JPS61280269A - Novel microorganism - Google Patents

Abstract

NEW MATERIAL:A novel microorganism Erwinia Nep5. Entirely different from Erwinia uredovora in that hydrogen sulfide is evolved from cysteine and casein is hydrolyzed. EXAMPLE:Erwinia Nep5 KS-5 (FERM-P No. 8261). USE:An edible meat softener, enzymic agent for brewing, anti-inflammatory enzymic agent, enzymic agent for addition of detergents, having the ability to produce heat-resistant protease. PREPARATION:An aqueous solution in an insectivorous sac of one species of the genus Nepenthes is uniformly spread on, e.g. a suitable plate culture medium, and cultivated at 37.5 deg.C for 24hr and further cultivated at room temperature for 24hr.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a new microorganism, and more particularly to a new bacterial species belonging to the genus Erwinia.

The microorganism according to the present invention is a microorganism isolated from the aqueous liquid in the insect bag of a plant belonging to the genus Nepenthes cultivated in Kagoshima City, Kagoshima Prefecture, and as described below, a new microorganism belonging to the genus Erwinia. It is a seed. The present inventors identified this bacterial strain as Erwinia Nep5.
It was named.

Erwinia Nep, which is a representative example of the new bacterial species of the present invention, is shown below.
The mycological properties of 5KS-5 are shown.

A. Morphological properties (1) Cell shape: Bacillus (2) Cell size: (0,5-1,0) x (1,
0-5,, 5) μ order (3) Presence or absence of cell pleocytosis: None 4) Motility: Yes (5) Epiphytic state of flagella bilayer flagella (6) Presence or absence of spores: None B, various media Growth status (1) Broth agar plate medium: Colony growth status is moderate;
The shape is circular, the ridges are semi-lens-shaped, the periphery is golden, and the color is pale yellow (2) Gravy agar slant medium: growth condition is moderate, surface condition is smooth, color is pale yellow, gloss is bright, shape is Smooth (3) Juicy liquid: Moderate moisture, no growth on the liquid surface, and some sediment.

(4) Meat juice gelatin puncture: Grows, liquefies (5) Lidomus milk: Peptonization, decolorization C1 Physiological properties (1) Acid-fastness: None 2) Durham staining: Negative (3) Nitrate reduction 20 (gravy) ) (4) Denitrification reaction: → - (5) MR test Ni (6) V P test: + (7) Indole production: Mo (8) Hydrogen sulfide production: + (9) Starch hydrolysis: + (10) Use of citric acid: Koser is 1 Chri
stensen is + (11) Use of inorganic nitrogen sources: ammonium salt is +, nitrate is - (12) Pigment production (diffusivity): Yes (light brown, King B medium) (13) Catalase: + (14) Urease - (15) Oxidase (16) PL that can grow (range: 5-9 (optimal pH 7,
5) (17) Temperature range for growth, 10-40°C (optimal temperature 32°C) (18) Attitude towards oxygen Bi-association anaerobic (19) OF test: Fermentation (glucose) (20) Acid from sugars and gas production Culture temperature 308C37℃ Acid gas Acid gas 1, L-arabinose + ++-2D-quinlose − ---3, D-glucose
+ ++-4D ~ Lamance + ++-5
, D~fructose + ++-6, D-galactose + 11-7, maltose
+ + Mo-8, sucrose +
++-9, lactose - -10 -10
, trehalose + ++-11, D~sorbitol ---12, D-mannitol
+ ++-13, Inojito -
---14, glycerin + ++-15
, starch + ++-16°D-ribose + ++-17, L-rhamnose
-----18, Salishin Sen
10--19, α-methylglucoside + ++-20
, raffinose - -1=21, zurcit -11122, melezitose
--1-23, melibiose --m-
24, Atonito -11125
Cellobiose = 26 Dextrin + 1,000 ±27, Aesculin
+ 10 ± -28, β-methylgelcond + +10-(21) Decomposition of casein: + (22) Decomposition of DNA + (23) Oxidation of gluconic acid: + (24) N a CC (5 %) resistance: + (25) Decomposition of arginine (26) Decarboxylation of lysine (27) Decarboxylation of ornithine (28) Deamination of phenylalanine (29)
Tolerance of potassium cyanide (30) Pectinase (31) Lipase (32) Auxotrophy: None (33) Utilization of carbon compounds 1, Acetic acid: + 2, Fumaric acid + 3, Gluconic acid: + 4, Malic acid: + 5 Succinic acid: 26, Benzoic acid: 7, Oxalic acid: 8, Propionic acid: -9, Formic acid: 10, Malonic acid: 11, Tartaric acid: +2. Lactic acid: + 13, Galacturonic acid: 14, Starch: + 15, Dextrin: + (34) Utilization of asparagine (single carbon and nitrogen source)
:+ (35) Oxidation of gluconic acid: + (36) Production of reducing substances from sucrose: + The above properties were determined according to Burge's Manual of Determinative Bacteriology, 8th edition (1974). A search revealed that this bacterium is a facultatively anaerobic, oxidase-negative, gram-negative bacillus with periflagella, and does not produce gas from glucose at 37°C, and does not produce gas from glucose, fructose, glucose, galactose, β-methylglucoside, or Acid is produced from loin but not from atonite, dulcitose, melezitose, acetic acid, fumaric acid,
It can assimilate gluconic acid, malic acid, and succinic acid as carbon sources, but cannot assimilate benzoic acid, oxalic acid, and propionic acid as carbon sources, so it belongs to the genus Erwinia.

