KR0140430B1 - Aspergillus fumigatus mutant strain and producing method of chitosan oligosaccharide - Google Patents

Abstract

The mycelia were harvested by culturing Aspergillus pumigatus mutants with high resolution of chitosan, and the method of providing the chitosan-oligosaccharide by reacting the harvested mycelium with chitosan was high in yield and meat concentration (3-5%). In most cases, oligosaccharides of more than 3 sugars are produced, and the process is simple and economical, and it is an excellent method for producing chitosan-oligosaccharides that can be produced without compromising environmental pollution.

Description

Aspergillus pumigatus mutant strain and production enzyme and preparation method of chitosan-oligosaccharide using the same

1 is a thin-film chromatogram of the ubiquinone system of Aspergillus fumigatus KB-1,

2 is the effect of temperature on the activity of the enzyme,

3 is the effect of pH on the activity of the enzyme,

4 is the production of chitosan-oligosaccharides by concentration,

5 is a high performance liquid chromatogram of chitosan-oligosaccharides produced.

[Industrial use]

The present invention relates to chitosan-oligosaccharides, and more particularly, in terms of useful use of natural resources, a method for preparing chitosan-oligosaccharides from chitosan, which is rich in crustaceans such as crabs and shrimp, and separated from the soil. The present invention relates to a chitosan-oligosaccharide producing enzyme produced by the Aspergillus fumigatus KB-1 strain, a chitosan decomposing bacterium.

[Prior art]

Chitin, or β-1,4-poly-N-acetyl-D-glucosamine, is a major structural polysaccharide of lower animals. Binding and present in the form of glycoproteins, these glycoproteins can be prepared by removing proteins with alkali. Such chitin is hydrolyzed with hydrochloric acid to obtain monosaccharide D-glucosamine hydrochloride, which is heated in a concentrated alkali solution and deacetylated to obtain chitosan.

On the other hand, chitosan is a basic polysaccharide in which D-glucosamine, or 2-amino-2-deoxy-D-glucose is polycondensed by β-1,4 bond, is abundantly contained in crustaceans such as crabs and shrimp. The molecular weight is slightly smaller than that of chitin because (Chitin) is heated and deacetylated in concentrated alkali solutions to produce chitosan. Since these chitosans have free amino groups with abundant reactivity in their molecules, they are being used as wastewater treatment as natural polymer flocculents, and research on their use is being actively conducted as a polymeric material of very interesting interest.

Chitosan-oligosaccharides are oligosaccharides in which D-glucosamine is bound by 2 to 10 by β-1,4 bonds, which are de-N-acetices of chitin-oligosaccharides, and can be obtained by partial hydrolysis of chitosan. Its properties are not clear, but it is known that it is a promising substance that can be used in medicine, food, cosmetics, agriculture, etc., and has a bioactive function, especially with both tumors. Chitosan is insoluble in water, but chitosan-oligosaccharides are soluble in water, and the chitosan-specific bitterness and astringent taste can not be detected at all, and chitosan-oligosaccharides have excellent physiological activity. For example, reports have shown that N-acetylchitonuclear iodine (GlcNAc6) or chitonuclear iodine (GlcN6), a chitosan 6-saccharide homologue, has a tumor metastasis inhibitory effect, an immune enhancing effect, and an infection defense effect on transplanted tumors in mice. (Carbohydr, Res., 151, 403 (1986)).

On the other hand, plants can be utilized in agriculture by inducing the reactivation of self-defense ability against pests and activation of plant cells, and there is a report that it is expected to be developed as a soil improving agent or a natural pesticide because it has a proliferation inhibitory effect on plant pathogens. (Reference: Japanese Chemical Industry 44. 10. 30-63. (1991)).

In addition, food preservatives such as hot cake syrup, yogurt, beverages, and pickled foods, and low calorie, low sugar, new sugars are expected to be developed (see Japanese Chemical Industry 44. 10. 30 ~ 63. (1991)).

In the case of food enzymes, the importance of functional foods in recent years is expected to accelerate the development of new enzyme markets. In Korea, hundreds of billions of dollars worth of oligosaccharides have already been commercialized and Choresterol Reductase is being studied in the United States. The commercialization of intake suppression studies using commercialization is expected to create a new market of more than $ 100 million annually (see Japanese Chemical Industry 44. 10. 30 ~ 63.

As a method for producing chitosan-oligosaccharides having such excellent bioactive properties, (1) acid hydrolysis, (2) enzymatic degradation, and (3) sugar transfer reaction are known.

