JPH09238676A - Vibrio SP SMY-2 strain - Google Patents

Abstract

(57) Abstract: An object of the present invention is to provide a microorganism having a high specific activity and ability to produce alkaline phosphatase. The present invention belongs to the genus Vibrio, and alkaline phosphatase (EC 3.1.3.
1) Vibrio sp. That has the ability to produce
The SMY-2 strain (FERM P-15017) is provided. As shown in FIG. 1, the growth of the Vibrio sp. SMY-2 strain of the present invention reached a maximum about 8 hours after the start of the culture, and the AlP activity in the cells rapidly increased about 5 hours after the start of the culture. However, it reached its maximum after about 8 hours, and its activity did not change until about 15 hours after the culture was completed.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel microorganism which produces alkaline phosphatase (EC 3.1.3.1) (hereinafter abbreviated as AlP) having a high specific activity.

[0002]

2. Description of the Related Art In recent years, enzymes have been widely used for analysis of various components in the medical field, food field, etc. because of their easiness of optical quantification, due to their stability of reaction and substrate specificity. There is. Among them, AlP is widely used for various diagnosis as a labeling enzyme in enzyme immunoassay. Al
When P is used as a labeling enzyme, AlP is bound to an antigen, an antibody of a substance to be analyzed or a substance that specifically binds to a target substance to be analyzed, and this is added to a test sample to bind to the substance to be analyzed. Then, the newly generated conjugate is separated and the AlP activity of this conjugate is measured, whereby the substance to be analyzed can be quantified.

[0003]

In such an analysis, the substance to be analyzed is often present only in a trace amount in the test sample, and therefore analysis conditions excellent in measurement sensitivity are desired. Since labeling with AlP is due to intermolecular binding between AlP and the above substances, in order to obtain high measurement sensitivity,
It is necessary that the AlP molecule show high activity. For that purpose, it is desirable that the labeling enzyme AlP has a high specific activity.

Conventionally, AlP is mainly produced from Escherichia coli or calf small intestine, and its supply source is limited. However, since AlP derived from Escherichia coli has a low specific activity, the labeling enzyme is mainly AlP derived from calf small intestine.
Is used. However, calf intestine-derived AlP
Upon crushing and extracting from organs, after complicated operations such as using protease or organic solvent, it was necessary to purify using several kinds of columns.

For these reasons, A showing a high specific activity
There has been a strong demand for means for efficiently obtaining 1P.
An object of the present invention is to provide a novel microorganism that produces AlP having a high specific activity.

[0006]

[Means for Solving the Problems] In order to solve the above problems, the present inventors have extensively screened natural microorganisms, and as a result, have selected AlP-producing microorganisms belonging to the genus Vibrio showing high specific activity. Heading out, the present invention has been completed.

[0007] That is, the present invention is a Vibrio
Vibrio sp. SMY-2 strain (FERM) characterized by belonging to the genus and having the ability to produce AlP
P-15017) is the main point.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.

First, the strain of the present invention, Vibrio sp. SMY-2 strain, and the existing Vibrio sp. Strains, which are considered to be related to it, are Vibrio splendidus biovar 1 and biovar 2 and Vibrio. The mycological properties of Vibrio tubiashii will be described. In addition, d is different depending on the strain, ND is not described, and FA is facultative anaerobic.

Vibrio sp. Vibrio splendidus Vibrio SMY-2 biovar 1 biovar 2 tubiashii (1) Morphological characteristics (a) Cell shape Bacillus Bacillus Bacillus Bacillus (b) Motility Yes Yes (c) Flagella Monopolar hair Pole Hair Polar hair Polar hair (d) Sporulation ability No No No No (e) Gram stainability Negative Negative Negative Negative (2) Growth state (a) ZoBell 2216b Shows circular shape ND ND ND Agar plate culture surface is smooth (b) Liquefaction of gelatin Yes Yes Yes Yes (3) Physiological properties (a) No reduction of nitrate Yes Yes Yes (b) VP test negative negative negative negative (c) oxidase activity positive positive positive positive (d) catalase activity positive positive positive positive (E) Dye formation No No No No (f) Luminous No Yes Yes No No (g) Starch Yes Yes Yes Yes Yes (h) Arginine Yes No Yes d Hydrolysis (i) Salt requirement 0% NaCl Growth under no ND ND ND under 2% NaCl Growth Yes ND ND ND (j) Range of growth No growth at 4 ° C No d No No Growth at 35 ° C Yes d No d (k) Behavior to oxygen FA FA FA FA (l) OF test F F F F (m ) PHB accumulation No No No ND (n) Use of PHB No No No d (o) Lipase activity Positive Positive Positive Positive Positive (p) Mannitol No Yes Yes Catabolism (q) Acid generation from various carbon sources D-Glucose Yes Yes Yes Yes ND Cellobiose No Yes d Yes D-Galactose No Yes No Yes Gluconic acid No d No Yes L-Glutamic acid Yes Yes d ND Glycine Yes Yes d Yes Putrescine No Yes No d Sucrose Yes d No Yes Trehalose Yes Yes Yes Yes (r) Production of gas without glucose from glucose (3) GC content of DNA 45 45 to 46 45 to 46 43 to 45 (mol%) Vibrio sp. SMY shown above
-2 based on the bacteriological characteristics of the strain, Barge's Manual of Systematic Bacteriology (Barg
ey's Mannual of Systematic Bacteriology, Vol. 1 (1984), Bargey's Mannual of Systematic Bacteriology (Bargey's Mann)
ual of Determinative Bacteriology) 9th edition (19
1994), International Journal of Systematic Bacteriology (International Jo
urnal of Systematic Bacteriology) Volume 34, 1-
As a result of examination from the properties of various bacteria described on page 4 (1984), this strain was found to belong to the genus Vibrio.

