JPS62198387A - Microorganism capable of producing chitinase and production of said chitinase and chitin hydrolyzate using said chitinase - Google Patents

Abstract

PURPOSE:To efficiently produce a chitin hydrolyzate, by cultivating a microorganism, belonging to the genus Streptomyces and capable of producing chitinase, collecting the chitinase and reacting the collected chitinase with raw material chitin. CONSTITUTION:A microorganism, e.g. any one of Streptomyces sp. KE406 (FERM-P No.8642), Streptomyces sp. KE-902 (FERM-P No.8643) and Streptomyces sp. KE-3332 (FERM-P No.8644), belonging to the genus Streptomyces and capable of producing chitinase is cultivated. The resultant chitinase is then collected from the culture or chitin is added to the culture fluid, etc., to adsorb the chitinase and the chitin adsorbing the above-mentioned chitinase is then added to raw material chitin to produce the aimed chitin hydrolyzate. In the reaction, the chitin hydrolyzate is concentrated using a membrane at the same time with or after the reaction.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a novel microorganism that produces a chitin-degrading enzyme, a method for producing a chitin-degrading enzyme using the microorganism, and a method for producing a chitin-degrading product using the enzyme. Regarding person D.

(Prior Art) Chitin and its derivatives have been studied for use in various fields such as films, fibers, medical materials, cosmetic materials, enzyme carriers, and flocculants, and have been put into practical use in some cases.

However, there is almost no research on the use of N-7 cetylglucosamine or chitooligosaccharides, which are decomposition products of chitin.

Therefore, it is hoped that an efficient method for producing such chitin decomposition products can be established.

[Problems to be solved by the invention] Actinomycetes such as Streptomyces, filamentous fungi such as Aspergillus, and bacteria such as Pseudomonas and Vibrio have been known as chitin-degrading enzyme-producing bacteria. For industrial production of products, we search for and use microorganisms that produce unique highly active chitin-degrading enzymes, which are different from conventional microorganisms. Ik, production of the enzyme υ, and development of a method for producing a chitin decomposition product using the enzyme are important.

Means for Solving Problem L] Therefore, the present inventors have conducted extensive research to search for microorganisms that produce enzymes that can decompose chitin and efficiently produce N-acetylglucosamine and chitooligosaccharides. the result,
We have discovered that a chitin-degrading enzyme can be obtained by culturing microorganisms belonging to the genus Streptomyces. Furthermore, it has been found that by using such IIP [4], it is possible to efficiently produce chitin decomposition products.
The present invention was completed based on this knowledge.

``The present invention will be described below.

(1) A microorganism that belongs to the genus Streptomyces and produces chitin-degrading enzyme JS.

(2) A method for producing a chitin-degrading enzyme, which comprises culturing a microorganism belonging to the genus Streptomyces and producing a chitin-degrading enzyme in a medium, planting the chitin-degrading enzyme in a culture medium, and collecting the enzyme from the culture. .

(3) Add chitin to the culture solution or culture fJj solution of a microorganism that belongs to the genus Streptomyces and produces chitin-degrading enzyme to adsorb the chitin-degrading enzyme, and add the chitin-adsorbed chitin to raw chitin to react. A method for producing a chitin decomposition product characterized by:

(4) A method for producing a decomposed chitin product, which comprises causing a chitin degrading enzyme to act on chitin and performing e-condensation using a membrane simultaneously with or after the reaction.

The mycological properties of the chitin-degrading enzyme-producing bacteria belonging to the genus Streptomyces that the present inventors isolated from soil are shown below.

1. Morphological properties 0A-Y agar IIIt plate medium (Kutsker White Auto 2
0g, yeast 11 extract Ig, agar 18g, pi (7,
0) To k! The morphology of each strain grown for 0.011 hours is shown below.

3. No. 406 due to mycological properties that are more than physiological properties. Strains No. 902, No. 3332 are classified into the genus Streptomyces. Bacterial strains related to each strain are listed in "Birge's Manual of Determinative Bacteriology,"
: JSa edition (1972) J and International Journal of Systematic Bacteriology, 18a, pp. 69-189 and 279-38.
2 (1988), 193, 391-512
Page (19H) and Volume 22, Nos. 265-334.
According to "Page (1972)", it is as follows.

No. 408 related bacteria: Streptomyces antimuticus, Streptomyces malachiticus.

Streptomyces viridobuiastateicus.

Streptomyces subvarsogenes, Streptomyces fasciculatus, Streptomyces toyorikiensis, Streptomyces viridiviolaceus No. 902*tM bacteria: Streptomyces parvarus.

