JPH0632605B2 - Chitinolytic enzyme producing bacteria - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel microorganism that produces a chitinolytic enzyme.

[Prior Art] Utilization of chitin and its derivatives in various fields such as films, fibers, medical materials, cosmetic materials, enzyme carriers, and aggregating materials has been studied, and some have been put to practical use.

However, there are almost no studies on the use of N-acetylglucosamine and chitooligosaccharide, which are decomposition products of chitin.

Therefore, establishment of an efficient production method of such a chitin degradation product is expected.

[Problems to be Solved by the Invention] As conventional chitin-degrading enzyme-producing bacteria, actinomycetes such as Streptomyces, filamentous fungi such as Aspergillus, and bacteria such as Pseudomonas and Vibrio have been known. For the industrial production of a product, it is necessary to search for a unique high-activity chitin-degrading enzyme-producing bacterium different from conventional microorganisms, and to develop a method for producing the enzyme using the microorganism and a method for producing a chitin-degrading product using the enzyme. is necessary.

[Means for Solving Problems] Therefore, the inventors of the present invention conducted extensive research to search for a microorganism that produces an enzyme that decomposes chitin and efficiently produces N-acetylglucosamine and chitooligosaccharide. as a result,
It was found that the desired chitinolytic enzyme can be obtained by culturing a microorganism belonging to the genus Streptomyces. Furthermore, they have also found that a chitin degradation product can be efficiently produced by using such an enzyme. The present invention has been completed based on such findings.

The present invention is shown below.

Streptomyces tsukubaensis or Streptomyces cobaciensis, which is a chitin-degrading enzyme-producing bacterium belonging to the genus Streptomyces.

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

1. Morphological Properties The morphology of each strain grown on OA-Y agar plate medium (Cooker white oat 20 g, yeast extract 1 g, agar 18 g, pH 7.0) for 10 days is shown below.

2. Growth condition on various media 3. Physiological properties Based on the above mycological properties, the strains No. 406, No. 902 and No. 3332 are classified into the genus Streptomyces. Strains that are closely related to each strain are "Virgin's Manual of Definite Native Bacteriology, 8th Edition (1972)" and "International Journal of Systematic Bacteriology, Vol. 18, 69-189. Pp. 279-392 (1968), Vol. 19, 391-512 (1969), and Vol. 22, 265-394 (1972).
Year) ”is as follows.

No.406 Related bacteria: Streptomyces antimuticus, Streptomyces malachiticus, Streptomyces viridodiastaticus, Streptomyces
Sparsogenes, Streptomyces fasiculatus,
Streptomyces toyocaensis, Streptomyces viridiviolaceus No. 902 related bacteria: Streptomyces parvalas, Streptomyces ambofaciens, Streptomyces plicarus, Streptomyces rochey No. 3332 Related bacteria: Streptomyces alboflavas ，
Streptomyces aerocolonigenes, Streptomyces mutaviris On the other hand, the 3 strains of the present invention have clear differences in morphological characteristics, culture properties and assimilation of carbon source from known strains including the above-mentioned related strains. is there. That is, the No. 406 strain has a spiny surface on the spores, aerial hyphae are gray strains, basal hyphae are brown or yellow strains, and no pigment is produced, and glucose, sucrose, inositol, and L-arabinose are characterized. , L-rhamnose and raffinose are well assimilated, and the present inventors recognized the species containing this bacterium as a new taxonomic species, and named it Streptomyces tsukubaensis. Also, N
The o.902 strain and the No. 3332 strain have a feature that the hyphae are hook-shaped or open-looped, and there are no aerial hyphae or almost no engraftment, and the former are glucose, sucrose,
Inositol, L-arabinose, L-rhamnose, and raffinose are characterized by being well assimilated, and the latter is characterized by not assimilating L-rhamnose alone.
The present inventors have recognized that species containing these strains are new taxonomic species, and Streptomyces cobaciensis (Streptomyces
kobashiensis).

The above 3 strains are all new strains belonging to the above new species, and among these strains, the No. 406 strain contained in Streptomyces tsukubaensis is the No. contained in Streptomyces sp. KE-406, Streptomyces covacciensis. The .902 strain and the No. 3332 strain were designated as Streptomyces sp. KE-902 strain and Streptomyces sp. KE-3332 strain, respectively. All of these strains have been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, and the deposit numbers are as follows.

Streptomyces sp.KE-406: FERM P-8642 Streptomyces sp.KE-902: FERM P-8643 Streptomyces sp.KE-3332: FERM P-8644 Depending on the culture conditions, chitin may be used even for the above-mentioned related fungi. May produce degrading enzymes.

