JPH0147996B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel chitinase and a method for producing the same. The present inventors have isolated a large number of microorganisms from natural soil and conducted various studies on their products. As a result, the present inventors have found that the bacteria isolated by the present inventors produce large amounts of chitinolytic enzymes. The present invention has been completed. That is, the present invention provides a novel chitinase and a method for producing the same. Bacteria producing the chitinase 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, containing 0.3% sodium chloride and 2.0% agar (PH7.0) in chitin-agar plate medium, 30
After 72 hours of incubation at °C, distinct colonies form. Grows at 37°C in broth liquid medium. Does not grow in broth liquid medium containing 7.5% sodium chloride. It is grown in an inorganic medium 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) Physical and chemical properties Nitrate reduction: Positive VP test: Negative Indole formation: Positive (in tryptone proth 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℃, 27-30℃
℃ Growth PH range: 5.5 to 9.0 Attitude towards oxygen: facultative anaerobic Assimilation of sugars, acid and gas production (those with and without assimilation, acid and gas production) (respectively indicated by + and -):

[Table] (4) Other properties Gluconic acid oxidation: Negative (gluconate oxidase test Lysine decarboxylation reaction: Negative [Moll
-er) method] Tolerance of sodium chloride: Positive (Maximum growth in prosthesis containing 1.0% Nacl) Tolerance of potassium cyanide: Positive (Molar method) Phosphatase: Positive Casein hydrolysis: Positive Gelatin solubility: Positive Deoxyribonuclease: Positive Ribonuclease: Positive Arginine dehydrogenase: Positive Glutamic acid decarboxylation reaction: Negative Reactivity to bibriostatic reagent, 2,4-diamino-6,7-diisopropyl pteridine (0/129): Negative Acetoin is removed from 2,3-butanediol produced, 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) Parahemolyticus, described by Cowan (1974).
V. (Chromobacterium) alg-inolyticus] such as V. cus] and Bauman (PL) [according to Bukiyanan et al. (1974), these two is classified as Vibrio parahaemolyticus]. Chitin-soluble bacteria have already been reported, including the Serratia genus [Monreal, J. et al. (1969), Reid, J.D. et al. (1981)], Vibrio genus [Uchida et al. (1979)], Venetcea [Takahashi et al. (1982)
) and the genus Streptomyces [Reynolds, DM (1954)] [According to Bukiyanan et al. (1974), 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 (IFO
13282). This is because this bacterium is V-P
reaction, gluconate oxidase test is negative, and no gas is produced from glycerin and glucose [Pukiyanan 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 inventor developed Aeromonas hydrophila subanaerogenes A52 (Aeromonas hydrophila
subsp.anaerogenes A52 (hereinafter sometimes abbreviated as Bacteria 52) and was deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology under the 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 for 72 hours at 30°C, colonies are collected and stored on slanted chitin-agar plates 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 6 times alternately on chitin-agar and ordinary prosthetic agar. Finally, among the 47 isolates, those that grew quickly on chitin-agar medium and formed large, clear colonies were selected as bacteria A52. The bacteria thus obtained were extracted from 1.0% meat juice extract,
Ordinary prosthetic slant medium containing 1.0% peptone, 0.5% sodium chloride, 2.0% agar (PH = 7.0 with 1N NaOH)
After culturing at 28°C for 3 days in a medium (adjusted to 28°C), store at room temperature, and transfer to a new medium every month for storage. The chitinase 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%. Examples include yeast extract, 0.7g/potassium dihydrogen phosphate, and 0.3% 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. Chitinase was isolated as shown in the Examples below.
This is carried out by appropriately combining various methods in consideration of the physicochemical properties of chitinase. That is, since chitinase 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 is dissolved in 0.1M Tris-HCl buffer (PH7.0), centrifuged, and the supernatant is used as a crude enzyme solution. The crude enzyme solution contains chitinase and chitobiase. Chitinase is isolated from the crude enzyme solution by utilizing the difference in the adsorption properties of chitinase and chitobiase to colloidal chitin. That is, the crude enzyme solution and colloidal chitin are mixed with a Tris-HCl buffer, and the chitinase is adsorbed onto the colloidal chitin, followed by centrifugation to obtain a precipitate. As shown in the Examples below, this precipitate is treated with a Tris-HCl buffer containing phenylmethylsulfonyl fluoride (PMSF) to dissolve colloidal chitin and obtain a chitinase fraction. Chitinase was separated and purified from the chitinase fraction by gel filtration and Bio-Gel P-200.
It is performed by column chromatography using DEAE A-50. The chitinase obtained as described above has the following physicochemical properties. Action: Acts on chitin and breaks it down. Optimal PH: PH7.0 Km ^* : Colloidal chitin amount is 1.35μg/ml. PH stability: PH5.2 when treated at 37℃ for 30 minutes