Streptomyces ampofaciens, Streptomyces pricarus, Streptomyces rotchei No. 3332 Related bacteria: Streptomyces albofulare, F bus, Streptomyces aerocolonita, Streptomyces mutabilis and higher related bacteria have culture properties, physiological Since there are some differences from each of the above-mentioned three strains used in the present invention in terms of properties, ability to assimilate carbon sources, etc., the present inventors recognize that all of the above-mentioned strains are new strains. sp, KE-4
They were named 08, KE-902 and 4KE-3332. All of these strains have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and their accession numbers are as listed below.

Streptomyces sp, KE-408: FERN P
-8θ42// KE-902: FERN
P-8643/l KE-3332: F
ERN P-8644 Note that even the above-mentioned related bacteria may produce chitinolytic enzymes depending on the culture conditions.

The desired chitinolytic enzyme can be produced by inoculating the novel chitinolytic enzyme producing bacterium of the present invention into a nutrient medium and culturing it. Here, the medium may be any medium as long as it contains chitin, which is the enzyme-inducing substrate, and nutrients that can be used by the microorganism, such as colloidal chitin sp.
0%, glucose sp. 0%, urea 0.2%, potassium dihydrogen phosphate sp. 0%! Magnesium acid 7 Wood salt 0.
A medium (pH 7.0) containing 0.05% yeast extract, 0.1% yeast extract and 0.5% constituency extract.

Shaking culture is suitable as a culture method, and the culture temperature is 30°C.
The number of days for culturing is preferably 37°C. As shown in Figure 1, the activity gradually increases until the 3rd day of culture, but after that it tends to decrease. Te1”
Culture conditions should be selected to maximize production of the target enzyme.

The culture solution itself obtained as described above or the supernatant obtained by separating the culture solution from the culture solution by a conventional method of centrifugation can be used as a crude enzyme solution, but if necessary, known methods can be applied. In addition, it is also possible to use a culture solution or supernatant in which colloidal chitin is added to adsorb the enzyme.In other words, chitin-degrading enzymes have the property of being adsorbed to colloidal chitin. Because there is
Colloidal chitin can be added to a culture solution to adsorb the enzyme, and this enzyme can be directly added to the raw material chitin to cause a decomposition reaction. Furthermore, it is also possible to use known chitinolytic enzymes alone or in combination with the enzyme according to the present invention.

When colloidal chitin is added to the culture solution in the method using the adsorbed enzyme described above, there are advantages in that contamination of impurities from the culture solution is suppressed and enzyme activity is stably maintained. However, it is also possible to use a mixture of bacterial cells and adsorbed enzymes obtained by adding colloidal chitin to the culture solution itself.
.

Next, as a raw material for producing a chitin decomposition product, any material containing chitin may be used, and suitable raw materials include crab, shrimp, krill, and the like. These raw materials can be used as is, but for example, they can be treated with acid! Colloidal chitin, acid added and then extrusion/cutting (high pressure, high temperature treatment), raw materials immersed in salt to demineralize, etc. The raw material may be processed and used as described above, but it is particularly preferable to use the raw material which has been deashed as described above and then treated at high pressure and high temperature. In this case, the I%I concentration should be increased and the packing conditions should be set to a higher pressure.
By raising the temperature to a high temperature, a product with a high content of N-7 cetylglucosamine and chitooligosaccharide can be obtained,
Depending on the purpose, this material can be used as a food material by simply undergoing neutralization treatment.

The reaction conditions for obtaining a chitin decomposition product can be set, for example, by adjusting the amount of enzyme, so that the reaction time i can be set in the range of 1 hour to several weeks. A temperature of about 41°C is appropriate. Practical reaction time is about 12 to 48 hours. For example, use 5% colloidal chitin aqueous solution 1 ra (!, 0.2 mol citrate phosphate buffer (pH 5,5) 11p and enzyme solution (separated from the culture medium) 1 mA+ and react at 37°C for 12 hours. By this, chitin decomposition products can be obtained efficiently.

The main components of the chitin decomposition product vary depending on the type of fermentation used, reaction conditions, etc. For example, Streptomyces s.
The enzyme derived from the KE-4013 strain Yamamai yields N-7 cetylglucosamine, while the enzyme derived from Streptomyces sp, KE-920, and KE-3332 yields products whose main component is chitooligosaccharides such as chitobiose. . It is also possible to obtain a decomposition product containing N-7 cetylglucosamine or chitooligosaccharide alone, or a decomposition product containing both in an appropriate ratio.

