JPH04187094A - Production of chitin decomposition product - Google Patents

Abstract

PURPOSE:To obtain the title decomposition product useful as a growth factor of useful bacteria in intestine, a sweetener, etc., by culturing a specific bacterium in a medium containing chitin and treating chitin with a filtrate component, etc., prepared from the culture mixture. CONSTITUTION:A bacterium [preferably Chromobacterium sp. N-41 (FERM P-11,779)] belonging to the genus Chromobacterium, capable of decomposing chitin, is cultured in a chitin-containing medium chitin is treated with a filtrate component and/or a cell component prepared from the culture mixture to give the objective decomposition product (preferably di-N-acetylchitobiose or N- acetylglucosamine).

Description

Detailed Description of the Invention "Field of Industrial Application" The present invention relates to a method for producing a chitin decomposition product that can be used as a growth factor for intestinal beneficial bacteria, a sweetener, etc. This invention relates to a production method using a chitinolytic enzyme.

"Prior Art" Conventionally, chitin decomposition products such as di-N-acetylchitobiose and N-acetylglucosamine have been mainly produced by chemical methods. For example, di-N-acetylchitobiose is made from crab and shrimp shells with concentrated hydrochloric acid.
It is prepared by hydrolyzing it at about 40°C for several hours, followed by separation and purification using activated carbon column chromatography. In addition, N-acetylglucosamine is
It is prepared by chemical synthesis by hydrolyzing the shells of crabs and shrimps with concentrated hydrochloric acid at about 100°C for about 1 hour, and then N-acetylating the obtained D-glucosamine hydrochloride with acetic anhydride. There is.

[Problems to be Solved by the Invention] However, in the method for producing chitin decomposition products by acid hydrolysis, a strong acid such as concentrated hydrochloric acid must be used, and special equipment is required because the reaction is carried out at high temperatures. and energy costs are high.

In addition, in the case of acid hydrolysis, chitin decomposition products are obtained as a mixture of monomers and various chitin oligosaccharides;
For example, to obtain di-N-acetylchitobiose as a single compound, it must be purified by column chromatography, and the yield is approximately 1% based on the chitin raw material.
It is about 0%, which is low.

Furthermore, when acid hydrolysis is performed, deacetylation occurs simultaneously with the decomposition of glucoside bonds, so in order to obtain N-acetylglucosamine, an N-acetylation reaction must be performed after hydrolysis. N produced in this way
-Acetylglucosamine is currently not used as a food material because it is a chemically synthesized product.

For this reason, various methods have been reported that utilize chitin-degrading enzymes produced by microorganisms as a method for easily producing chitin decomposition products that can be used as food materials in good yields without relying on chemical methods. Microorganisms that decompose chitin include Serratia [5erracial genus (Monreal
, J et al., 1985), Vibrio fVibriol
Genus (Naimaen et al., 1979), Pseudomonas iPseu
Domonasl (Takiguchi et al., 1985), Aniromonas (Aeroa+onasl (Yabuki et al., 1983)), Bacillus (Takiguchi et al., 1983),
1989) etc.6 The purpose of the present invention is to search for a microorganism in nature that has a chitin-degrading ability that has not been reported so far, and to utilize the chitin-degrading enzyme produced by this microorganism. An object of the present invention is to provide a method for producing a chitin decomposition product with good yield that can be used as a food material.

"Means for Solving the Problems" In line with the above objectives, the present inventors searched various samples for bacterial strains that efficiently decompose chitin, and as a result, they isolated Chromobacterium from soil.
A specific microorganism belonging to the genus Teriuml has an excellent ability to decompose chitin, and when its culture filtrate is applied to chitin, di-N-acetylchitobiose is produced at a high yield. -We have discovered that N-acetylglucosamine is produced when acetylchitobiose is reacted with, and have completed the present invention.

That is, the method for producing a chitin decomposition product of the present invention is as follows.

Chromobacterium fchromobacterium
The method is characterized in that a microorganism belonging to the genus I and having the ability to decompose chitin is cultured in a medium containing chitin, and a filtrate component and/or bacterial cell component prepared from this culture is allowed to act on chitin.

Hereinafter, the present invention will be described in more detail by citing preferred embodiments.

Chromobacterium
As a microorganism that belongs to the genus I and has the ability to decompose chitin, Chromobacterium sp., which the present inventors isolated from soil,
, N-41 (Chromobacterium Sp.
, N-411 are preferably used. The mycological properties of this bacterium are as follows.

fa) Morphology (11 Cell shape: Cancer fungus (2) Cell size: 0.55X 1.4 μm (3)
Motility: Yes (4) Flagella: Very single hair (5) Durham staining, negative (BL growth condition (11) Flesh agar plate culture The periphery is transparent and the center is white, flat and smooth, slightly glossy 1 Does not produce pigment .