In addition, this bacterium decomposes deoxyribonucleic acid, oxidizes gluconic acid, reduces nitrate, and can grow at 36°C.
'Erwinia ure
dovora). However, this bacterium is distinctly different from Erwinia uredvora in that it generates hydrogen sulfide from cysteine and hydrolyzes casein. Furthermore, this bacterium differs from Erwinia uredvora in that it decomposes arginine, cannot assimilate formic acid and tartaric acid, and has an optimum growth temperature of 32°C.

Based on the above, the present inventors recognized this bacterium as a new bacterial species belonging to the genus Erwinia, and named and identified it as Erwinia Nep5.

Erwinia N, one of the representative examples of Erwinia Nep5
ep5KS-5 was granted accession number 8261 (FERM-P826) to the Institute of Microbial Technology, Agency of Industrial Science and Technology.
It has been deposited as 1).

The novel microorganism of the present invention belonging to the genus Erwinia produces thermostable protease in the culture solution by culturing it. This protease is characterized by its activity even at high temperatures, and is used in meat tenderizers, brewing enzymes, anti-inflammatory enzymes, etc.
It is useful as an enzyme additive for detergents.

As a medium for culturing the microorganism of the present invention, any medium including a synthetic medium or a natural medium can be used as long as it appropriately contains a carbon source, a nitrogen source, inorganic substances, and other nutrients.

As a carbon source, glucose, soluble starch, sucrose, dextrin, cornmeal, etc. can be used. As nitrogen sources, use inorganic and organic ammonium salts or other nitrogen-containing substances, such as peptone, meat juice extract, yeast extract, cornstarch liquor, skim milk, casein, soybean flour, proteinaceous organic matter such as defatted soybean meal. Can be done.

Furthermore, as inorganic substances, sodium dihydrogen phosphate, phosphoric acid-
Potassium hydrogen, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, calcium carbonate, and the like can be used. Nutrient sources such as vitamins can also be used as appropriate.

Cultivation may be carried out according to conventional methods, for example, culture medium 1) H
When the microorganism is inoculated into a medium and cultured aerobically at 10 to 40°C, a significant amount of thermostable protease is produced and accumulated in 1 to 5 days.

After culturing, purification is performed using conventional separation and purification methods, salting out using ammonium sulfate, organic solvent precipitation, chromatography using ion exchange or adsorption resin, precipitation using a protein precipitant, isoelectric focusing, or electrical focusing. Highly pure protease can be obtained by purifying it by applying a method such as dialysis. Alternatively, the cultured cells can be simply separated from the medium and the cells or culture solution can be used as the crude enzyme.

