JP3026312B2 - Production method of chitin degradation products - Google Patents

Description

The present invention relates to a method for producing a chitin hydrolyzate which can be used as, for example, a growth factor of a useful intestinal bacterium, a sweetener, etc., and more specifically, a method for producing a specific microorganism. The present invention relates to a production method using a chitinolytic enzyme.

[Background Art] Conventionally, chitin degradation products such as di-N-acetylchitobiose and N-acetylglucosamine are mainly produced by a chemical method. For example, di-N-acetylchitobiose is obtained by using a crab or shrimp shell as a raw material, hydrolyzing with concentrated hydrochloric acid at about 40 ° C. for several hours, and then separating and purifying the same by activated carbon column chromatography. Has been prepared. N-acetylglucosamine is obtained by hydrolyzing crusts and shrimp crusts with concentrated hydrochloric acid at about 100 ° C. for about 1 hour, and converting the obtained D-glucosamine hydrochloride into N-acetyl with acetic anhydride. It is prepared by chemical synthesis.

[Problems to be Solved by the Invention] However, in the above-mentioned method for producing a chitin hydrolyzate by acid hydrolysis, a strong acid such as concentrated hydrochloric acid must be used, and a special device is required because the reaction is performed at a high temperature. Energy costs.

In addition, in the case of acid hydrolysis, a decomposition product of chitin is obtained as a mixture of a monomer and various chitin oligosaccharides. For example, to obtain di-N-acetylchitobiose as a single compound, a column is required. It must be purified by chromatography or the like, and the yield is as low as about 10% based on the chitin raw material.

Further, when acid hydrolysis is performed, deacetylation occurs simultaneously with decomposition of glucoside bonds. Therefore, when N-acetylglucosamine is obtained, N-acetylation must be performed after hydrolysis. Since N-acetylglucosamine produced in this way is a chemically synthesized product,
At present it is not used as a food material.

Therefore, various methods using a chitin-degrading enzyme produced by a microorganism have been reported as a method for easily producing a chitin-decomposed product that can be used as a food material in good yield without depending on a chemical method. As a microorganism that degrades chitin, genus Serratia (Monreal. J et al., 1985)
Year), genus Vibrio (Uchida et al., 1979), genus Pseudomonas (Takiguchi et al., 1985),
The genus Aeromonas (Yabuki et al., 1983) and the genus Bacillus (Takiguchi et al., 1989) have been reported.

An object of the present invention is to search for a microorganism having a chitin-degrading property which has not been reported so far from the natural world, and to obtain a chitin-decomposed product that can be used as a food material in good yield by using a chitin-degrading enzyme produced by this microorganism. It is to provide a manufacturing method.

[Means for Solving the Problems] In accordance with the above object, the present inventors searched for strains capable of efficiently degrading chitin from various samples, and found that Chromobacterium isolated from soil was obtained.
m) A specific microorganism belonging to the genus is excellent in chitin-decomposing ability, and when a culture filtrate thereof is allowed to act on chitin, di-N-acetylchitobiose is produced in a high yield. It has been found that N-acetylglucosamine is produced when acting on -N-acetylchitobiose, and the present invention has been completed.

That is, the method for producing a chitin hydrolyzate according to the present invention comprises a method of culturing a microorganism belonging to the genus Chromobacterium and having a chitin-degrading ability in a chitin-containing medium, and preparing a filtrate component and / or a bacterium prepared from the culture. Body components,
It is characterized by acting on chitin.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments.

Belongs to the genus Chromobacterium,
Examples of the microorganism having chitin decomposability include Chromobacterium sp.N-41 (Chromosome) isolated from the soil by the present inventors.
bacterium sp. N-41) is preferably used. The mycological properties of this bacterium are as follows.

(A) Morphology (1) Cell shape: Bacillus (2) Cell size: 0.55 × 1.4 μm (3) Motility: Yes (4) Flagella: polar monochaete (5) Gram staining: negative (b) Growing state (1) Gravy agar plate culture: The periphery is transparent, the center is white,
Flat and smooth, slightly glossy and does not produce pigments.