[0011] Further, the present strain showed properties very similar to those of Vibrio splendidas (Vibrio splendidus biovar 1 and biovar 2) and Vibrio tubiashii among bacteria described in the above-mentioned literature. However, while this strain has no ability to reduce nitrate and assimilate mannitol, Vibrio splendidus (Vibrio splendidus biovar 1 and biovar 2) and Vibrio tubiashii both have the ability, This strain was judged to be different from these species. Therefore, it can be judged that this strain is a new strain. Therefore, Vibrio sp.
It was named MY-2 strain and deposited on June 29, 1995 at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry. Its deposit number is FERM P-15017.

As the nitrogen source added to the nutrient medium for culturing the strain of the present invention, inorganic or organic substances such as ammonium sulfate, ammonium chloride, peptone, meat extract, yeast extract and casamino acid can be used. Further, as the carbon source, glucose, sucrose, starch or the like can be used. Furthermore, as inorganic salts, potassium, sodium, iron, magnesium, manganese, and other salts,
You may use vitamins.

The culture conditions are aerobic or microaerobic conditions, and the culture temperature is 10 to 35 ° C, preferably 20 to 30 ° C, most preferably 25 to 30 ° C.
Done below. The pH of the medium is 6.0 to 9.5,
It is preferably 7.0 to 9.0, and most preferably 7.4 to 8.6. Under such conditions, 3 to 40 hours, preferably 4
By culturing for about 20 hours, optimally for 5 to 15 hours, it is possible to obtain cells having AlP production ability.

As a method for isolating and purifying AlP from the strain thus obtained, for example, after the completion of the culture, the cells are recovered from the culture solution by an operation such as centrifugation or filtration, and the crude cells are recovered from the cells. The enzyme solution may be extracted and purified. As the extraction method, any of ultrasonic crushing, pressure crushing, surfactant treatment, lysozyme treatment and the like may be used, and after such treatment, cell debris is removed by centrifugation to obtain a crude enzyme solution. With respect to the crude enzyme solution, the desired AlP can be isolated and purified by combining chromatography such as ion exchange chromatography, affinity chromatography, hydrophobic chromatography, gel filtration chromatography and the like. As an ion exchange resin, Q
AE-Toyopearl 550C (manufactured by Tosoh Corporation), DEAE-
As a resin for affinity chromatography such as Sepharose FF (Pharmacia), Red Sepharose C
Examples of resins for hydrophobic chromatography such as L-6B and Blue Sepharose CL-6B column (all manufactured by Pharmacia) include octyl-sepharose CL-4B column (manufactured by Pharmacia) and phenyl sepharose 6FF (manufactured by Pharmacia). As the carrier for filtration or the resin,
Sephadex G-100, Ultrogel ACA34 and the like can be mentioned. In addition to these column chromatography, nucleic acid removal by streptomycin sulfate or protamine sulfate treatment and salting out of protein by ammonium sulfate treatment may be performed.

The activity was measured with 10 mM sodium p-nitrophenyl phosphate (hereinafter abbreviated as PNPP .: purchased from Nacalai Tesque, Code No. 250-
19GR), 1.0 mM magnesium chloride hexahydrate (purchased from Wako Pure Chemical Industries, Code No. 135-0016)
5M) containing 1M diethanolamine (hereinafter abbreviated as DEA .: purchased from Wako Pure Chemical Industries, code No. 093-
(03115) The enzyme solution was added to the buffer solution (pH 10.0), gently mixed, and then measured by the change in absorbance at 405 nm. The measurement was performed at 37 ° C., and the amount of enzyme that produced 1 μmol of p-nitrophenol per minute was 1 unit (U).