After completion of the reaction or during the reaction, the chitin decomposition product can be collected by a conventional method. For example, the reaction solution containing N-7 cetylglucosamine and chitooligosaccharide can be taken out and concentrated using a reverse osmosis membrane to produce a product. Furthermore, simultaneously with the reaction, the chitin decomposition product can be taken out from the ultra-1 filtration membrane and concentrated through the membrane to produce a product. In this case, a dynamic membrane can be used as a model, and if an acid-resistant membrane or an exclusion type membrane is used, the production of chitin decomposition products by acid decomposition will also be effective.

[Advantageous Effects of the Invention 1] According to the present invention, highly active chitin-degrading enzyme-producing microorganisms are provided, and chitin-degrading enzymes can be efficiently produced using the microorganisms. Furthermore, various aspects of the method for producing chitin decomposition products using the enzyme have been developed, and it is expected that N-7 cetylglucosamine and chitooligosaccharides, which are chitin decomposition products, will be used for various purposes. For example, this chitin decomposition product can be used alone as a food material, such as a 1F flavoring agent, or it can also be used in combination with sugar, maltooligosaccharide (as a base or a mixture), and the like. 1. When the chitin decomposition product is blended with sugar.

Former: *Former = 1: 10 to 1-9: Approximately 1 can be mixed.

In addition, this food material can also be used to improve the physical properties of foods, such as increasing the body feel.

In this case, use:,:: should be added at a ratio of about 1 to 5% to the food. Note that [r] is 80 to 70 N-7 cetylglucosamine when sugar is 100, It has a mild refreshing feeling and has a chitobiose content of about 30%. Furthermore, the quality of each of these 11 tastes is superior to that of glucosamine.

Furthermore, this food material suppresses the growth of harmful bacteria such as Clostridium, which is an intestinal bacterium, and promotes the growth of useful bacteria such as Bifidobacterium, and is non-digestible.

Since various physiological activities have been observed, such as preventing IE from cholesterol accumulation, it is expected to be used as foods, special foods, and pharmaceutical products. It may also have antibacterial effects. In addition, when used to suppress the growth of harmful bacteria, 0-1 (
M G! The effect can be achieved by using the food material at a ratio of about 1.

The enzyme-adsorbed chitin described above may also be used as feed for livestock; for example, it can be added to the feed of chickens, pigs, cows, etc. to increase the production of eggs and meat.

[Example] Next, examples of the present invention will be shown.

Example 1 Streptomyces rp, KE-408 (FERN P-
8642) on Bennett agar (1% glucose, 0.8642).
1% meat extract, 0.1% fermented IIJ extract, 0.2% NZ
Amine.

sp. Inoculate 5-2.0% agar, PI(7,0),
After culturing for 5 to 7 days at 37°C, take the 1 platinum sample and add it to sp. 0% colloidal chitin, sp. 0% glucose, 0.2% urea, sp. 0% potassium dihydrogen phosphate.

0.05% magnesium sulfate heptahydrate, 061% PPJr
t extract, O65 corn staple liquor, pH 7,
The cells were transferred to a liquid medium (80μR medium 150Gmf Erlenmeyer flask) with a composition of 0.0 and cultured with aeration and shaking at 37°C for 3 hours. After the culture was completed, the cells and insoluble matter in the culture solution were removed by centrifugation or dilution. It was removed by Ji filtration to obtain a 1: clear solution, which was used as crude enzyme.

Enzyme activity was measured by the following method.

2% colloidal chitin aqueous solution sp. 0m1J, 0.
2 M citrate phosphate buffer (P)I 5.5)
sp. 0 rin! , fermentation solution (culture solution) sp. 0ml) was mixed and reacted at 37℃ for 3 hours with gentle shaking.After the 0 reaction route, the mixture was heated at 100℃ for 5 minutes to increase the reaction by +1.Next, centrifugation was performed. Regarding the obtained Nisumi part, N-7 cetylglucosamine H and H were measured by the Morgan-Nilson method to determine the colloidal chitin saccharification activity.

In addition, for the precipitate, add 1% SDS 3.0, stir and mix thoroughly to disperse the precipitate, and then add the wavelength H
, 0nII, the degree of p:J was set at fl+1, the angle decrease rate was calculated, and this was taken as the colloidal chitin liquefaction activity. The results are shown in Figure 1.

Example 2 Colloidal chitin was added to the ji';a filtrate obtained in Example 1, stirred at low temperature overnight, filtered through lji, washed with cold water,
The obtained enzyme preparation was applied to colloidal chitin to obtain a chitin decomposition product containing 50% or more of N-7 cetylglucosamine.

