JPS625598B2 - - Google Patents

Description

[Detailed description of the invention]

BACKGROUND OF THE INVENTION Technical Field The present invention relates to the production of chitinases by a new species of the genus Streptomyces. In general, chitinolytic enzymes refer to a complex enzyme system consisting of various enzyme components such as chitinase and chitobiase, and their action is to degrade chitin and saccharify it into N-acetylglucosamine or oligosaccharides. Can be mentioned. Chitin is a type of polysaccharide belonging to amino sugars, which is abundant in lower animals, especially arthropods.
The amount of biosynthesized chitin is enormous, estimated to be several billion tons per year, and for this reason chitin is one of the unused natural resources that has recently been in the spotlight. Since chitin has poor reactivity and is more stable than cellulose, it is conceivable to decompose it with an enzyme, ie, chitinase, to reduce its molecular weight. Prior Art Chitinase is contained in the digestive fluid of snails, and is also known to be produced by filamentous fungi and bacteria (for example, "Chemistry Encyclopedia", Vol. 2, p. 745). (Kyoritsu Shuppan Co., Ltd.).According to the catalog of commercial products, microorganisms are obtained from Streptomyces griseus and Serratia marcescens, and according to recent publications, bacteria of the genus Aeromonas Summary of the Invention The present inventors conducted a wide search in soil for chitinase-producing bacteria that have an excellent ability to decompose powdered chitin and have a strong ability to saccharify it into N-acetylglucosamine.
The present invention was completed based on the discovery that Streptomyces sp. WAK-83 strain produces and accumulates chitinase in its medium, which has a strong chitin-degrading activity and an ability to produce N-acetylglucosamine. Therefore, the method for producing chitinase according to the present invention includes:
This method is characterized by culturing the species of microorganisms to which the WAK-83 strain of the genus Streptomyces belongs. Effects The chitinase provided by the present invention has high chitinolytic activity and high N-acetylglucosamine production ability. Therefore, this chitinase can advantageously degrade chitin. DETAILED DESCRIPTION OF THE INVENTION Microorganisms The microorganisms involved in the present invention belong to the species to which WAK-83 strain of the genus Streptomyces belongs. A representative example of this species of microorganism is Streptomyces sp. WAK-83 strain. This bacterium was isolated from the soil of Koji, Koda-cho, Takada-gun, Hiroshima Prefecture in December 1983, and was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology on July 13, 1983. It has been entrusted as FERMP-7714, and its mycological properties are as follows.In addition, below, the color descriptions are based on the color name book of the Japan Color Research Institute (Shinjudo). ) Morphological properties This strain forms aerial hyphae on various media, and the hyphae are branched. Sporangia are not formed. Spores are not motile. Mature on aerial hyphae. The color of the spores is yellow,
Its spore chains are straight to slightly curved (belongs to Rectus Flexibilis). The spores are cylindrical (2
~3 μ x 0.8-1.2 μ), and 20 or more spores are formed in a spore chain. The surface of the spore is smooth. In addition, the remarkable feature of this strain is that
Pycnidium is produced by culturing at ℃/14 days. Properties on various media 1. Sucrose/nitrate agar medium Growth: Spore formation is common. The colony surface is cream colored. The back side is rust red. 2 Glucose-asparagine agar medium Growth: Very good sporulation. The surface of the colony is flesh-colored, and the underside is salmon-colored. 3 Glycerin/asparagine agar medium Growth: Spores are formed, but colonies do not spread much. Pale yellow colony, salmon colored underside. 4 Starch/Inorganic Salt Agar Medium Growth: Good spore formation. The surface of the colony is pale yellow, and the underside is gray-pink. 5 Tyrosine/agar medium Growth: Spore formation is low, but aerial mycelia are formed. Colonies do not wrinkle. The upper surface is white to pale yellow, and the lower surface is dull reddish-yellow-orange. Soluble pigment: None 6 Nutrient agar medium Growth: White aerial hyphae spread on yellow basal hyphae. Spore formation is not very good. The back side is yellow-orange. 7 Yeast/malt agar medium Growth: Grows in a wrinkled shape and becomes curled. The surface of the colony is flesh-colored to pale yellow, and the underside is dull reddish-yellow-orange. 8 Oatmeal/agar medium Growth: Does not grow in wrinkled form. The surface of the colony is white to pale yellow, and the underside is pale yellow. 9 Peptone Yeast Iron Medium Growth: Grows in a wrinkled shape, but hardly forms aerial mycelia and does not form spores. The surface of the colony is light orange-brown, and the underside is the same color. Physiological properties 1 Growth temperature range: 20-37℃. The optimal growth temperature is 28
℃. Not so good at 37℃. 2 Liquefaction of gelatin: Positive 3 Hydrolysis of starch: Positive 4 Effect on milk: No growth 5 Production of melanin-like pigment: Negative Carbon source assimilation The results of culturing on Pridham-Gottlieb agar medium are as follows. It is as follows. Growth ^* L-arabinose + D-xylose + D-glucose + D-fructose + sucrose + i-inositol + L-rhamnose - raffinose - D-mannitol + no additives - * +Grows, -Does not grow Identification Above Based on various characteristics, strain WAK-83 is thought to belong to the genus Streptomyces, with spore chains ranging from straight to slightly curved, and aerial hyphae being white to yellow.