[0016]

EXAMPLES Next, the present invention will be specifically described with reference to examples.

[Example 1] Yeast extract 0.1% (% by weight)
Represents The same shall apply hereinafter) (Oriental Yeast Co., Code N
o. 41207000), casamino acid (Nippon Pharmaceutical Co., Ltd., casamino acid "Daigo", Code No. 398-00657).
0.5%, iron (III) citrate n-hydrate (Wako Pure Chemical Industries, Ltd., Code No. 097-02415) 0.01%, sodium chloride (Wako Pure Chemical Industries, Ltd., Code No. 194-01677)
2.0%, magnesium chloride hexahydrate (Wako Pure Chemical Industries, code No.135-00165) 0.88%, sodium sulfate decahydrate (Wako Pure Chemical Industries, code No.193-).
03325) 0.32%, calcium chloride hexahydrate (Nacalai Tesque, Code No. 067-32EP)
0.18%, potassium chloride (Nacalai Tesque, code N
o. 285-13EP) 0.055%, sodium carbonate (Wako Pure Chemical Industries, Ltd., Code No. 196-01595) 0.0
16%, iron (II) sulfate heptahydrate (Nacalai Tesque, Code No. 195-31EP) 0.001%, potassium bromide (Nacalai Tesque, Code No. 285-02E)
P) 0.008%, strontium chloride hexahydrate (Nacalai Tesque, Code No. 325-08EP) 0.0
034%, boric acid (Wako Pure Chemical, Code No. 021-0
2195) 0.022%, sodium silicate (Wako Pure Chemical Industries, Ltd., Code No. 190-03215) 0.0004
%, Sodium fluoride (Wako Pure Chemical Industries, Code No. 196
-01975) 0.0024%, sodium nitrate (Wako Pure Chemical Industries, Ltd., Code No. 192-02555) 0.0001
6%, disodium phosphate dodecahydrate (Nacalai Tesque, Code No. 317-22EP) 0.0008%,
20 liters of a medium having a pH of 7.6 was placed in a jar fermenter having a volume of 30 liters, sterilized by autoclaving at 121 ° C for 15 minutes, and then vibrio sp.
io sp.) SMY-2 strain (FERM P-15017) was inoculated, and at 28 ° C. for 15 hours, 0.5 vvm, 300 rpm.
Cultured with aeration and stirring. At this time, the growth of the bacterium and the AlP activity in the bacterium were examined over time.

The growth of the bacteria depends on the turbidity of the culture solution (OD660).
It was investigated by measuring. The AlP activity in the cells was measured by the following procedure. That is, 1 ml of culture solution
Were centrifuged, and the precipitated bacterial cells were suspended in 1 ml of a 20 mM phosphate buffer (pH 7.5) and sonicated at 200 W for 10 minutes to disrupt the cells. After crushing, the cell debris was removed by centrifugation, and AlP in the obtained supernatant was
Activity was measured.

FIG. 1 shows the results. FIG. 1 shows the results of culturing the Vibrio sp. SMY-2 strain of the present invention,
FIG. 3 is a diagram showing the growth of bacteria and the time course of AlP activity in the bacterial cells, wherein the vertical axis represents bacterial growth and Al of bacterial cells in 1 ml of the culture solution.
The P activity is shown on the horizontal axis, and the culture time is shown.

As can be seen from FIG. 1, about 8 from the start of culture.
After a lapse of time, the growth of the bacteria reached the maximum, and the AlP in the bacterial cells reached
The activity rapidly increased from about 5 hours after the start of the culture, reached the maximum after about 8 hours, and did not change until about 15 hours after the end of the culture.

After completion of the culture, cells were collected by centrifugation to obtain about 70 g of cells. When the AlP content in the bacterial cells was measured, the value was about 2.6 million U / kg wet bacterial cells. The obtained bacterial cells were stored in a frozen state.

Reference Example 1 About 5 frozen cells obtained in Example 1
0 g was suspended in 0.4 liter of 20 mM phosphate buffer (pH 7.5), and the cells were disrupted by sonicating at 200 W for 10 minutes. After crushing, the cell debris was removed by centrifugation to obtain a crude enzyme solution containing AlP. This crude enzyme solution was previously equilibrated with 20 mM phosphate buffer (pH 7.5) to DEAE-Sepharose FF (Pharmacia, Code No. 17-0709-01) column (column size, inner diameter 3.2 cm, height. 12 cm)
20 mM phosphate buffer containing 0.5 M NaCl (p
H7.5), the concentration of NaCl was 0.2-
The activity eluted near 0.3M.

