]

[0036]

[Table 2]

[0037]

The Aspergillus fumigatus mutant bacterium of the present invention produces an enzyme excellent in the decomposition of chitosan into chitosan-oligosaccharide, and the mycelium of the bacterium is used to decompose chitosan-oligosaccharide. The sugar hydrolysis rate was high, and most of the sugars were 3 sugars or more even at a high substrate concentration of 3 to 5%. The present invention provides a method for producing chitosan-oligosaccharide from chitosan which is simple in process, economical, and does not cause environmental pollution problems.

[Brief description of drawings]

FIG. 1 is an Aspergillus fumigatus KB-1.
2 is a photograph showing a thin-layer chromatogram of the ubiquinone system of FIG.

FIG. 2 shows the relationship between enzyme activity and temperature (horizontal axis).

FIG. 3 shows the relationship between enzyme activity and pH (horizontal axis).

FIG. 4 shows the relationship between chitosan concentration (horizontal axis) and chitosan-oligosaccharide production.

FIG. 5A shows (Glc1), (Glc2), and (Glc).
lc3), (Glc4), (Glc5), (Glc6)
Shows the HPLC standard. FIG. 5B shows the HP of the produced chitosan-oligosaccharide at a chitosan concentration of 1%.
LC is shown. FIG. 5C shows HPLC of chitosan-oligosaccharide produced at a chitosan concentration of 3%.

Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display area A01N 63/04 A01N 63/04 A A23L 3/3562 A23L 3/3562 A61K 31/73 ABD A61K 31/73 ABD ADU ADU ADX ADX (C12N 1/14 C12R 1:68) (C12N 9/24 C12R 1:68) (C12P 19/26 C12R 1:68)

Claims (9)

[Claims] 1. An Aspergillus fumigatus mutant strain deposited under accession number KCTC0139BP at the Institute of Genetic Engineering, Korea Institute of Science and Technology, which has the property of degrading chitosan into chitosan-oligosaccharide. 2. The enzyme produced by the Aspergillus fumigatus mutant strain according to claim 1, which decomposes chitosan into chitosan-oligosaccharide. 3. A method for producing chitosan-oligosaccharide, which comprises culturing the Aspergillus fumigatus mutant bacterium according to claim 1 to harvest mycelium, and reacting the obtained mycelium with chitosan. 4. The cultivation of Aspergillus fumigatus mutant bacterium in chitosan 5 to 1 per 1 liter of distilled water.
5 g, yeast extract 0.25 to 1.0 g, ammonium nitrate 0.5 to 2.0 g, sodium chloride 0.25 to 1.5 g, dibasic potassium phosphate 0.5 to 2.0 g, dibasic sodium phosphate 2 4. Chitosan according to claim 3, characterized in that it is carried out in a medium consisting of 0.0 to 5 g, magnesium sulfate 0.25 to 1.0 g, calcium chloride 0.02 to 0.1 g, agar 10 to 20 g, and nitric acid 10 ml. A method for producing an oligosaccharide. 5. A culture of Aspergillus fumigatus mutant in a liquid medium at a temperature of 20 to 30 ° C. and 70 to 1
The method for producing a chitosan-oligosaccharide according to claim 3 or 4, wherein the shaking culture is performed at 50 rpm for 3 to 6 days. 6. The reaction between mycelium and chitosan is carried out at a temperature of 50.
6. The method according to claim 3, wherein the temperature is 60 to 60 ° C.
A method for producing the described chitosan-oligosaccharide. 7. The reaction between mycelium and chitosan is carried out at pH 4.
The method for producing a chitosan-oligosaccharide according to claim 3, wherein the method is performed at 0 to 5.0. 8. The reaction time between the mycelium and chitosan is 10
8. The method for producing chitosan-oligosaccharide according to claim 3, wherein the method is for 60 to 60 minutes. 9. The method for producing a chitosan-oligosaccharide according to claim 3, wherein the concentration of chitosan in the reaction between the mycelium and chitosan is 1 to 5% by weight.