The target chitin-degrading enzyme can be produced by inoculating the nutrient medium with the novel chitin-degrading enzyme-producing bacterium of the present invention and culturing. Here, the medium may be any medium as long as it contains chitin which is the enzyme-inducing substrate and nutrients that can be utilized by the microorganism. For example, colloidal chitin 1.0
%, Glucose 1.0%, urea 0.2%, potassium dihydrogen phosphate 1.0%, magnesium sulfate heptahydrate 0.05%, yeast extract
Medium containing 0.1% and corn steep liquor 0.5% (p
H7.0).

Shaking culture is suitable as the culture method, and the culture temperature is 30 to
37 ° C is preferred. The number of days of culture was as shown in FIG.
The activity gradually increases until the third day of culture, but thereafter tends to decrease, so that the culture for 2 to 4 days is appropriate.
The culture conditions should be selected in consideration of the properties of the microorganisms used so that the production amount of the target enzyme is maximized.

The culture solution obtained as described above or the supernatant obtained by separating the culture solution from the culture solution by a conventional method such as centrifugation can be used as a crude enzyme solution, but if necessary, a known method may be applied. It may be used after purification. In addition, it is also possible to use a culture medium or a supernatant obtained by adding colloidal chitin to adsorb the enzyme. That is, since the chitin-degrading enzyme has a property of being adsorbed by colloidal chitin, colloidal chitin is added to a culture solution to adsorb the enzyme,
This enzyme can be directly added to the raw material chitin for a decomposition reaction.

When colloidal chitin is added to the culture medium by the method using the adsorbed enzyme, there is an advantage that impurities are prevented from being mixed from the culture medium and the enzyme activity is stably maintained. However, it is also possible to use a mixture of bacterial cells and adsorbed enzyme obtained by adding colloidal chitin to the culture solution itself.

Next, as a raw material for producing a decomposed product of chitin, any raw material containing chitin may be used, and examples thereof include crab, shrimp, krill and the like. These raw materials can be used as they are, but for example, those obtained by acidifying the raw materials (colloidal chitin) or adding an acid and then extrusion cooking (high pressure,
It may be used after being subjected to a high temperature treatment), or further, after the raw material is dipped in hydrochloric acid to be deashed and processed into a product such as extrusion cooked. In particular, it is preferable that the raw material is decalcified as described above and then subjected to high pressure and high temperature treatment. In this case, a high content of N-acetylglucosamine and chitooligosaccharides can be obtained by increasing the acid concentration and making the cooking conditions higher in pressure and temperature. Can be used as a food material.

Regarding the reaction conditions for obtaining the chitin degradation product, the reaction time can be set in the range of about 1 hour to several weeks by adjusting the amount of enzyme, and the reaction temperature is usually about 35 to 40 ° C. Appropriate. The practical reaction time is
It is about 12 to 48 hours. For example, 1 ml of 5% colloidal chitin aqueous solution, 0.2 mol of citrate phosphate buffer (pH 5.
5) By using 1 ml and 1 ml of enzyme solution (supernatant separated from the culture solution) at 37 ° C. for 12 hours, a chitin degradation product can be efficiently obtained.

The main component in the degradation product of chitin varies depending on the type of enzyme used, reaction conditions, etc.
The enzyme derived from p.KE-406 is obtained from N-acetylglucosamine, and the enzyme derived from Streptomyces sp. KE-920 or KE-3332 is obtained from a product containing chito-oligosaccharide such as chitobiose as a main component. It is also possible to obtain a degradation product containing N-acetylglucosamine or chitooligosaccharide alone or a degradation product containing both of them in an appropriate ratio.

After completion of the reaction or during the reaction, the chitin degradation product may be collected by a conventional method. For example, a reaction solution containing N-acetylglucosamine or chitooligosaccharide can be taken out and concentrated by a reverse osmosis membrane to obtain a product. Further, at the same time as the reaction, a chitin degradation product is taken out from the ultrapermeaton membrane and the membrane is concentrated to produce a product. You can also do it. As the membrane used in this case, a dynamic membrane can be used, and if an acid resistant membrane or an exclusion membrane is used, it is possible to produce a chitin decomposition product by acid decomposition.

[Effect of the Invention] According to the present invention, a highly active chitin-degrading enzyme-producing bacterium is provided, and a chitin-degrading enzyme can be efficiently produced using the microorganism. Further, a chitin degradation product can be produced using the enzyme. It is expected that N-acetylglucosamine and chitooligosaccharides, which are degradation products of chitin, will be used for various purposes. For example, the decomposed product of chitin can be used alone as a food material, for example, as a sweetener, and can also be used in combination with sugar, maltooligosaccharide (single or mixture) and the like.

Furthermore, this food material suppresses the growth of harmful bacteria such as intestinal bacteria Clostridium, promotes the growth of useful bacteria such as bifidobacteria, and has various indigestible properties, and various physiological activities such as the prevention of cholesterol accumulation are also observed. It can be expected to be used for health foods, special foods, medicines, etc. It may also have an antibacterial effect.

