JPH0257917B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel method for producing chitobiase. The present inventors isolated a large number of microorganisms from natural soil and conducted various studies on their products. As a result, the present inventors discovered that the bacteria isolated by the present inventors produced large amounts of chitinolytic enzymes. , completed the invention. That is, the present invention provides a novel method for producing chitobiase. Bacteria producing chitobiase of the present invention have the following mycological properties. (1) Morphology Straight, rod-shaped with a spherical end, size 1.0~
2.4 μm, motile, extremely monociliated, with flagella, Gram negative (2) Growth status 0.2% colloidal chitin, 0.1% peptone, 0.1
% meat juice extract, 0.3% sodium chloride and 2.0%
After incubation for 72 hours at 30°C in chitin-agar plates containing % agar (PH 7.0), distinct colonies are formed. Grows at 37°C in broth liquid medium. Does not grow in broth liquid medium containing 7.5% sodium chloride. Grow in mineral media containing ammonia as the sole nitrogen source and glucose, L-arginine, L-asparagine, L-histidine, L-glutamic acid, L-serine or L-alanine as the sole carbon source. Does not produce brown water-soluble pigment on trypticase soy agar. It grows in a mixed medium containing arginine, asparagine, leucine and methionine. (3) Physiological properties Nitrate reduction: Positive V-P test: Negative Indole formation: Positive (in tryptone broth containing 0.1% tryptophan) Hydrogen sulfide formation: Positive (in 2.5% peptone water) Starch hydrolysis: Positive Urease: Negative Oxidase: Positive Catalase: Positive Cytochrome Oxidase: Positive Growth temperature range: Grows at 13-42℃, 27-42℃
Grows best at 30℃ Growth PH range: 5.5-9.0 Attitude towards oxygen: Facultative anaerobic Assimilation of sugars, production of acids and gases (those with assimilation, production of acids and gases,
Items that are not available are indicated by + and -, respectively):

【table】

[Table] (4) Other properties Gluconic acid oxidation: Negative (gluconate oxidase test) Lysine decarboxylation reaction: Negative [Mora Kg
(Moller) method] Sodium chloride resistance: Positive (1.0% NaCl
Potassium cyanide resistance: Positive (Molar method) Phosphatase: Positive Casein hydrolysis: Positive Gelatin solubility: Positive Deoxyribonurease: Positive Ribonuclease: Positive Arginine dehydrogenase: Positive Glutamate decarboxylation: Negative Vibriostatic reagent, 2,4-diamino-6,7-diisopropyl Pteridine (0/129) Reactivity: Negative Acetoin is produced from 2,3-butanediol, but not from glucose. Buchanan, P. et al. (1974),
Based on the above properties investigated by the systematic method of Cowan, ST. (1974) and Gerhardt, P. et al. (1981) and the chitin solubility according to the present invention, When you search for
An example of an organism capable of decomposing chitin is Vibrio (Beneckea) parahaemolyticus [Vibrio (Beneckea)] by Cowan (1974).
parahemolyticus] and Bauman (P.
Vibrio (Chromobacterium) such as L.) and V. (Chromobacterium)
[According to Bukiyanan et al. (1974), these two species are Vibrio alginolyticus].
It is classified as Parahaemolyticus]. Chitinolytic bacteria have already been reported, and they belong to the Serratia genus [Monreal,
J.) et al. (1969), Reid, J.D. et al. (1981)
)], Vibrio [Uchida et al. (1979)], Venetskea [Takahashi et al. (1982)], and Streptomyces [Reynolds, DM (1954)].
[Bukiyanan et al. (1974)
According to the 2011 study, Venetcea is considered to belong to the genus Vibrio]. Based on these, this bacterium is considered to belong to the family Vibrionaceae. Incidentally, this bacterium is completely different from the genus Vibrio in that it has the ability to produce hydrogen sulfide from peptone and is not sensitized to Vibriostatic reagent 0/129. Based on the evidence presented above, this bacterium is a strain of Aeromonas.