(2) Gravy agar slant culture: the whole is slightly transparent and colonies are not clear.

(3) Broth liquid culture: no surface growth, the whole medium is turbid.

(4) Broth gelatin stab culture: gelatin is not liquefied. It grows along the puncture hole.

 (5) Litmus milk: acidic, does not liquefy milk.

(C) Physiological properties (1) Nitrate reduction: positive (2) Denitrification reaction: negative (3) MR reaction: positive (4) VP test: negative (5) Indole formation: negative (6) Hydrogen sulfide Production: Negative (7) Starch hydrolysis: Positive (8) Use of citric acid: Positive (9) Inorganic nitrogen source: Negative (10) Pigment production: Negative (11) Urease: Negative (12) Oxidase: Positive ( 13) Catalase: positive (14) Growth temperature range: 10-39 ° C (15) Growth pH range: 5.0-9.0 (16) Attitude to oxygen: facultative anaerobic (17) Acid and gas production from saccharides (Saccharide) (Generation of acid) (Generation of gas) L-arabinose--D-xylose--D-glucose +-(Saccharide) (Generation of acid) (Generation of gas) D-mannose + -D- Fructose +-D-galactose--maltose--sucrose --Lactose--Trehalose--D-sorbitol--D-mannitol--Inosit--Glycerin--Starch--(d) Other properties Oxidation of gluconic acid: Negative degradation of Esculin: Negative degradation of arginine: Negative Decarboxylation of lysine: Negative Decarboxylation of ornithine: Negative Deamination of phenylalanine: Negative Sodium chloride tolerance: Does not grow in broth containing 6% sodium chloride Potassium cyanide resistance: Negative Phosphatase: Negative Pectinase: Negative lecithinase : Negative Liquefaction of gelatin: Negative Decomposition of casein: Negative DNase: Negative Oxidation of lactic acid: Positive Generation of hydrogen cyanide: Negative Reactivity with vibriostatic reagent 2,4-diamino-6,7-diisopropylpteridine (0/129): Negative GC content: 63.1 mol% This bacterium is facultative anaerobic In bacilli, there are motile a pole single hair, oxidase and catalase positive, generates an acid by fermentation from glucose. The barge aids manual of systematic
When searched by Biotechnology (1984), Vibrio, Aeromonas, Plesiomonas, and Chromobacterium are applicable.

However, comparing this genus with these genera for other properties, they differ from the genus Vibrio in that they are not resistant to 6% sodium chloride and are not sensitized to the vibriostatic reagent (0/129). It differs from Aeromonas in that indole is produced, gas is produced from D-glucose, D-mannitol is fermented, and DNase is negative. Furthermore,
Plesiomonas is a vibriostatic reagent (0 /
129) in that it does not sensitize and does not produce indole.

Furthermore, a search revealed that this bacterium had pH
You can not grow below the 5, to produce a CO ₂ by oxidizing a lactic acid, reducing nitrates to assimilate citric acid and GC content
It is similar to the genus Chromobacterium because it has properties such as 63.1 mol%. Although this bacterium does not produce the purple pigment that is characteristic of the genus Chromobacterium, the non-pigment producing bacterium in the genus Chromobacterium is in the Barges Manual of Systematic Biotechnology (1984). It is described. For this reason, the bacterium was identified as a bacterium belonging to the genus Chromobacterium.

Next, when examining the species, this bacterium produced purple pigment, produced hydrogen cyanide, liquefied gelatin, egg yolk reaction,
Casein degradation, arginine degradation, acid production from trehalose and starch are all negative, and the MR test is positive, which is different from Chromobacterium violaceum. It does not grow at 30 ° C, grows at 30 ° C or more, and has a different GC content.
Also different from Chromobacterium flavia tiles. Therefore, this bacterium was determined to be a new species belonging to the genus Chromobacterium, and the present inventors named this strain Chromobacterium sp. N-41. This strain has been deposited with the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology under the name of No. 11779.