Quantification of protease is carried out using the Kanifu method, “Enzyme Research Methods,” Vol. 2, 239, using Hammerstein casein as a substrate.
p., Asakura Shoten (1956)], the amount of enzyme that gives a trichloroacetic acid soluble product equivalent to 1 μ gtyrosine per minute is defined as 1 unit, and after reacting at 30°C for 30 minutes, the wavelength Calculate titer from absorbance at 275 nm.

Next, the method for pure isolation of microorganisms of the present invention and the reproducibility by subculture of the microorganisms will be described in detail with reference to Examples.

Example 1 The aqueous liquid in the insect trap bag of a type of Nepenthes sp. cultivated in Kagoshima City, Kagoshima Prefecture was diluted 100 times, and 0.1 m12 of the solution was uniformly spread onto a broth agar plate medium using a conrage stick. Spread out, 37.5°C
The cells were incubated for 24 hours at room temperature and then for 24 hours at room temperature.

Colonies generated by this culture were transferred to a broth agar slant medium using a platinum loop and cultured at 30°C for 2 days.

One platinum loopful of the generated bacteria was diluted 10,000 times with physiological saline, 0.1 ml (l) of it was evenly spread on a broth agar plate medium with a Conrage stick, and cultured at 30°C for 2 days. It was confirmed visually and microscopically that the colonies were not different from each other.

Ten of the above colonies were inoculated onto broth agar slants, cultured at 28°C for 3 days, and it was confirmed that the bacteria on the 10 slants were macroscopically and microscopically the same. It was confirmed that the properties and physiological properties on each medium of these 10 cultured bacteria were the same. The properties and physiological properties of each of the above-mentioned media are as described above.

As a result of the above test, it was found that all 10 cultured bacteria were pure monobacteria isolated from nature.

Next, a protective agent (10% skim milk and 1% aqueous solution of sodium glutamate) was added to the pure cultured bacteria on the broth agar slant as described above to prepare a bacterial suspension on the broth agar slant. Approximately 1 mfl of this finely suspended liquid was dispensed into ampoules for freeze-drying, and freeze-drying was performed. The freeze-drying was carried out by quickly freezing the ampoule containing the bacterial suspension in dry ice/acetone, setting it in a freeze dryer, and setting the vacuum to 0.03 torr or less. Then, it was vacuum sealed with a gas burner and stored at 4°C. After storing the freeze-dried bacteria (standard sample) obtained in this way for 3 months, open the ampoule and transfer it to a sterile test tube using a sterile mini-spartil. Aqueous solution) was added and left to stand for over 1 hour, and the properties and physiological properties on each culture medium were examined under the same conditions as described above. As a result, no changes were observed compared to the bacteria before freezing (standard sample). I couldn't.

In addition, when the properties and physiological properties of the bacteria on each culture medium were similarly examined after the above-mentioned freezing and regeneration was repeated five times every month, no changes were observed. From these results, it is understood that the microorganism of the present invention can reliably reproduce the same results by subculture.

Reference example 1 Erwinia Nep5KS-5 with 2HP0.0.7%,
KH, Po, 0.2%, Mg5O, 7HtOO.

01%, sodium citrate 0.05%, glucose 0
．． Liquid medium (pH 7.0) containing 2% skim milk and 1% skim milk.
) 100J! Planted in a flask containing No. 12 and heated to 35°C.
The cells were cultured for 72 hours. The amount of protease produced at this time was 17.6 units/good.

Reference Example 2 In the same manner as Reference Example 1, 40 flasks were used.
A culture solution of Q was obtained. Bacterial cells were removed from this culture solution by centrifugation. The obtained supernatant crude enzyme solution was subjected to 50% ammonium sulfate precipitation, ion exchange with DEAE-cellulose, and gel filtration with Sephadex G75 according to conventional methods to obtain purified protease. The optimal temperature for the action of this purified protease was investigated and found to be 55°C.

Furthermore, after treatment at 75°C for 15 minutes, 90% of the activity at 55°C was expressed.

Claims (1)

[Claims] 1. Erwinia uredovora is a new bacterial species Erwinia Nep that differs in mycological properties in at least (1) generating hydrogen sulfide from cysteine and (2) hydrolyzing casein.
5.