Hydrophila subanerogenes ATCC15467
Similar to (IFO13282). This is because this bacterium is V
-P reaction, gluconate oxidase test is negative, and no gas is produced from glycerin and glucose [Bukiyanan et al. (1974)]. However, while this bacterium has a strong chitinolytic activity, the above-mentioned strain is completely different in that it does not show any chitinolytic activity. Therefore, in order to distinguish this bacterium from known strains, the present inventors investigated Aeromonas hydrophila A52 (Aeromonas hydrophila subanaerogenes).
subsp.anaerogenesA52 (hereinafter sometimes abbreviated as bacterium 52) and was deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology under accession number FERM P-7206. This bacterium is isolated and purified as follows. Separation: 0.2% colloidal chitin, 0.1% peptone, 0.1%
The test was carried out by streaking one platinum loop of the sample suspension on a chitin-agar plate medium (PH7.0) containing meat juice extract, 0.3% sodium chloride, and 2.0% agar (streaking plate method). After incubation at 30° C. for 72 hours, colonies are picked and stored on slanted chitin-agar medium with the same composition as above. A distinct area of dissolved colloidal chitin forms around the colony. Purification of the bacteria from isolated colonies is carried out by plating six times alternately on chitin-agar and ordinary broth-agar. lastly
Among the 47 isolates, those that grew quickly and formed large, well-defined colonies on chitin-agar medium were selected as bacteria A52. The bacteria thus obtained were extracted from 1.0% meat juice extract,
Ordinary broth-slant medium containing 1.0% peptone, 0.5% sodium chloride, 2.0% agar (PH7.0 with 1N-NaOH)
After culturing at 28°C for 3 days in a medium (adjusted to 100°C), the cells were kept at room temperature and transferred to a new medium every month for storage. The chitobiase of the present invention is produced by inoculating the above-mentioned bacterium into a nutrient medium and culturing it.
The culture medium may be any medium as long as it contains chitin, which is an enzyme-inducing substrate, and a nutrient source used by the bacteria, such as 1.0% shrimp shell chitin, 0.2% glucose, 0.5% peptone, or 1.0%. Yeast extract, 0.7g/potassium dihydrogen phosphate, 0.3%
Examples include those containing sodium chloride (adjusted to pH 7.0 with 1N-NaOH). As a culture method, shaking culture is suitable. The appropriate temperature for culturing is 25-30℃, but in most cases it is 28℃.
Cultivate nearby. After culturing for 2 to 3 days, the culture solution is subjected to the following operation. Chitobiase is isolated by appropriately combining various methods in consideration of the physicochemical properties of chitobiase, as shown in Examples below. That is, since chitobiase is usually present in the culture solution, after separating bacteria from the culture by centrifugation or other means, ammonium sulfate is added to the culture solution to perform salting out. Next, the protein precipitate precipitated by salting out was mixed with 0.1M Tris.
Dissolve in hydrochloric acid buffer (PH7.0), centrifuge, and use the supernatant as the crude enzyme solution. The crude enzyme solution contains chitobiase and chitinase. Chitobiase is isolated from the crude enzyme solution by utilizing the difference in adsorption properties of chitobiase and chitinase to colloidal chitin. That is, the crude enzyme solution and colloidal chitin are mixed with a Tris-HCl buffer solution, chitinase is adsorbed to the colloidal chitin, and then centrifuged to obtain a supernatant, which is used as the chitobiase fraction.
Note that chitinase is obtained from the precipitate obtained by centrifugation. Chitobiase is separated and purified from the chitobiase fraction by CM-cellulose chromatography and
DEAE-Sephadex A-50 column chromatography is used. The chitobiase obtained as described above has the following physicochemical properties. Action: Works together with chitinase to degrade chitin. Optimal PH: PH7.0 Km ^* : p-nitrophenyl-N-acetylglucosaminide 0.17mM PH stability: PH6.0 when treated at 37℃ for 30 minutes
Shows residual activity of 90% or more at ~PH9.0. Optimum temperature: At pH 7.0, when p-nitrophenyl-N-acetylglucosaminide is used as a substrate, it is around 50°C. Temperature stability: p-nitrophenyl-N-acetylglucosaminide substrate at pH 7.0;
Shows residual activity of 80% or more after treatment at 0-40°C for 30 minutes. Isoelectric point ^** : Around PH6.8 Molecular weight ^*** : 115000 * [Measurement of Km value] 5.0mM p-nitrophenyl-N-acetylglucosaminide 0.2ml, 50mM Tris-maleic acid buffer (PH7.0) A reaction solution consisting of 0.7 ml of enzyme solution and 0.1 ml of enzyme solution is incubated at 37°C for 10 minutes. Next, 0.25M sodium carbonate solution was added to this reaction solution.