This eluate fraction was previously equilibrated with 20 mM phosphate buffer (pH 7.5) to QAE-Toyopearl 5
When it was passed through a 50C (Tosoh Corp., Code No. 014026) column (column size, inner diameter 3.2 cm, height 5 cm) and eluted by gradually increasing the NaCl concentration, N
The active fraction was eluted near the aCl concentration of 0.6 to 0.8M. Ammonium sulfate (Wako Pure Chemical Industries, Ltd., Code No. 019-06735) was added to this elution fraction so as to have a concentration of 1.0 M, which was previously equilibrated with a 20 mM phosphate buffer solution (pH 7.5) containing 1 M ammonium sulfate. Phenyl Sepharose 6FF (Pharmacia, Code No. 17)
-0973-05) Column (column size, inner diameter 1.6
cm, height 5 cm), the ammonium sulfate concentration of the same buffer solution was gradually lowered to elute, and the active fraction eluted near the ammonium sulfate concentration of 0.3 to 0.2 M.

This active fraction was collected, desalted, and equilibrated with 20 mM phosphate buffer (pH 7.5), Red Sepharose CL-6B (Pharmacia, Code No. 17).
-0961-02) column (column size, inner diameter 1.6
cm, height 5 cm), the NaCl concentration was gradually increased to elute, and the active fraction was eluted near the NaCl concentration of 0.6 to 0.8 M. AlP thus obtained
Was detected as a single band in polyacrylamide gel electrophoresis, and a purified enzyme preparation could be obtained. The activity yield was about 15%, and the specific activity was about 8,300 U per mg of enzyme. The obtained AlP had the following physicochemical properties.

(1) Action and substrate specificity: The phosphate monoester is hydrolyzed under alkaline conditions. The Michaelis constant of the reaction when the phosphoric acid monoester is p-nitrophenylphosphoric acid is about 1 mM [1M DEA buffer (pH 1
0.0) at 37 ° C.].

(2) Optimum pH: around 10.5 (temperature 37
(3 ° C) (3) Stable pH: 7-8 (4) Optimum temperature range: 30 ° C to 45 ° C (diethanolamine buffer pH 10.0) (5) Molecular weight: about 90,000 (Ultgel ACA34 gel filtration chromatography) Reference Example 2 Vibrio SP obtained in Reference Example 1 (Vibr
io sp.) SMY-2 strain-derived AlP and calf small intestine-derived AlP (purchased from Boehringer Mannheim, code No. 567744), E. coli-derived AlP (purchased from Sigma, code No. P-5931) The specific activity per 1 mg was determined as follows.

That is, each AlP solution was prepared so that the protein concentration was 0.2 to 0.3 mg / ml, and the AlP activity in this solution was measured. The protein was quantified using bovine serum albumin (Boehringer Mannheim, albumin fraction V, code No. 735094) as a standard, and a protein assay stain solution (Code No. 500-0006) manufactured by Bio-Rad Laboratories. Table 1 shows the results.

[0028]

[Table 1] As is clear from the results in Table 1, the AlP derived from the Vibrio sp. SMY-2 strain has a specific activity about 2.4 times that of AlP derived from the calf small intestine and about 88 times the specific activity of AlP derived from Escherichia coli. showed that.

[0029]

EFFECTS OF THE INVENTION Since the strain of the present invention has the ability to produce AlP having a high specific activity, it is possible to easily produce AlP having a high specific activity by culturing this strain.

[Brief description of drawings]

FIG. 1 Vibrio sp. S of the present invention
Growth of the MY-2 strain and microbial Al in the MY-2 strain
It is a figure which shows P activity over time.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location C12R 1:63) (72) Inventor Kazue Kawahara 23 Uji Kozakura, Uji City, Kyoto Prefecture Unitika Stock Company Central In the laboratory (72) Takuma Yano, 23 Uji Kozakura, Uji City, Kyoto Prefecture Unitika Central Corporation, Central Research Institute (72) Inventor, Ken Iwata, 23 Uji Kozakura, Uji City, Kyoto Unitika Central Company, Central Laboratory (72) Invention Person Koji Kondo 23 Uji Kozakura, Uji City, Kyoto Prefecture Unitika Ltd. Central Research Institute

Claims (1)

[Claims] 1. Vibrio s, which belongs to the genus Vibrio and has an alkaline phosphatase (EC 3.1.3.1) -producing ability.
p.) SMY-2 strain (FERM P-15017).