As described above, microorganisms having chitin decomposability that have been reported so far include the genus Serratia, the genus Vibrio, and the genus Pseudomonas.
Genus, Aeromonas, Bacillus
s) There are no reports on microorganisms belonging to the genus Chromobacterium which have the ability to degrade chitin.

This strain was isolated as follows. That is, a soil sample was prepared by adding 1.0% colloidal chitin and 0.1% KH ₂ PO ₄
When added to a liquid medium containing _{0.02% MgSO 4 · 7H 2 O} , 30
After shaking and culturing at 24 ° C. for 24 hours, the cells on which the bacteria had grown were applied to a plate medium obtained by adding 2% agar to the same medium as described above, and cultured at 30 ° C. for 72 hours to dissolve the colloidal chitin. A colony having a large transparent ring was collected. Furthermore,
Culture was repeated using a similar medium to purify the target microorganism.

Microorganisms used in the present invention are not limited to those isolated by the above method, but may belong to the genus Chromobacterium and have a chitin-degrading ability. Any strain such as a strain mutated to increase the strain may be used.

Next, the method of culturing the microorganism will be described. As the medium, a liquid medium containing chitin and other nutrients is preferably employed. As chitin, colloidal chitin is preferably used. Colloidal chitin can be prepared by treating shrimp and crab shells with hydrochloric acid or alkali, dissolving the resulting crude chitin in cold hydrochloric acid, and then dispersing it in a large amount of water. The concentration of colloidal chitin in the medium is preferably 1.0 to 10.0%. As other nutrient sources, for example, nitrogen sources, inorganic salts and the like are used. As the nitrogen source, it is preferable to add 0.01 to 0.5% of peptone, and further, 0.01 to 0.5% of yeast extract.
It is preferred to add. Further, as inorganic salts, KH ₂ P
It is preferable to add O ₄ and MgSO ₄ .7H ₂ O, and CaCl ₂ may be added as necessary. The pH of the medium is preferably adjusted to 6 to 8. The culture is preferably performed by shaking culture or by using a jar fermenter. The culture temperature is preferably in the range of 25 to 37 ° C, and the culture time is preferably in the range of 1 to 3 days.

After culturing in this manner, the culture solution is separated into a filtrate and cells by filtration, centrifugation, or the like. In the present invention, the filtrate and the cells can be directly used as a crude enzyme in a chitin decomposition reaction. However, the filtrate can be further purified and used by dialysis, salting out, ultrafiltration and the like. Also,
Cells can be lysed by lytic enzymes such as lysozyme,
Alternatively, it is preferable to use one crushed by an ultrasonic crusher or the like. In the filtrate, a chitinase activity for decomposing chitin to produce di-N-acetylchitobiose was observed,
In the cells, N-acetylglucosaminidase activity (chitobiaase activity) that decomposes di-N-acetylchitobiose to produce N-acetylglucosamine is observed. In the present invention, each of the active fractions can be further purely separated from the filtrate and / or the cells by a known purification means, and purified enzymes can be used. Therefore, the filtrate component and / or the bacterial cell component in the present invention are:
The filtrate and / or cells may be used as they are, and the filtrate and / or
Alternatively, the enzyme may be a crude enzyme prepared from cells, or a pure enzyme separated and purified from a filtrate and / or cells.

Next, the decomposition reaction of chitin will be described.When di-N-acetylchitobiose is produced by allowing the above-mentioned filtrate component to act on chitin, colloidal chitin is preferably used as a substrate, and the concentration of colloidal chitin is 1.0 to 1.0. 10.0%
Is preferred. The chitinase activity in the reaction solution is preferably 1 to 20 U per 1 g of the substrate. The reaction conditions are pH4 ~
It is preferably carried out at a temperature of 30 to 60 ° C. for 2 to 24 hours. In the reaction solution, di-N-acetylchitobiose is produced, and, if necessary, after concentration, can be powdered using a freeze dryer, a vacuum dryer, a spray drier, or the like.