Example 3 The reaction of Example 2 was carried out in an ultrafiltration model 1 (UF5000) container, and the reaction product N-acetylglucosamine that had passed through the ultra7 filtration membrane was concentrated using a reverse osmosis membrane to obtain pure 1 piece 92 %
An N-acetylglucosamine product was obtained.

Example 4 Crab and shrimp shells were immersed in 2N hydrochloric acid at room temperature for 2 days, drained, and extruded with an extruder at 150 to 200°C to swell the chitin. 406 (FERMP-864
2) Chitin decomposition lvl derived from! A chitin decomposition product containing 30% or more of N-acetylglucosamine was obtained by the action of JG.

Example 5 The product obtained in Example 3 was added to sugar in a tr (rli ratio of 1:
A soup seasoning agent was prepared by mixing the ingredients in a ratio of 1/1. This IT flavoring agent can be used in food and drink products such as juice, coffee, lactic acid drinks, cakes, and candies.

[Brief explanation of drawings]

Fig. 1 is a graph showing the culture progress of chitinolytic enzyme production, and Fig. 2 is a graph showing the culture progress of the chitinolytic enzyme production fraction.
Fig. 3 is a scanning electron micrograph of the same strain (x to, ooo); Fig. 4 is a photomicrograph of Streptomyces sp, KE-902 and KE-902.
Optical micrograph of E-3332 (X600)', No. 5
The figure is a scanning electron hjtJ micrograph (X4,0
00). 1fI:'=; Title of the invention: Bacterium producing chitin-degrading enzyme, method for producing the enzyme, and method for producing a chitin-decomposed product using the enzyme 3. Relationship with the case of the person making the amendment Patent applicant: Director, Food Research Institute, Ministry of Agriculture, Forestry and Fisheries Mitsui Sugar Co., Ltd. 4. Agent Address: 1-1-10-5 Kyobashi, Chuo-ku, Tokyo 104 Date of amendment order: March 310, 1985 April 22, 1988 (Shipping port) 6. Drawings of the specification subject to amendment Brief explanation column, Drawing 7, and contents of amendment (11 Specification cylinder page 19, lines 12 to 18, “Figure 1 is...
・ ...is. ” is corrected as follows. ``Figure 1 is a graph showing the culture progress of chitinolytic enzyme-producing bacteria, Figure 2 is an optical micrograph (X 600) of Streptomyces sp, KE-406 (organism form), and Figure 3 is a graph of the same strain (organism Scanning electron micrograph (XI
O,000), Figure 4 shows Streptomyces sp.
-902 (biological form) and E-3332 (biological form) at an optical microscope (x600), and Figure 5 is a scanning electron micrograph (x4,
000). (2) Correct Figure 2 as shown in the attached sheet (color photo changed to black and white photo). (that's all)

Claims (7)

[Claims] (1) A microorganism that belongs to the genus Streptomyces and produces chitin-degrading enzymes. (2) A microorganism that belongs to the genus Streptomyces and produces a chitinolytic enzyme is Streptomyces sp. KE-406
(FERM P-8642), Streptomyces sp.
KE-902 (FERM P-8643) and Streptomyces sp. KE-3332 (FERM P-86
44) The microorganism according to claim 1, which is any one of 44). (3) A method for producing a chitin-degrading enzyme, which comprises culturing a microorganism belonging to the genus Streptomyces and producing a chitin-degrading enzyme in a medium, accumulating the chitin-degrading enzyme, and collecting the enzyme from the culture. (4) A microorganism that belongs to the genus Streptomyces and produces a chitinolytic enzyme is Streptomyces sp. KE-406
(FERM P-8642), Streptomyces sp.
KE-902 (FERM P-8643) and Streptomyces sp. KE-3332 (FERM P-86
44) The manufacturing method according to claim 3, which is any one of 44). (5) Adding chitin to the culture solution or culture filtrate of a microorganism that belongs to the genus Streptomyces and producing a chitin-degrading enzyme to adsorb the chitin-degrading enzyme, and adding the chitin-degrading enzyme-adsorbed chitin to the raw chitin to react. A method for producing a chitin decomposition product characterized by: (6) A microorganism that belongs to the genus Streptomyces and produces a chitinolytic enzyme is Streptomyces sp. KE-406
(FERM P-8642), Streptomyces sp.
KE-902 (FERM P-8643) and Streptomyces sp. KE-3332 (FERM P-86
44) The manufacturing method according to claim 5, which is any one of 44). (7) A method for producing a decomposed chitin product, which comprises allowing a chitin degrading enzyme to act on chitin and concentrating it using a membrane at the same time or after the reaction.