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

(3) Meat juice liquid culture 0 No surface growth, whole medium becomes cloudy.

(4) Meat juice gelatin puncture culture: gelatin is not liquefied.

puncture hole grow along Ru.

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

fcl Physiological properties il + nitrate reduction, positive (2) Denitrification reaction Negative i31MR reaction: Positive +41VP test: Negative (5) Indole formation: Negative (6) Hydrogen sulfide formation: Negative (7) Starch hydrolysis: Positive (8) Utilization 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℃ (15) pH range of growth = 5.0-9.0 (16) Attitude towards oxygen Binary anaerobic (17) Presence or absence of acid and gas production from sugars (saccharides) (acid (Production of acid) (Production of gas) L-arabinose -- 〇-Xylose - -D-Glucose 10 - (Saccharide) (Production of acid) (Production of gas) Romannose + -D-
Fructose + - D-galactose - - Maltose - Sucrose - Lactose - Trehalose - - Sorbitol - D-Mannit - Inosit - - Glycerin - Starch - FDL Other properties Oxidation of gluconic acid Degradation of negative esculin :Negative arginine decomposition゛Negative lysine decarboxylation reaction:Negative ornithine decarboxylation reaction:Negative phenylalanine deamination reaction:Negative sodium chloride resistance, potassium cyanide resistance that does not grow in broth medium containing 6% sodium chloride:Negative phosphatase : Negative pectinase - Negative lecithinase/Negative gelatin liquefaction, Negative casein decomposition: Negative DNase: Negative lactic acid oxidation/Positive hydrogen cyanide generation: Negative vibriostatic reagent 2.4-diamino-6,7-
Reactivity/negative for ditubrovir buteridine fO/1291 GC content: 63.1 mo1% Water spores are facultative anaerobic rods that are motile with extremely single hairs, and positive for oxidase and catalase. Acid is produced from glucose through fermentation.

When searching for such properties using the Purge Aids Manual Systematic Biotechnology (19841), the genera Vibrio, Aeromonas, Plesiomonas, and Chromobacterium fall under the category.

However, when comparing other properties with these genera, the genus Vibrio differs from the genus Vibrio in that it does not tolerate 6% sodium chloride and is not sensitized to the vibriostatic reagent (0/129). In addition, Aeromonas produces indole, produces gas from D-glucose, and produces D-glucose.
The difference is that mannitol fermentation and DNase are negative. Furthermore, it differs from the genus Plesiomonas in that it is not sensitized to vibriostatic reagents 10/129) and does not produce indole.

Furthermore, when searching, water locks have, in addition to the above properties, p
Cannot grow below H5, oxidizes lactic acid to produce CO□, reduces nitrate, assimilates citric acid, GC
It has properties such as a content of 63.1 mo1%, so it is similar to the genus Chromobacterium. The water lock is
Although it does not produce the purple pigment characteristic of the genus Chromobacterium, the Purge Aids Manual Systematic Biotechnology (1984) states that there are non-pigment producing bacteria within the genus Chromobacterium. has been done. For these reasons, the water droplet was identified as a bacterium belonging to the genus Chromobacterium.

Next, when considering the species, water locks were negative for the production of purple pigment, production of hydrogen cyanide, liquefaction of gelatin, egg yolk reaction, decomposition of casein, decomposition of arginine, and production of acid from trehalose and starch. Chromobacterium violaceum is different from Chromobacterium violaceum because the MR test is positive, and Chromobacterium violaceum does not produce pigment, does not grow at 4°C, grows above 30°C, and has a different GC content. It is also different from Bacterium Flaviatile. Therefore, it was determined that Chromobacterium is a new species belonging to the genus Chromobacterium, and the present inventors identified this strain as Chromobacterium sp, N-41 (Chro+mobacterium
It was named teriuw+ sp, N-411. This strain has been deposited with the National Institute of Microbial Technology, Agency of Industrial Science and Technology, as Fiber Science and Technology Research Institute No. 11779.

As mentioned above, the microorganisms that have the ability to degrade chitin that have been reported so far include the genus Serratia f.
Vibrio fVibriol, Pseudomonas iPs
eudomonasl genus, Aeromonas fAero+n
Onasl genus, Bacillus fBacillus genus, etc., but there are no reports on microorganisms having chitin-degrading ability belonging to the Chromobacterium genus.

Isolation of this bacterial strain was performed as follows. That is,
Soil samples were mixed with 1.0% colloidal chitin and 0.1% H
, PO4 and 002% MgSO4・7H20, and cultured with shaking at 30°C for 24 hours.
For those in which the bacteria grew, they were spread on a plate medium containing the same medium as above with 2% agar added, cultured at 30°C for 72 hours, and colonies with large transparent rings formed by dissolving colloidal chitin were collected. Furthermore, culturing was repeated using the same medium to purify the target microorganism.

