JPH0712306B2 - Culture medium - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention is directed to 1,5-anhydroglucitol (hereinafter, “1,5-
"AG") relates to a culture medium for culturing a microorganism capable of producing an oxidase. 1,5-AG oxidase is useful for quantifying 1,5-AG, which is expected as a diagnostic marker for diabetes.

[Conventional technology]

1,5-AG is present in human cerebrospinal fluid and plasma, and it is known that plasma 1,5-AG level is lowered in certain diseases, especially in diabetes, and is effective as a diagnostic marker for diabetes. Is a reported compound.

This quantification of 1,5-AG is currently mainly performed by chromatography.

[Problems to be solved by the invention]

Since the conventional method for quantifying 1,5-AG is complicated in operation, a 1,5-AG oxidase capable of simplifying the operation is useful. However, the existence of this enzyme is unknown and therefore no means for the production of this enzyme has been proposed.

[Means for solving problems]

Therefore, as a result of various studies by the present inventors, when a microorganism having the ability to produce 1,5-AG oxidase is cultured in a medium containing 1,5-AG, a microorganism having high 1,5-AG oxidative activity is obtained. It was found that 1,5-AG oxidase can be collected from this microorganism.

The present invention has been completed based on the above findings.

1,5-AG oxidase is a new enzyme that catalyzes the following reaction,
It is present in the membrane fraction of microorganisms.

Microorganisms capable of producing 1,5-AG oxidase have the ability to convert 1,5-AG into the corresponding ketone bodies, and any microorganisms having such ability can be used in the present invention. It can be cultured in a medium. As a carbon source, 1,5-A
When a medium containing only G is used, only microorganisms capable of producing 1,5-AG oxidase will grow, so by using that medium, 1,5-AG from the natural world such as soil
Microorganisms capable of producing oxidase can be isolated.

As a microorganism having a 1,5-AG oxidase-producing ability to which the medium of the present invention can be applied, a microorganism belonging to the genus C. pseudomonas,
For example, Pseudomonus spNK-85001
(Microtechnology Research Institute No. 1037), microorganisms belonging to the genus Enterobacter, for example, Enterobacter cloacae (Entero
bactor cloacae) IFO3320, a microorganism belonging to the genus Citrobacter, for example, Citrobac
tor freundii) IFO12681, a microorganism belonging to the genus Serratia,
For example, Serratia marcescen
s) IFO12648 and the like.

The medium of the present invention contains 1,5-AG as a carbon source. The amount is 0.05 to 1%, preferably 0.1 to 0.5, based on the total amount of the medium.
%. Note that other carbon sources such as glucose, glycerin, and succinic acid may be contained in a range that does not substantially inhibit the ability of the microorganism to produce 1,5-AG oxidase.

The medium of the present invention contains a nitrogen source and salts in addition to 1,5-AG. Examples of the nitrogen source include inorganic nitrogen such as ammonium sulfate and ammonium chloride, and organic nitrogen such as peptone, casamino acid, meat extract, corn steep liquor and yeast extract. As salts, sodium, potassium,
Examples include salts of magnesium, calcium and iron.
The pH of the medium is preferably 6 to 7, the culturing temperature of the microorganism using this medium is 25 to 35 ° C., and the culturing time is 16 to 24 hours. In the case of a liquid medium, aerobic conditions such as aeration stirring culture and shaking culture are preferable.

Collection of 1,5-AG oxidase from the culture cultivated under the above conditions is performed by a conventional method. An example is as follows. That is, since this enzyme exists in the membrane fraction of bacterial cells,
First, bacterial cells are separated from the culture, the bacterial cells are destroyed in an appropriate buffer solution, and a membrane fraction is obtained from the treated solution.