Because the spore surface is smooth and does not produce soluble pigments, Virgie's Manual of Determinative Bacteriology, 8th edition (1975)
When searching from within, 46 bacterial species of the Yellow series are found. However, none of the 46 bacterial species forms pycnidium, which is one of the distinctive characteristics of this bacterial species, and therefore, this bacterial species is different from the 46 bacterial species. The bacterial species that form Pycnidium are classified into the known strains described in Burgess' Manual of Determinative Bacteriology, 8th edition (1975), Shearling and Gottlieb's ISP strains, and Watzmann's ``The Actinomycetes.'' When searching among the bacterial strains, only one strain, Streptomyces syndenensis (ISP5255), was listed.
However, Streptomyces syndenensis forms pink pycnidium, but WAK-
83 strains produce pale yellow pycnidium,
The two are completely different bacterial species. From these results, it is recognized that there are no known bacterial strains that correspond to this bacterial strain, and that this bacterial strain is a new bacterial species.
The present inventors have identified this as Streptomyces sp.
It was named strain WAK-83. Cultivation of microorganisms For culturing this bacterial strain, a normal method for culturing actinomycetes is used. Various carbon sources can be used for the culture medium, such as glucose, starch, fructose,
Glycerin, mannitol, etc. can be used alone or in combination. As the nitrogen source, ammonium chloride, ammonium sulfate, sodium nitrate, soybean flour, yeast extract, peptone, meat extract, casamino acid, etc. are used alone or in combination. In addition, inorganic salts such as common salt, nitrate, calcium carbonate, potassium chloride, cobalt chloride, iron sulfate, and trace amounts of heavy metals may be added as necessary. Furthermore, an organic substance that aids the growth of bacteria and promotes the production of chitinase can be added as appropriate. As a culture method, although culture on a solid medium is also possible, it is preferable to use liquid culture as in the general enzyme production method. Cultivation is carried out under aerobic conditions, and the culture temperature is generally 20-30°C, with a temperature of 28°C.
Preferably it is around ℃. Production of chitinase can be carried out, for example, by tank culture. In that case, chitinase is produced and accumulated in the culture solution by culturing for 2 to 4 days. When the production amount in the culture medium reaches the maximum, the culture is stopped, and the chitinase of interest is isolated and purified from the culture. A crude enzyme solution can be obtained by centrifuging the obtained culture solution or by adding a supernatant. The culture solution obtained by subjecting the obtained culture solution to centrifugation, filtration, etc. to separate the bacterial cells can be used as is as a crude enzyme solution, but the filtrate can be concentrated and mixed with an organic solvent such as acetone or ethanol. A crude enzyme preparation having strong activity can be obtained by adding a solvent to precipitate it or by precipitating it with a salting-out agent such as ammonium sulfate. The crude enzyme preparation obtained in this way is
A purified enzyme can be obtained by purification and crystallization using a known method. Produced Enzyme The crude enzyme preparation obtained as described above has the following properties. (1) Action and substrate specificity It acts on powdered chitin, chitin flakes, colloidal chitin, ethylene glycol chitin, etc., decomposing them and producing N-acetylglucosamine. Please also refer to Table 1 below. (2) Optimal PH and stability The optimal PH at 37°C is 5.5 to 6.0. Also,
This enzyme is stable in the pH range of 3.5 to 8.0. (3) Optimum temperature of action and stability The optimum temperature of action was 37°C in a 3 hour reaction at PH5.5. Furthermore, it was stable for more than 24 hours at temperatures below 30°C. (4) Purification Contaminants in the crude enzyme can be removed by adsorption chromatography using a chitin column, conventional ion exchange chromatography, gel filtration using biogel, etc., thereby producing a highly purified enzyme. can be obtained. The chitinase produced according to the present invention has the characteristic that it acts better on chitin powder than conventional enzymes. For example, commercially available chitinase manufactured by Sigma produces reducing sugars slowly in a 48-hour reaction, but this enzyme can produce the same amount of reducing sugars in a 3-hour reaction. Table 1 shows the activity of chitinases obtained according to the present invention against various substrates. The activity of ethylene glycol chitin was measured by the method of Tsukamoto et al. (Agriculture of Biological Chemistry, Vol. 48, Vol.
931, 1984), and 1% chitin powder and chitin gel were used as substrates. Enzyme activity was measured by the following method of measuring the increase in N-acetylglucosamine due to an enzymatic reaction (for example, the Morgan-Elson method). Use 1 ml of 1% powdered chicken (manufactured by Hanui Chemical) as a substrate, add 1 ml of enzyme solution of an appropriate concentration, and then
Add 3 ml of 0.025M phosphate-acetate buffer PH5.5,
The reaction was carried out at ℃ for 3 hours. Under these conditions, 1μ per minute
The amount of enzyme that produces mol of N-acetylglucosamine was defined as 1 unit.