2.0 ml was added to stop the reaction, and at the same time color development of p-nitrophenol occurred. Since the generated N-acetylglucosamine is equimolar to p-nitrophenol, the absorbance at 405 nm was measured and the amount was determined from the calibration curve of p-nitrophenol. For chitobiase activity, one unit was defined as the amount of enzyme that produced 1 μmol of N-acetylglucosamine from p-nitrophenyl-N-acetylcosaminide per minute. The Km value was obtained from the Lineweaver-Burk plot. A purified chitobiase preparation was used as the enzyme solution, and the amount of enzyme in one reaction solution was 0.054 U. ** [Isoelectric focusing] Polyacrylamide gel disk isoelectric focusing is performed by Davis, B. J.A. Ann.NYAcad.
Sci., 121 , 404 (1964) in 7.5% gel, Tris-glycine buffer (PH8.4). A current of 2 to 4 mA was applied to each tube, and electrophoresis was performed at 5°C. *** [Molecular weight] Based on SDS-polyacrylamide gel electrophoresis. The chitobiase of the present invention thus obtained can be used as a cell wall lytic enzyme for the formation of protoplasts, etc. Next, examples and reference examples will be given and explained. Reference Example 1 (1) Preparation of shrimp shell chitin Thaw frozen shrimp shells, process in a Waring blender for 10 minutes, and wash with tap water at least 3 times. The obtained shrimp shell flakes were soaked in 1N sodium hydroxide overnight to remove protein.
Rinse at least 3 times with tap water. Next, the obtained deproteinized shrimp shell flakes are soaked in 1N hydrochloric acid overnight to remove calcium. After washing the purified shrimp shell flakes obtained by the above procedure with tap water,
After adjusting the pH to 7.0 with 1N sodium hydroxide, process in a Waring blender for 10 minutes, adjust the dry matter amount to 2%, and sterilize in an autoclave at 120°C for 20 minutes. (2) Preparation of colloidal chitin The shrimp shell chitin obtained in (1) was ground in a ball mill for about 24 hours to obtain ball milled chitin. This ball milk chitin was subjected to the following operations to obtain colloidal chitin. a. Moisten a cooled mortar with acetone and thoroughly mix ball milk chitin and concentrated hydrochloric acid. b Disperse dropwise into a large amount of cold water while stirring well. c Wash with water by centrifuging at 18000g for 10 minutes. d Process in a Waring blender for 10 minutes. e Centrifuge at 18000g for 10 minutes to collect colloidal chitin, and further wash with 0.025M Tris-HCl buffer (PH7.2). The colloidal chitin thus obtained is used after being dispersed in an appropriate buffer. Example 1 (1) Culture for enzyme production 1 part tap water, 15 g peptone, 5 parts yeast extract
g, add 0.68 g of potassium dihydrogen phosphate, 1N
- Pour 5 ml of the preculture medium adjusted to pH 7.0 with sodium hydroxide into test tubes with cotton plugs and sterilize according to the usual method. This is then followed by bacteria from the storage medium.
A loopful of A52 was inoculated and cultured with shaking at 28°C for 24 hours. For the main culture, 1 part of tap water, 10 g of shrimp shell chitin, 2 g of glucose, 5 g of peptone, 10 g of yeast extract, 0.68 g of potassium dihydrogen phosphate,
Add 3 g of sodium chloride and adjust the pH to 7.0 with 1N sodium hydroxide, then add 500 ml of main culture medium.