Shows residual activity of 80% or more at ~PH8.2. Optimum temperature: At pH 7.0, when colloidal chitin is used as a substrate, it is around 45°C. Temperature stability: Colloidal chitin substrate exhibits over 95% residual activity after treatment at 0 to 50°C for 30 minutes at pH 7.0. Isoelectric point ^** : around PH7.4 Molecular weight ^*** : 153.000 * [Measurement of Km value] 0.4ml of 50mM acetate buffer (PH5.2) containing 1.3% colloidal chitin, 0.4ml of 50mM acetate buffer (PH5.2) 1.6ml,
A reaction solution consisting of 2.0 ml of enzyme solution was incubated at 37° C. for 45 minutes, and the activity was measured from the decrease in turbidity at this time. The decrease in turbidity was determined from the decrease in absorbance at 610 nm, and the amount of enzyme that reduced the turbidity by 1% in 1 minute was defined as 1 unit. The result is Km by Lineweaver-Burk plot.
I found the value. The Km value was expressed as the dry weight of colloidal chitin. A purified chitinase preparation was used as the enzyme solution, and the amount of enzyme in one reaction solution was 0.075 U. ** [Isoelectric focusing] Polyacrylamide gel disk isoelectric focusing was performed by Davis, B., J.A. Ann.NYAcad.
Sci... 121 , 404 (1964), 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 chitinase of the present invention thus obtained can be used as a cell wall lytic enzyme for the formation of protoplasts, etc. Next, an example 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 are soaked in 1N sodium hydroxide overnight to remove protein, and then washed with tap water three or more times. 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, adjusting the pH to 7.0 with 1N sodium hydroxide, processing in a Waring blender for 10 minutes, adjusting the dry matter amount to 2%, and autoclaving. Sterilize at 120℃ 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 wash with 0.025M Tris-hydrochloride buffer (PH7.2). The colloidal chitin thus obtained is used after being dispersed in an appropriate buffer. Example 1 (1) Culture for enzyme production 5 g of peptone, 5 g of yeast extract, 1 part of tap water,
Add 0.68 g of potassium dihydrogen phosphate and adjust the preculture medium to pH 7.0 with 1N sodium hydroxide.
Pour 5 ml each into test tubes with cotton plugs and sterilize according to standard methods. Next, a loopful of bacteria A52 was inoculated from the storage medium into this, and cultured with shaking at 28°C for 24 hours. For the main culture, 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, and 3 g of sodium chloride were added to tap water 1, and the pH was adjusted to 7.0 with 1N sodium hydroxide. Dispense 70 ml of the main culture medium into 500 ml flasks and sterilize according to a conventional method. 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 the completion of culture, after removing the bacterial cells from the main culture medium by centrifugation at 18,000g for 20 minutes, add sodium azide to a final concentration of 0.02% to prevent contamination by 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, the culture solution
The pH was maintained around 7.0 with 1N sodium hydroxide to prevent the pH from becoming acidic. Salting out was carried out for over 1 hour while cooling to 0 to 4°C. The protein precipitate precipitated by salting out weighed 18,000 g.
Collected by centrifugation for 20 minutes. This precipitate was dissolved in 0.1M Tris-HCl buffer (PH7.0) of 1/10 volume of the culture solution, cooled to 0-4℃ and left to stand for 1 hour, and then centrifuged at 18,000g for 20 minutes to remove insoluble matter. Remove the supernatant, use it as a crude enzyme solution, and store at -20°C.
It was stored frozen. (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. Mix 75 ml of crude enzyme solution (approximately 887 U of chitinase) and 300 ml of 25 mM Tris-HCl buffer (PH7.2) containing 2.5 g (dry weight) of colloidal chitin, and add colloidal chitinase for 1 hour under ice cooling with occasional stirring. Adsorbed to chitin. Then, centrifugation was performed at 18,000g for 20 minutes to collect the precipitate. In this precipitate
Add 400ml of 25mM Tris-HCl buffer (PH7.2) containing 0.5M sodium chloride, disperse well on ice, wash thoroughly for 1 hour, and centrifuge at 18,000g for 20 minutes to remove impure proteins other than chitinase. was excluded. Contain 1mM PMSF in the resulting precipitate
When 300 ml of 25 mM Tris-HCl buffer (PH7.2) was added and the mixture was well dispersed and incubated at 32°C for 12 hours, the colloidal chitin was almost completely dissolved and chitinase was liberated. Here, the mixture was centrifuged again at 18,000 g for 20 minutes to remove indigested matter, and the supernatant was used as a chitinase fraction. (4) Purification of chitinase After concentrating the chitinase fraction obtained in (3) of Example 1 using an evaporator, it was purified using a Bio-Gel P-200 column (2.6 100 cm). The flow rate is 28
ml/hour. Next, the chitinase fraction obtained by gel filtration was concentrated with sucrose in a dialysis tube, and then applied to a DEAE-Sephadex A-50 column chromatograph equilibrated with 25mM Tris-HCl buffer (PH7.2). (Column size: 2.6×
45cm) Purified chitinase was obtained. The elution was with a sodium chloride concentration gradient of 0 → 0.3M, and the flow rate was 42ml/
Sometimes. Table 1 shows the purification degree and recovery rate of chitinase in each of the above-mentioned operation steps, and FIG. 1 shows a column chromatogram using DEAE-Sephadex A-50. As is clear from Table 1, the chitinase in the crude enzyme solution was purified 171 times by the above operation.