[Table] As is clear from the table, the chitinase produced according to the present invention effectively degrades ethylene glycol chitin and chitin gel. Experimental Examples Streptomyces sp WAK-83 strain was cultured on a Bennett's medium slant at 28°C for 4 days.
Add 2.5 ml of sterile water to the slant to create a spore suspension. 2.5 ml of the mixture was inoculated into a medium having the following composition in a 100 ml/500 ml flask, and cultured with reciprocal shaking at a temperature of 28° C. for 3 days. Medium composition Yeast extract 3.0g/liter Polypeptone 3.0g/liter Glucose 2.0g/liter Coloidal chitin 4.0g/liter The crude enzyme solution obtained by centrifuging this culture solution to remove the residue is 1.12% of the chitin powder. mu/
ml of enzyme activity. Reference Example The method for preparing colloidal chitin is as follows. Prepare 4 g of powdered chitin in 100 ml of distilled water and 100 ml of concentrated sulfuric acid, and pre-chill each in ice for 3 hours. 3 hours later
Gently mix both. Use a mouthful filled with glass wool. Add the filtered mixture to 180ml of distilled water. After that, 3000rpm
Add distilled water to the residue and centrifuge again.
Repeat this operation until the cleaning solution reaches pH6.

[Brief explanation of the drawing]

Figure 1 shows this bacterium, Streptomyces. sp.WAK―
83 spores, Figure 2 is an optical micrograph of Pycnidium produced by this fungus, and Figure 3 is an optical micrograph of spore chains of this fungus. FIG. 4 is a graph showing the optimum pH of the produced enzyme, FIG. 5 is a graph showing the optimum temperature for action, and FIG. 6 is a graph showing the stable temperature range.

Claims (1)

[Scope of Claims] 1. A method for producing chitinase, which comprises culturing a microorganism of the species to which the WAK-83 strain of the genus Streptomyces belongs. 2. The microorganism is Streptomyces sp. WAK.
There are 83 stocks. A method according to claim 1.