In (1) acid hydrolysis, Rupley isolated degradation products in 1964 using activated carbon-zeolite columns and obtained N-acetylchitopentataose (GlcNAc4) from monosaccharide to five sugars. Chitin-oligosaccharides were prepared in combination with a column for HPLC (Biochem. Biophys Acta. 83, 245. 1964). (1) As an acid hydrolysis-improving method, a concentrated hydrochloric acid of 10% isoi was added to chitosan at 5 to 30 times the weight of chitosan, and partially hydrolyzed chitosan at a temperature of 60 to 100 for 1 to 4 hours to obtain a high polymerization oligosaccharide. There are also reports that it can be produced (see Japanese Patent Application Laid-Open No. 61021102). However, such acid hydrolysis does not only increase the cost by using more fuel for acid and heating than necessary, but also causes secondary environmental pollution in the treatment of waste hydrochloric acid. There is a problem that can cause.

(2) Enzymatic digestion of N, N'-acetate by shaking medium temperature bacteria Vibrio anguillarum E-383a isolated from seawater in liquid medium containing medium (Medium A) for 35 or 16 days. It has been reported that chilcitobiose (GlcNAc 2) (yield 53%) was obtained (Agric. Biol. Chem. 53. 1537-1541, (1989)). However, enzymatic digestion using mesophilic bacteria Vibrio anguillarum E-383a isolated from seawater has a relatively low yield of 53% and requires incubation for 16 days at medium temperature for a long time.

In addition, N, N-acetylchitotriose (GlcNAc3) was cultured by rotating shaking culture in liquid medium (medium B) for 50 to 3.5 days using Bacillus licheniformis X-7u, a thermophilic bacterium isolated from a hot spring. (Yield 72%) has been reported (Nippon Nogeikagaku Kaishi 63, 7, 1199-1205, (1989)). However, the enzymatic digestion method using Bacillus licheniformis X-7u isolated from the hot spring bacteria, Bacillus licheniformis, was relatively high in yield of 72%, and the chitin-oligosaccharide produced was 3 sugars. There is a problem that there is inconvenience.

Bacillus sps. Chitosan-oligosaccharides have been successfully synthesized from 2 to 5 sugars using chitosanase derived from 7-M, but there is a problem in that they almost produce only monosaccharides (see Ahric. Biol. Chem. 51). (1989).

Meanwhile, Pseudomonas spp. Isolated from soil was cultured (Pseudomonas spp.) And cultured for 3 to 10 days at 25 ° C at room temperature, and then seed germs were made. Chitosanase was secreted and the highest amount of enzyme in the medium was reported after 4 days (Agric. Biol. Chem. 54. 12, 3341 to 3343, 1990). However, the method using Pseudomonas spp., Which is a chitosan degrading bacterium isolated from soil, has a problem in that yield and product are unclear.

(3) As a sugar transfer reaction method, meat concentration (5-10%) of N-acetylkytonuclear iodine (4 sugars) was derived from Nocardia orientalis or Trichoderma reesi. Chitinase was used to obtain a white precipitate of six sugars in acetic acid buffer, and seven sugars were synthesized using five sugars as a substrate. However, the reaction is highly influenced by substrate concentration, reaction temperature and pH, but yield is 34%. However, this method of sugar transfer reaction also synthesizes the existing chitin-oligosaccharides 4 sugars and 2 sugars to obtain 6 sugars. Therefore, this method uses the oligosaccharides that have already been produced, which has a drawback that the yield (35%) is still very low.

In order to solve the above problems, many researchers have recently conducted research on a method for synthesizing oligosaccharides having high efficiency and a desired degree of polymerization in large quantities, but the results are still insignificant.

[PROBLEMS TO BE SOLVED BY THE INVENTION]

In order to solve the problems of the above-described maritime technology, the present invention first provides an Aspergillus fumigatus mutant having the ability to decompose chitosan into chitosan oligosaccharide, and secondly, the chitosan produced by the Aspergillus fumigatus mutant. The present invention provides a method for preparing chitosan oligosaccharides having high yield, economical efficiency and environmental pollution by using the Aspergillus pumigatus mutant.

[Means for solving the problem]

In order to achieve the object of the present invention as described above, the present invention provides Aspergillus pumigatus having the properties of microorganisms deposited with No. KCTC 0139BP to the Genetic Engineering Research Institute of Korea Advanced Institute of Science and Technology to decompose chitosan into chitosan-oligosaccharide.

The present invention also provides an enzyme for decomposing chitosan produced by Aspergillus pumigatus of the present invention into chitosan-oligosaccharides.

And the present invention provides a method for producing chitosan-oligosaccharides comprising the step of culturing the Aspergillus pumigatus of the present invention described above to harvest these mycelium, and reacting the harvested mycelium with chitosan.