Further, the enzyme-adsorbed chitin described above can be used as a feed for livestock, and it can be mixed with feed for chickens, pigs, cattle, etc. to increase the production of eggs and meat.

[Examples] Next, examples of the present invention will be described.

Example 1 Streptomyces sp. KE-406 (FERM P-8642) was treated with Bennett agar medium (1% glucose, 0.1% meat extract, 0.1% yeast extract, 0.2% NZ amine, 1.5 to 2.0% agar, pH 7.0). After culturing for 5 to 7 days at 37 ° C, 1 platinum loop was taken, and 1.0% colloidal chitin, 1.0% glucose,
Liquid medium with the composition of 0.2% urea, 1.0% potassium dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate, 0.1% yeast extract, 0.5 corn steep liquor, pH 7.0 (80 ml medium / 500 ml
The mixture was transferred to an Erlenmeyer flask and cultured at 37 ° C for 3 days with aeration and shaking. After completion of the culture, the bacterial cells and insoluble matter in the culture solution were removed by centrifugation or filtration to obtain a supernatant, which was used as a crude enzyme.

The enzyme activity was measured by the following method.

A 2% colloidal chitin aqueous solution (1.0 ml), a 0.2 M citrate phosphate buffer (pH 5.5) (1.0 ml) and an enzyme solution (culture liquid) (1.0 ml) were mixed, and the reaction was carried out at 37 ° C. for 3 hours while gently shaking. After completion of the reaction, the reaction was stopped by heating at 100 ° C. for 5 minutes. Next, regarding the supernatant obtained by centrifugation,
The amount of N-acetylglucosamine was measured by the Morgan-Erson method and used as the colloidal chitin saccharification activity. Also,
For the precipitation part, 3.0 ml of 1% SDS was added, and the mixture was thoroughly stirred and mixed to disperse the precipitate, and then the turbidity was measured at a wavelength of 660 nm to calculate the reduction rate of turbidity, which was liquefied to colloidal chitin. It was made active. The results are shown in Fig. 1.

Application Example 1 Colloidal chitin was added to the culture solution obtained in Example 1, and the mixture was stirred overnight at a low temperature and then washed with cold water to obtain an enzyme-adsorbed chitin. This enzyme agent acts on colloidal chitin to give N-
A chitin degradation product containing 50% or more of acetylglucosamine was obtained.

Application Example 2 The reaction of Application Example 1 is carried out in an ultrafiltration membrane (UF5000) container,
The reaction product N-acetylglucosamine that passed through the ultrafiltration membrane was concentrated by a reverse osmosis membrane to obtain an N-acetylglucosamine product having a purity of 92%.

Application Example 3 Crab and shrimp shells were immersed in 2N hydrochloric acid at room temperature for 2 days, drained, and then extruded at 150 to 200 ° C. in an extruder to obtain swollen chitin. A chitin-degrading product containing 30% or more of N-acetylglucosamine was obtained by allowing a chitin-degrading enzyme derived from Streptomyces sp. KE-406 (FERM P-8642) to act on this chitin.

Application Example 4 The product obtained in Application Example 2 was mixed with sugar at a weight ratio of 1: 1 to prepare a sweetener. This sweetener can be used in foods and drinks such as juice, coffee, lactic acid drinks, cakes and candy.

[Brief description of drawings]

Fig. 1 is a graph showing the culture process of a chitin-degrading enzyme-producing bacterium, Fig. 2 is a photomicrograph (× 600) of Streptomyces sp. KE-406 (morphology of organism), and Fig. 3 is the same strain (of organism). Form) scanning electron micrograph (x10,000),
FIG. 4 is an optical micrograph (× 60) of Streptomyces sp. KE-902 (biological form) and KE-3332 (biological form).
0) and FIG. 5 are scanning electron micrographs (× 4,000) of the same strain (form of organism).

Front Page Continuation (72) Inventor Kazumasa Suzuki 1327 Fukaya, Ayase City, Kanagawa Prefecture (56) References JP 61-40790 (JP, A) JP 60-22916 (JP, B2) JP 54- 36113 (JP, B2)

Claims (3)

[Claims] 1. Streptomyces tsukubaensis or Streptomyces cocciensis, which is a chitin-degrading enzyme-producing bacterium belonging to the genus Streptomyces. 2. The chitin-degrading enzyme-producing bacterium according to claim 1, wherein the Streptomyces tsukubaensis is Streptomyces sp. KE-406 (FERM P-8642). 3. The chitin according to claim 1, wherein the Streptomyces cocciensis is Streptomyces sp. KE-902 (FERM P-8643) and Streptomyces sp. KE-3332 (FERM P-8644). Decomposing enzyme producing bacterium.