When the above-mentioned cell components are allowed to act on the thus obtained di-N-acetylchitobiose, N-acetylglucosamine can be produced. In this case, the substrate di-
The concentration of N-acetylchitobiose is preferably 1.0 to 20%, and the chitobiase activity in the reaction solution is 1 to 10% per 1 g of the substrate.
U is preferred. The reaction temperature is preferably 20 to 50 ° C., and the reaction time is preferably 2 to 24 hours. The reaction solution containing N-acetylglucosamine thus obtained is
After concentration, if necessary, it can be dried by a freeze dryer, a vacuum dryer, a spray drier or the like to be powdered.

In the above reaction, the filtrate component and the bacterial cell component may be immobilized on a suitable carrier and used. In addition, a filtrate component and a bacterial cell component can be simultaneously reacted with chitin to produce a chitin degradation product. Further, the resulting chitin degradation product is treated with a combination of separation and purification means such as activated carbon column chromatography, gel filtration, and high performance liquid chromatography to obtain highly purified di-N-acetyl chitobi. Oose and / or N-acetylglucosamine can also be obtained.

In the present invention, the chitinase activity of the culture filtrate,
The N-acetylglucosaminidase activity of the cells was measured as follows.

(1) Chitinase activity measurement 0.2 M acetate buffer at pH 5.2 containing 0.2% colloidal chitin
1 ml of a sample (culture filtrate) is added to 1 ml, and reacted at 50 ° C. for 20 minutes. Next, 3 ml of the Schar reagent is added to the reaction solution, and the mixture is heated in a boiling water bath for 15 minutes. After cooling, the impurities are removed by centrifugation, and the absorbance at 420 nm is measured. From the absorbance, the amount of reducing sugars generated is determined from a standard curve using N-acetylglucosamine. 1 μmo per minute
The amount of enzyme that produces 1 N-acetylglucosamine is defined as 1 unit (U) of enzyme activity.

(2) Measurement of N-acetylglucosaminidase (chitobiaase) activity 5 mM p-nitrophenyl-N-acetyl-β-D-
Glucosamide 0.2 ml and pH 7.0 0.1 M phosphate buffer 0.5 ml
And 0.3 ml of the sample (cell lysate) at 37 ° C,
After reacting for 2 minutes, the reaction is stopped by adding 2.0 ml of a 0.25 M sodium carbonate solution, and the absorbance at 405 nm is measured to quantify the released p-nitrophenol. 1 μmo per minute
The amount of enzyme that produces 1 p-nitrophenol is defined as 1 unit (U) of enzyme activity.

According to the present invention, a microorganism having a chitin-degrading property belonging to the genus Chromobacterium which has not been reported,
By culturing in a medium containing chitin and allowing a filtrate component and / or a cell component prepared from the culture to act on chitin, a chitin degradation product such as di-N-acetylchitobiose and N-acetylglucosamine is obtained. It can be produced in high yield.

The chitin hydrolyzate obtained in this way is produced under relatively mild reaction conditions using enzymes produced by microorganisms, and is safer for the human body than one decomposed by chemical methods. Yes, it can be freely used in the food field, pharmaceutical field, and the like, for example, as a growth factor for useful enteric bacteria, a sweetener, and the like.

"Example" Reference example (preparation of culture filtrate and cell crushed product) As a preculture, 10 ml of a 0.8% solution of Nutrient Broth (manufactured by Difco) was placed in a test tube, sterilized, and then subjected to chromobacterium sp. Inoculate one platinum loop with 41
Cultured for days.