The microorganisms used in the present invention are not limited to those isolated by the above method, but any microorganisms that belong to the genus Chromobacterium and have the ability to decompose chitin can be isolated from nature, strains preserved at depository institutions, or strains with the ability to decompose chitin. Any strain may be used, such as a strain that has been mutated to increase

Next, the method for culturing the microorganism will be described. As the medium, a liquid medium containing chitin and other nutrient sources is preferably employed. Colloidal chitin is preferably used as chitin.

Colloidal chitin is produced by treating the shells of shrimp and crabs with hydrochloric acid or alkali, and dissolving the resulting crude chitin in cold hydrochloric acid.
It can be prepared by dispersing it in a large amount of water. The concentration of colloidal chitin in the medium is 11 to 1
Other nutrient sources, which are preferably 0.0%, include nitrogen sources, inorganic salts, and the like. As a nitrogen source, it is preferable to add peptone in an amount of 0.01 to 0.5%, and further add yeast extract in an amount of 0.01 to 0.5%. It is preferable to add 1 to 0.5% of O. Furthermore, the inorganic salts include KH, PO,...
It is preferable to add Mg5044HJ, and CaC1z may be added if necessary. The pH of the medium is 6-8
It is preferable to adjust to The culture is preferably carried out by shaking culture or 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 bacterial cells by filtration, centrifugation, or the like. In the present invention, this filtrate and bacterial cells can be used as crude enzymes in the chitin decomposition reaction as they are. However, the filtrate can be further purified by dialysis, salting out, ultrafiltration, etc. before use. Furthermore, it is preferable to use cells that have been lysed by a lytic enzyme such as lysozyme or crushed using an ultrasonic crusher or the like. In the filtrate, chitinase activity that decomposes chitin to produce di-N-acetylchitobiose was observed, and in the bacterial cells, di-N-acetylchitobiose was decomposed to produce N.
- N-acetylglucosaminidase activity (chitinase activity) that produces acetylglucosamine is observed. In the present invention, it is also possible to use enzymes that have been purified by separating each active fraction from the above-mentioned filtrate and/or bacterial cells using known purification means. Therefore, the filtrate component and/or bacterial cell component in the present invention may be the above-mentioned filtrate and/or bacterial cells, crude enzyme prepared from the filtrate and/or bacterial cells, or even filtrate and/or bacterial cells. It may also be a pure enzyme separated and purified from.

Next, to explain the decomposition reaction of chitin, when di-N-acetyl chitobiose is produced by reacting the above-mentioned filtrate components on chitin, colloidal chitin is preferably used as the substrate, and the concentration of colloidal chitin is 10 to 10. l010
% is preferred. The chitinase activity in the reaction solution is 1g of substrate.
It is preferable to set it as 1-20U. The reaction conditions are preferably pH 4 to 8 and temperature 30 to 60°C for 2 to 24 hours. Di-N-acetylchitobiose is produced in the reaction solution, and if necessary, it can be concentrated and then powdered using a freeze dryer, vacuum dryer, spray dryer, or the like.

When the above bacterial cell components are allowed to act on the di-N-acetylchitobiose thus obtained, N-acetylglucosamine can be produced. In this case, the substrate di-N
-The concentration of acetyl chitobiose is preferably 1.0 to 20%, and the chitinase activity in the reaction solution is 1 to 1 per gram of substrate.
1 reaction temperature is preferably 20 to 50°C
The reaction time is preferably 2 to 24 hours.

The reaction solution containing N-acetylglucosamine thus obtained can be concentrated, if necessary, and then dried and powdered using a freeze dryer, a vacuum dryer, a spray dryer, or the like.

In the above reaction, the filtrate components and bacterial cell components can also be used after being immobilized on a suitable carrier. Furthermore, a chitin decomposition product can also be produced by simultaneously reacting chitin with a filtrate component and a bacterial cell component. Furthermore, the obtained chitin decomposition product is subjected to, for example, activated carbon column chromatography, gel filtration,
Di-N is purified to a high degree of purity through a combination of separation and purification methods such as high-performance liquid chromatography.
-Acetylchitobiose and/or N-acetylglucosamine can also be obtained.

In the present invention, the chitinase activity of the culture filtrate and the N-acetylglucosaminidase activity of the bacterial cells were measured as follows.

fil Chitinase activity measurement 0.2M containing 0.2% colloidal chitin, pH 5.2
Add 1 ml of the sample (culture filtrate) to acetate buffer 1IiI2 and react at 50°C for 20 minutes. Next, 3 mβ of Schall's reagent was added to the reaction solution, and the mixture was heated in a boiling bath for 1
Heat for 5 minutes. After cooling, it is centrifuged to remove impurities, and the absorbance at 420 r+m is measured. From this absorbance,
The amount of reducing sugar produced is determined from a standard curve using N-acetylglucosamine. The amount of enzyme that produces Iua+ol of N-acetylglucosamine per minute is defined as 1 unit (U) of enzyme activity.