Dispense 70 ml into volumetric flasks and sterilize according to standard methods. Next, 1 ml of the preculture medium after completion of culture was inoculated into this, and cultured with shaking (240 rpm) at 28°C for 72 hours. (2) Preparation of crude enzyme solution After culturing, after removing the bacterial cells from the main culture medium by centrifugation at 18,000 g for 20 minutes, add sodium azide to a final concentration of 0.02% to prevent contamination with bacteria. It was added as a culture solution. Next, solid ammonium sulfate was gradually added to the culture solution to achieve 80% saturation while cooling to 0° C. and stirring gently with a stirrer to perform salting out. At this time, make sure that the pH of the culture solution does not lean toward the acidic side.
The pH was maintained around 7.0 with 1N sodium hydroxide. Salting out is carried out at 1°C while cooling to 0 to 4°C.
I went for more than an hour. The protein precipitate precipitated by salting out was collected by centrifugation at 18,000 g for 20 minutes. Add this precipitate to 0.1M in one-tenth volume of the culture solution.
Dissolved in Tris-HCl buffer (PH7.0), 0-
After cooling to 4 and standing for 1 hour, remove 18000g of insoluble matter.
The supernatant was removed by centrifugation for 20 minutes, and the supernatant was used as a crude enzyme solution and stored frozen at -20°C. (3) Separation of chitinase and chitobiase by chitin adsorption Based on the difference in affinity of both enzymes for colloidal chitin, they were separated as follows. 25mM containing 75ml of crude enzyme solution (approximately 887U of chitinase) and 2.5g of colloidal chitin (dry weight)
Mix 300ml of Tris-HCl buffer (PH7.2),
Chitinase was adsorbed onto colloidal chitin for 1 hour under ice cooling with occasional stirring. then 18000
Centrifugation was performed for 20 minutes at 100 g for 20 minutes, and the supernatant was used as the chitobiase fraction. (4) Purification of chitobiase The chitobinase fraction obtained in (3) of Example 1 was concentrated in an evaporator in the presence of 1mM PMSF, and then dialyzed overnight against 5mM Tris-HCl buffer (PH7.2). The product was ion-exchanged on a CM-cellulose column (2.6 x 45 cm) equilibrated with 50 mM acetate buffer (PH5.2). The elution was performed using a sodium chloride concentration gradient from 0 to 0.3M, and the flow rate was 40 ml/hour. Next, the eluate was concentrated and desalted by ultrafiltration, and then added to a 25mM Tris-HCl buffer (PH7.2) containing 0.05M sodium chloride.
DEAE equilibrated with SEFAEDEX A-50
column chromatogram (column size
2.6×45 cm) Purified chitobiase was obtained. In addition,
Elution was with a sodium chloride concentration gradient of 0.05→0.2M.
The flow rate was 44 ml/hour. The purification degree and recovery rate of tokibiase in each of the above operation steps are shown in Table 1, and the column chromatogram using DEAE-Sephadex A-50 is shown in Table 1.
As shown in the figure. As is clear from Table 1, the chitobiase in the crude enzyme solution was purified to 144 medium by the above procedure.

【table】 [Brief explanation of the drawing]

Figure 1 shows the DEAE of chitobiase eluate obtained by ion exchange with a CM-cellulose column.
- It is a drawing showing a column chromatogram using Sephadex A-50.

Claims (1)

[Scope of Claims] 1. Cultivating Aeromonas hydrophila subanerogenes A25 (Feikoken Bacteria No. 7206) in a medium,
A method for producing chitobiase, which comprises collecting chitobiase having the following physicochemical properties from the culture. Action: Works together with chitinase to degrade chitin. Optimal PH: PH7.0 Km: p-nitrophenyl-N-acetylglucosaminide 0.17mM PH stability: PH6.0 when treated at 37℃ for 30 minutes
Shows residual activity of 90% or more at ~PH9.0. Optimum temperature: At pH 7.0, when p-nitrophenyl-N-acetylglucosaminide is used as a substrate, it is around 50°C. Temperature stability: p-nitrophenyl-N-acetylglucosaminide substrate at pH 7.0;
Shows residual activity of 80% or more after treatment at 0-40°C for 30 minutes. Isoelectric point: around PH6.8 Molecular weight: 115000