As a method for destroying the bacterial cells, a physical method such as Dynomill, French press, ultrasonic wave or the like, a chemical method such as Triton X-100, EDTA or the like and an enzymatic method such as lysozyme or the like can be used alone or in combination. The membrane fraction can be obtained in a suspended state, which is separated from cell wall components, nucleic acids, intracellular soluble proteins, etc. by utilizing different centrifugal forces from the cell disruption solution multiple times.

In addition, the suspension was treated with Triton X-100 (polyoxyethylene octylphenyl ether), cholic acid, deoxycholic acid and other membrane component solubilizers to extract the active ingredient, and the insoluble matter was removed by centrifugation to remove the active ingredient. A 1,5-AG oxidase extract can be obtained, and the 1,5-AG oxidase can be isolated from the extract using a method generally used for purifying enzymes such as ammonium sulfate fraction.

Next, as an example, various properties of the 1,5-AG oxidase obtained from C. spp.

1. Oxidize the working 1,5-AG to produce a ketone body.

2. Reference specificity Acts specifically on 1,5-AG.

3. Optimum pH pH 6 to 7.5 4. Optimum temperature 25 to 35 ° C. 5. Stable pH 6.5 to 8 [Effect] Next, the effect of the present invention will be described with reference to experimental examples.

Experimental Example 1,5-AG Oxidizing capacity measurement (1) Experimental method The microorganisms shown in Table 1 below were analyzed by the method of Example (2) described below.
The cell suspension obtained by culturing in A medium containing 5-AG was used as a sample.

In addition, B medium in which 1,5-AG in A medium was replaced with glucose was used, and the bacterial suspension obtained in the same manner as above was used as a control sample.

1,5-AG was added to this sample or control sample solution so that the concentration was 2 mg / ml, and the mixture was shaken in 100 ml triangular Kolben at 27 ° C. After reacting for 2 hours, the presence or absence of 1,5-AG in the solution was measured by the chromatographic method. That is, the reaction solution was spotted onto a silica gel plate, developed with a solvent of iso PrOH-H ₂ O (95: 5), dried well, and sprayed with an anisaldehyde sulfuric acid reagent to 90-100.
When heated to ℃ for 5-10 minutes, 1,5-AG can be observed as blue spot near Rf0.6.

(2) Experimental results The results are shown in Table 2.

As is clear from this table, in the case of the microorganisms cultured in the medium containing no 1,5-AG, most of the 1,5-AG remained, whereas in the microorganisms cultured in the medium containing 1,5-AG, If 1,5-
AG has disappeared.

Therefore, by using the medium of the present invention, a microorganism having a high 1,5-AG oxidative activity can be obtained, and a 1,5-AG oxidase can be obtained from this microorganism.

Example (1) Isolation of 1,5-AG oxidase-producing bacterium A soil sample obtained from a field in Yoshino-cho, Omiya city was soaked in sterilized saline, and the supernatant was 100 m of A medium having the following composition.
The mixture was added to a 500 ml Erlenmeyer flask containing l and cultured at 27 ° C. for 2 days on a rotary shaker. A medium composition: 1,5-AG0.2
%, Ammonium sulfate 0.1%, dipotassium phosphate 0.1%,
Magnesium sulfate 0.02%, salt 0.1%, yeast extract 0.1%
pH7, tap water. After the completion of the culture, the culture solution was centrifuged to collect the bacterial cells, which were washed with sterilized saline, diluted, spread on an agar plate prepared by adding 2% agar to the A medium, and cultured at 27 ° C. for 2 days in a thermostat. The colonies that appeared were purified by a conventional method and separated into slant medium having the same composition.

(2) Cultivation of 1,5-AG oxidase-producing bacterium S. cerevisiae spNK-85001 isolated by the above method
A slant culture of the strain (Ferm P8100) was inoculated into an Erlenmeyer flask containing A medium with 1 platinum loop and 27 ° C on a rotary shaker.
It was cultured for 16 hours. Collect the cells from the culture medium by centrifugation to 50 mM
Wash with phosphate buffer pH 7 and apply in the same buffer. 10 ml of the washed bacterial cell suspension was obtained from 100 ml of the culture solution.