(2) Activity measurement of N-acetylglucosaminidase (chitobiose) 5IIIM p-nitrophenyl-N-acetyl-β-
D-gelcosamide 0.2III2 and 0 at pH 7.0
．． Mix 1M phosphate buffer 0.5111ε and sample (disintegrated cell solution) 0.3+12, react at 37°C for 10 minutes, and then add 0.25M sodium carbonate solution 2.011E.
is added to stop the reaction, and the absorbance at 405Em is measured to quantify liberated p-nitrophenol. The amount of enzyme that produces 1 μmol of p-nitrophenol per minute is defined as 1 unit (U) of enzyme activity.

"Action/Effect" According to the present invention, a microorganism that has the ability to decompose chitin belonging to the genus Chromobacterium, which has not been reported so far, is cultured in a medium containing chitin, and a filtrate component and/or Alternatively, chitin decomposition products such as di-N-acetylchitobiose and N-acetylglucosamine can be produced in high yield by allowing fungal cell components to act on chitin.

The chitin decomposition products obtained in this way are manufactured under relatively mild reaction conditions using enzymes produced by microorganisms, so they are safer for the human body than those decomposed by chemical methods. It can be freely used in the food and pharmaceutical fields, for example, as a growth factor for beneficial intestinal bacteria and as a sweetener.

"Example" Reference example (preparation of culture filtrate and crushed bacterial cells) As a preculture, a 0.8% solution of Nutriendo broth (manufactured by Difco) was placed in a test tube at 10 m℃, and after sterilization, Chromobacterium sp. , N-41 was seeded with one platinum lug and heated at 30°C.
It was cultured for 1 day.

Next, as a main culture, colloidal chitin 2.5%, peptone 0.1%, yeast extract 0.05%, KH, PO40,
A liquid medium 1β containing 1% Mg5O, .7H200.02% was placed in a 52-volume flask, inoculated with the pre-culturing solution, and cultured with shaking at 30° C. for 1 day.

Obtained culture medium II solution 1! , IO, [l[10 rotations,
The mixture was centrifuged for 10 minutes to separate the culture filtrate and the bacterial cells.

The bacterial cells were suspended in water at 100 m℃ and added to egg white lysozyme at 100 mC.
mg was added and treated for 1 hour while cooling with water.

Next, this solution was treated with an ultrasonic crusher to completely crush the bacterial cells, and then 100 mE of water was added and 12, (li
) (1 rotation for 60 minutes to remove the residue, and the supernatant was used as a bacterial cell disruption solution.

Example 1 (Production of di-N-acetyl chitobiose) To 200 g of a 10% colloidal chitin solution, 200 U of the culture filtrate prepared in Reference Example was added with chitinase activity, and the mixture was reacted at 50°C for 12 hours. I let it happen.

After the reaction was completed, 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 for high performance liquid chromatography are as follows.

Column: YMC-Pack PA-0314, 6X
250 mml mobile phase acetonitrile, water = 72:28
Flow rate = 08 mβ/min Temperature 30°C Detection: UV 215r++n The analysis results are shown in Figure 1.6 In Figure 1, 1 indicates the elution position of N-acetylglucosamine, and 2 indicates the elution position of di-N-acetylchitobiose. The elution position is shown. From FIG. 1, it can be seen that the reaction solution obtained in Example 2 contains di-N-acetylchitobiose as a main component.

In addition, from 200 g of 10% colloidal chitin solution, that is, 20 g of chitin, 11 g of di-N-acetyl chitobiose.
was obtained, indicating a high yield.

Example 2 (Production of N-acetylglucosamine) lOg of di-N-acetylchitobiose obtained in Example 1,
After dissolving in 100 mβ of water, the cell suspension prepared in the reference example was added to
After the reaction was completed, 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. Ta.

After concentrating this reaction solution, it was freeze-dried to obtain an N-acetylglucosamine log.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the results of analyzing the composition of a reaction solution obtained by reacting chitin with a culture filtrate 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.

Claims (3)

[Claims] (1) Chromobacterium
A chitin decomposition product characterized by culturing a microorganism belonging to the genus Ium) and having the ability to decompose chitin in a medium containing chitin, and allowing a filtrate component and/or bacterial cell component prepared from this culture to act on chitin. Manufacturing method. (2) The microorganism is Chromobacterium sp. N-41
(Chromobacterium sp.N-41)
The method for producing a chitin decomposition product according to claim 1. (3) Claim 1, wherein the chitin decomposition product is di-N-acetylchitobiose and/or N-acetylglucosamine.
Or the method for producing a chitin decomposition product according to 2.