【table】 [Brief explanation of drawings]

FIG. 1 is a drawing showing a DEAE-Sephadex A-50 column chromatogram of the chitinase fraction obtained by gel filtration with a Bio-Gel P-200 column.

Claims (1)

[Claims] 1. A chitinase having the following physicochemical properties. Action: Acts on chitin and breaks it down. Optimal PH: PH7.0 Km: Colloidal chitin amount is 1.35μg/ml. PH stability: PH5.2 when treated at 37℃ for 30 minutes
Shows residual activity of 80% or more at ~8.2. Optimum temperature: At pH 7.0, when colloidal chitin is used as a substrate, it is around 45°C. Temperature stability: Colloidal chitin substrate exhibits over 95% residual activity after treatment at 0 to 50°C for 30 minutes at pH 7.0. Isoelectric point: around PH7.4 Molecular weight: 153000 2 Chitinase-producing bacteria belonging to the genus Aeromonas are cultured in a medium, and the culture has the following physical and chemical properties: Action: Acts on chitin to decompose it. Optimal PH: PH7.0 Km: Colloidal chitin amount is 1.35μg/ml. PH stability: PH5.2 when treated at 37℃ for 30 minutes
Shows residual activity of 80% or more at ~8.2. Optimum temperature: At pH 7.0, when colloidal chitin is used as a substrate, it is around 45°C. Temperature stability: Colloidal chitin substrate exhibits over 95% residual activity after treatment at 0 to 50°C for 30 minutes at pH 7.0. A method for producing chitinase, which comprises collecting chitinase having an isoelectric point: around PH7.4 and a molecular weight: 153,000. 3 The chitinase-producing bacterium is Aeromonas hydrophila subanerogenes A52 (Feikoken Bacterial Serial No. 7206)
A method for producing chitinase according to claim 2.