In the method for producing chitosan-oligosaccharide of the present invention, the medium for culturing Aspergillus pumigatus is 5-15 g of chitosan, 0.25-1.0 g of yeast extract, and 0.5-2.0 ammonium nitrate per 1 L of distilled water. mg, sodium chloride 0.25-1.5mg, dipotassium phosphate

It is preferable to include 2.0-5mg, magnesium sulfate 0.25-1.0mg, calcium chloride 0.02-1.0mg, agar 10-20g, acetic acid 10% by volume, and the culture of Aspergillus fumigatus is carried out in a liquid medium at a temperature of 20- Shaking incubation at 30 ° C. at 70-150 rpm for 3-6 days is preferred.

And in the method for producing chitosan-oligosaccharide of the present invention, the reaction of the mycelium and chitosan is in the temperature range of 50 to 60 ℃, pH range of pH 4.0 to 5.0, the reaction time for 10 to 60 minutes The reaction is preferably carried out at a concentration of 1 to 5% by weight of chitosan.

EXAMPLE

Hereinafter, representative examples, preferred examples and comparative examples of the present invention are described. However, the following examples are only one preferred embodiment of the present invention to aid in understanding the present invention, and the present invention is not limited to the following examples.

Representative Example

The Aspergillus pumigatus KB-1 strain was cultured to recover the mycelium produced at this time, and the mycelium was reacted with chitosan for 30 minutes at 60 ° C., compared to the conventional acid hydrolysis method, without using hydrochloric acid and heating. The oligosaccharide can be produced, and among the enzymatic digestion methods, the present invention uses mycelium cultured at 25 ° C. for 3 to 5 days, so that it is not necessary to manipulate salts in the enzyme purification process and to produce high yields of chitosan-oligosaccharides in a short time. This is possible.

Isolation of enzyme producing bacteria

Aspergillus pumigatus strain, which decomposes chitosan into chitosan-oligosaccharides, is a commercially available method, for example, after agitated processed chitosan, which is made from crab shells that are deteriorated by bacteria when stored for a long time, in sterile saline solution. CH Collins et al., Microbial, 4th. (1976) and WF Harrigan et al., Laboratory Methods in food and diary microbiology (1976). Agar medium of medium D (chitosan 10g, 0.5% acetic acid 100ml, yeast extract 0.5g, KNO ₃₎ of medium D described in Table 1 as a carbon source, using general chitosan produced from crab shell as raw material having a deacetylation degree of 70% or more. 1.0 g, Na ₂ HPO ₄ 1.0 g, NaCl 0.5 g, MgSO ₄ 7H ₂ O 0.5 g, FeSO ₇ H ₂ O 0.05 g, CaCl ₂ 0.05 g, 20 g agar, 900 mL deionized water, pH 5.5).

Then, after culturing for 3 to 7 days at a temperature of 20 to 30 ° C. for intensively separating and culturing fungi (fungus), colonies that appeared at this time were observed. When the enzyme produced by the bacterium decomposes chitosan, the periphery of the transparent ring formed around the colony changes vividly, so the presence or absence of the transparent ring generated around the cell was confirmed. One strain with the largest and clearest clear ring was selected and purified. In this way, Aspergillus fumigatus, which is a bacterium of the present invention capable of decomposing chitosan to chitosan-oligosaccharide, was cultured and selected.

Using the liquid medium containing chitosan (except agar in the separation medium containing chitosan: ph 5.5) as the enzyme production medium was inoculated with the Aspergillus fumigatus strain selected above, and incubated at 25 ℃ 5 Daily shake culture (120 rpm stroke 5.4 cm). At this time, the mycelium collected by filtration with filter paper was used to use the mycelium in the culture medium.

The identification of bacteria was observed in the colony of Czapeck agar plate culture and the development of colonies, and the size and shape of the conidia, conidia, and conidia disease of the fungi were observed through slide cultures (GC Ainsworth). , et al., The Fungi, Vol 4 A. Academic Press, New York, pp 45-68 (1973): KB Paper et al., The Genus Aspergillus, Robert E. Kriege Pub. Co. Huntington.Now York, pp 13-577 (1973).

As a result, as shown in Table 2 below, compared to Aspergillus pumigatus of the buggy's manual, the morphological properties were vertically branched with hyphae (4-6 μm thick) and the conidia surface was smooth. It was. In the Chapek agar plate culture, colony color was initially yellowish brown and changed to brown as the incubation period increased.

And it was confirmed by the ubiquinone system like FIG. 1, and it confirmed that this selection bacterium was Aspergillus pumigatus. The present inventors deposited the strain Aspergillus pumigatus KB-1 of the present invention with the accession number KCTC 0139BP to the Gene Bank under the Korea Advanced Institute of Science and Technology on December 15, 1994.