Next, as the main culture, 2.5% of colloidal chitin, 0.1% of peptone, 0.05% of yeast extract, 0.1% of KH ₂ PO ₄ , MgSO ₄ .7H ₂
Liquid medium 1 containing 0.02% of O was placed in a 5-volume flask, inoculated with the preculture-completed solution, and cultured at 30 ° C. for 1 day with shaking.

The obtained culture solution 1 was centrifuged at 10,000 rpm for 10 minutes to separate a culture filtrate and cells.

The cells were suspended in 100 ml of water, 100 mg of egg white lysozyme was added, and the cells were treated with ice cooling for 1 hour. Next, the solution was treated with an ultrasonic crusher to completely crush the cells,
After adding 100 ml, the mixture was centrifuged at 12,000 rpm for 60 minutes to remove the residue, and the supernatant was used as a cell lysate.

Example 1 (Production of di-N-acetylchitobiose) The culture filtrate prepared in Reference Example was added to 200 g of a 10% colloidal chitin solution so that the chitinase activity became 200 U,
The reaction was performed at 50 ° C. for 12 hours.

After completion of the reaction, the composition of the reaction solution was confirmed by high performance liquid chromatography, and di-N-acetylchitobiose was detected. After concentrating this reaction solution, it was freeze-dried to obtain 11 g of di-N-acetylchitobiose.

The analysis conditions of the high performance liquid chromatography are as follows.

 Column: YMC-Pack PA-03 (4.6 x 250 mm) Mobile phase: acetonitrile: water = 72:28 Flow rate: 0.8 ml / min Temperature: 30 ° C Detection: UV215 nm The analysis results are shown in Fig. 1.

In FIG. 1, 1 indicates the elution position of N-acetylglucosamine, and 2 indicates the elution position of di-N-acetylchitobiose. FIG. 1 shows that the reaction solution obtained in Example 2 contains di-N-acetylchitobiose as a main component.

Also, 200 g of a 10% colloidal chitin solution, that is, chitin
From 20 g, 11 g of di-N-acetylchitobiose was obtained,
It can be seen that the yield is high.

Example 2 (Production of N-acetylglucomisan) Di-N-acetylchitobiose 10 obtained in Example 1
g was dissolved in 100 ml of water, and the cell lysate prepared in Reference Example was treated with 50 U as N-acetylglucosaminidase activity.
And reacted at 37 ° C. for 20 hours.

After completion of the reaction, the composition of the reaction solution was confirmed by high performance liquid chromatography in the same manner as in Example 1, and N-acetylglucosamine was detected.

After concentrating this reaction solution, it was freeze-dried to obtain 10 g of N-acetylglucosamine.

[Brief description of the drawings]

FIG. 1 is a view showing the result of analyzing the composition of a reaction solution obtained by allowing a culture filtrate to act on chitin by high performance liquid chromatography. In the figure, 1 indicates the elution position of N-acetylglucosamine, and 2 indicates the elution position of di-N-acetylchitobiose.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12N 1/20 C12R 1:01) (C12N 9/24 C12R 1:01) (72) Inventor Kazuo Sakai 5-2 Asahigaoka, Fujieda-shi, Shizuoka Prefecture (72) Inventor Ryosuke Katsumi 54 Nibancho, Shizuoka City, Shizuoka Prefecture (72) Inventor Masato Ishikawa 3-18-16, Otowacho, Fujieda City, Shizuoka Prefecture

Claims (3)

(57) [Claims] 1. Chromobacterium
A method for producing a chitin-decomposed product, comprising: culturing a microorganism belonging to the genus and having chitin-degrading ability in a medium containing chitin, and allowing a filtrate component and / or a cell component prepared from the culture to act on chitin. . 2. The microorganism according to claim 1, wherein said microorganism is Chromobacterium sp.
2. The method for producing a chitin hydrolyzate according to claim 1, which is (Chromobacterium sp. N-41). 3. The method for producing a chitin hydrolyzate according to claim 1, wherein the chitin hydrolyzate is di-N-acetylchitobiose and / or N-acetylglucosamine.