(3) Collection of 1,5-AG oxidase 100 ml of the washed bacterial cell suspension of the C. pseudomonas spNK85001 strain obtained by the method of (2) above was sonicated to destroy the bacterial cells and centrifuged (10,000 xg, The residue was removed by 10 minutes). The supernatant was further centrifuged (80,000 xg, 1h) to obtain a precipitate, and the supernatant was discarded. The precipitate was washed with 50 mM phosphate buffer pH 7 and centrifuged, and then 10 ml of the buffer was used to obtain an enzyme solution. (Protein content 10 mg / ml) Example 2 casamino acid 1%, 1,5-AG0.2%, (NH 4) 2 SO 4 0.1%, K 2
A 100 ml medium consisting of HPO ₄ 0.1%, NaCl 0.1%, MgSO ₄ / 7H ₂ O 0.02%, yeast extract 0.1%, pH 7 distilled water was dispensed into a 500 ml Erlenmeyer flask and sterilized at 115 ° C for 15 minutes to sterilize Pseudomonas. sp.
One platinum loop of a slope culture of NK-85001 [Microtechnological Research Institute No. 8100 (Ferm p-8100)] is inoculated and cultured at 30 ° C for 16 hours on a rotary shaking culture machine (220 rpm). The cells are separated from the culture solution by centrifugation and washed with Tris / HCl buffer (0.05M, pH7) to give a 1/10 volume of the starting cell suspension. The cell suspension was cooled and a cell disruption solution was obtained by French press. This was centrifuged for 10 minutes (10,000 xg) to remove the precipitating cell wall, and then further centrifuged for 1 hour (100,000 x 100,000 x g) to obtain a precipitate, which was washed with Tris-HCl buffer (0.05M, pH7) and suspended in the same buffer to give a specific activity.
0.23 of 1,5-AG oxidase suspension is obtained. Triton X-100 was added to this suspension at a concentration of 1% (w / v), and the temperature was 4 ° C.
After stirring for 1 hour, insoluble matter is centrifuged (100,000 x
g) and removed to obtain a 1,5-AG oxidase extract having a specific activity of 0.47.

Also, while cooling the obtained 1,5-AG oxidase extract solution, ammonium sulfate powder was added to make it 40% ammonium sulfate saturation, and the precipitated protein was separated by centrifugation (10,000 xg, 10 minutes) to give a specific activity of 0.85. Obtain 1,5-AG oxidase.

The value of specific activity is 1 unit of enzyme and 2μmo of ferricyanide.
It is the value calculated as the activity of reducing les for 10 minutes and converted per 1 mg of protein.

Reference Example 1,5-AG quantification by 1,5-AG oxidase 0.1 ml of the enzyme solution obtained in Example 1 (3), 50 mM phosphate buffer pH 7 0.5 ml, distilled water 0.2 ml, 0.1 M potassium ferriciyanide 0.1 ml, a known concentration of 1,5-
0.1 ml of AG solution was added and reacted at 34 ° C for 10 minutes. After the reaction, ferric sulfate-depropanol reagent (5 g of ferric sulfate, 3 g of sodium lauryl sulfate, 95 ml of 85% phosphoric acid, 1 with distilled water) 0.5 m
l and 3.5 ml of distilled water were added, and the mixture was allowed to stand at room temperature for 10 minutes and then the absorbance at 660 nm was measured to prepare a calibration curve.

Next, for a solution containing 1,5-AG 500 mg / ml, glucose 1 mg / ml and sorbitol 1 mg / ml as a sample, the absorbance at 660 nm was measured by the same operation as the calibration curve creation, and the concentration of 1,5-AG was determined from the calibration curve. I asked.

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Claims (1)

[Claims] 1. A culture medium for culturing a microorganism having an ability to produce 1,5-anhydroglucitol oxidase, which contains 1,5-anhydroglucitol.