Aspergillus pumigatus KB-1 was stored in 100 ml of potato dextrose broth and inoculated with 6 layers of cheese clad aseptically with bacterial suspension obtained by shaking incubation at 25 ° C and 120 rpm for 5 days. Mycelia were removed by filtration (cheese cloth), and then centrifuged at 1,500 rpm to obtain conidia. When the hyphae suspension is washed again with sterile distilled water and the concentrated condensate suspension is stored in the refrigerator and used for the production of enzymes, the culture medium is cultured in sludge for 5 days at 25 ° C. and 120 rpm for 5 days in a liquid medium excluding agar in medium D of Table 1 above. Used.

Another preferred embodiment of the present invention is described next.

Example 1

Effect of temperature on enzyme activity

Aspergillus fumigatus KB-1 mycelium was added to chitosan, and the effects of the optimum temperature on the enzyme activity were examined.

After adding chitosan and mycelium, enzyme activity was measured at each temperature by changing the temperature from 30 ℃ to 80 ℃ at 10 ℃ intervals. As shown in FIG. 2, the optimum temperature was 60 ℃ and about 87% at 50 ℃. Enzyme activity was observed, but the enzyme activity was sharply decreased below 40 ° C or 70-80 ° C. Therefore, the optimum temperature was found to be 60 ℃.

Example 2

Effect of pH on Enzyme Activity

As shown in FIG. 3, the optimum pH of enzyme activity was examined. As shown in FIG. 3, the optimum pH of the enzyme produced by Aspergillus fumigatus KB-1 was 4.0-5.0, and there was little enzymatic activity below pH 3.5 and above pH 6.5.

Example 3

Changes in Production of Chitosan-oligosaccharides by Concentration

The concentration of chitosan was dissolved in 1% acetic acid (5-8% sms at 60 ° C.) in the range of 1-8% and then incubated with 50 ml of each chitosan solution. 1 mycelium was mixed and reacted by shaking slowly for 30 minutes in a 60 ℃ water bath. At this time, the chitosan remaining without reacting with or decomposing the enzyme is centrifuged by adding the same amount of ethanol to convert the supernatant except the precipitate into chitosan-oligosaccharide.

Chitosan-oligosaccharides produced by mixing the mycelia of Aspergillus pumigatus KB-1 of the present invention with chitosan were produced at 95% or more in the range of 1 to 3% and 74% at the 5% concentration. At higher concentrations, however, the production of chitosan-oligosaccharides was significantly reduced.

In addition, the chitosan-oligosaccharides produced at this time were analyzed by high-performance liquid chromatography. As shown in FIG. 5, (A) is a high performance liquid chromatogram for the standard materials of (Glc 1), (Glc 2), (Glc 3), (Glc 4), (Glc 5), (Glc 6) The chitosan-oligosaccharide produced when the concentration of chitosan in (B) is 1% is 43% or less. More than three sugars accounted for 57%. When the chitosan concentration was 3% or more, 22% per 2, 34% for 3 sugars and 44% for 4 sugars.

Therefore, chitosan-oligosaccharides produced using the mycelia of Aspergillus pumigatus KB-1 strain used in the present invention had little production of monosaccharides compared to acid hydrolysis, and 78% 3 or more chitosan- in 3% concentration range. The production of oligosaccharides was confirmed.

[effect]

Aspergillus pumigatus mutants of the present invention as described above produces enzymes with high resolution of chitosan, and the yield of chitosan-oligosaccharides is high when meat is degraded using mycelia of these strains, and meat concentration (3-5%). In most cases, more than 3 sugars are produced, and the process is simple, economical and can be produced without causing environmental pollution.

Claims (7)

Aspergillus pumigatus having the properties of microorganisms deposited with KCTC 0139BP in the Genetic Engineering Research Institute of Korea Advanced Institute of Science and Technology to decompose chitosan to chitosan-oligosaccharide. Cultivating the Aspergillus pumigatus of claim 1 to harvest these mycelium: reacting the harvested mycelium with chitosan; Chitosan-oligosaccharide production method comprising a process. The medium for culturing Aspergillus pumigatus according to claim 2, wherein 5 to 15 g of chitosan, 0.25 to 1.0 g of yeast extract, 0.5 to 2.0 mg of ammonium nitrate, 0.25 to 1.0 mg of magnesium sulfate, Method containing 0.02 to 0.1 mg of calcium chloride, 10-20 g of agar, 10% by volume of acetic acid. The method of claim 2, wherein the culture of Aspergillus pumigatus is a method of shaking culture for 3 to 6 days at 70 to 150 rpm at a temperature of 20 to 30 ℃ in a liquid medium. The method according to claim 2, wherein the reaction between the mycelium and chitosan is carried out at a temperature range of 50 to 60 ° C. The method according to claim 2, wherein the reaction between the mycelium and chitosan is performed at a pH range of pH 4.0 to 5.0. The method according to claim 2, wherein the concentration of chitosa is reacted at 1 to 5% by weight.