JPH0838169A - Production of 3-hydroxybutyric dehydrogenase - Google Patents

Abstract

PURPOSE:To obtain the enzyme useful as an enzyme for measuring 3-butyric acid by culturing a bacterium belonging to the genus Alcaligenes, capable of producing 3-hydroxybutyric dehydrogenase. CONSTITUTION:A bacterium belonging to the genus Alcaligenes, capable of producing 3-hydroxybutyric dehydrogenase, preferably Alcaligenes sp. No.981 (FERM BP-2,570), is cultured in a medium and 3-hydroxybutyric dehydrogenase is collected from its culture product. The bacterium is cultured under aerobic conditions usually at 15-37 deg.C.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention is directed to Alcaligenes (Alc
3-hydroxybutyrate dehydrogenase (3-hydroxybutyrate deh) belonging to the genus
The present invention relates to a method for producing 3-hydroxybutyrate dehydrogenase, which comprises culturing a hydrogenase) -producing bacterium and collecting 3-hydroxybutyrate dehydrogenase from the culture.

[0002]

BACKGROUND OF THE INVENTION 3-Hydroxybutyrate dehydrogenase (EC.1.1.1.10) acts on 3-hydroxybutyrate in the presence of NAD to consume 1 mol of NAD, and 1 mol of acetoacetic acid. Also, it is known as an enzyme having a catalytic action of converting 1 mol of reduced NAD.

Up to now, regarding 3-hydroxybutyrate dehydrogenase, those derived from animals include, for example, rat brain (Bi
ochem. Cell Biol. , 68,980-9
83 (1990)), rat liver (Biochem. C).
ell Biol. , 68, 1225-2230 (19
90)), bovine heart (Arch. Biochem. Bi
ophys. , 262, 85-98 (1988)).

[0004] Further, as a microorganism-derived one, Rhodospirillum ( Rhodospirillum)
rubrum ) (J. Biol. Chem. 237, 6 ).
03-607 (1962)), Pseudomonas lemoigne
i ) (J. Biol. Chem. 240, 4023-4.
029 (1965)), Mycobacterium phlei (J. G.
en. Microbiol. 104, 123-126
(1978)), Paracoccus denitrificans (Paracoccusdenitrificans)
(Biochim. Biophys. Acta839,
300-307 (1985)), Zoogloea ramigera ( J. Bi ).
ochem. 89, 625-635 (1981)), Rhodops.
eudomonas spheroides ) (Bio
chem. J. 241,297-300 (198
7)), Azospirillum Brasilens ( Azospi
rillum brasilense ) (J. Gen.
Microbiol. 136, 645-649 (199
0)) and the like are known.

[0005]

However, it has been desired to develop a method for producing the enzyme using a strain having the ability to produce 3-hydroxybutyrate dehydrogenase other than these strains.

[0006]

[Means for Solving the Problems] The inventors of the present invention have earnestly studied for a production method capable of industrially producing 3-hydroxybutyrate dehydrogenase, and as a result, soil of a potato field in Gojo City, Nara Prefecture. Alcaligenes sp. NO. 981
The present invention was completed by first discovering that the strain has the ability to produce 3-hydroxybutyrate dehydrogenase.

The present invention has been completed on the basis of the above findings. Cultivating a 3-hydroxybutyrate dehydrogenase-producing bacterium belonging to the genus Alcaligenes and collecting 3-hydroxybutyrate dehydrogenase from the culture. Is a method for producing 3-hydroxybutyrate dehydrogenase. The present invention will be described in detail below.

First, the 3-hydroxybutyrate dehydrogenase-producing bacterium of the present invention is not limited as long as it is a microorganism capable of producing 3-hydroxybutyrate dehydrogenase belonging to the genus Alcaligenes. Variants and mutants capable of producing hydroxybutyrate dehydrogenase may be used, and preferably NO. 98
1 strain is listed, and this strain is a technical laboratory of the Agency of Industrial Science and Technology (currently;
It has been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology) with the deposit number, Micro Engineering Research Article No. 2570 (FERM BP-2570). The mycological properties, identification and nomenclature of this strain are described in detail in JP-A-3-127985 and US Pat. No. 5,173,416.

In carrying out the present invention, either liquid culture or solid culture can be used as the culture form, but industrially it is advantageous to carry out aeration stirring culture. As a culture source to be used, in addition to carbon sources, nitrogen sources, inorganic salts and other trace nutrients generally used for culturing microorganisms, any nutrient source that can be utilized by microorganisms belonging to the genus Alcaligenes can be used.

As the carbon source, glucose, fructose, saccharose, xylose, maltose, glycerol, dextrin, starch, amino acids, etc., as well as fatty acids, oils and fats, organic acids, etc. may be used alone or in combination. Either an inorganic nitrogen source or an organic nitrogen source can be used as the nitrogen source, and examples of the inorganic nutrient source include ammonium sulfate, ammonium nitrate, urea, sodium nitrate, ammonium chloride and the like. Examples of the organic nitrogen source include soybean, rice, corn, wheat flour and the like, corn steep liquor, peptone, meat extract, casein, amino acid, yeast extract and the like. Inorganic salts and micronutrients include phosphates, magnesium, potassium, iron, calcium, zinc, and other salts, as well as vitamins, nonionic surfactants, antifoaming agents, fungal growth, and 3-hydroxybutyrate dehydrogenase. Any material that promotes production can be used as needed.

The culture is carried out under aerobic conditions, and the culture temperature may be within the range where the bacteria grow and the 3-hydroxybutyrate dehydrogenase produces, and is usually 15 ° C to 37 ° C, preferably 25 ° C to.
It is 35 ° C. Although the culturing time varies depending on the conditions, it may be cultivated until the time when 3-hydroxybutyrate dehydrogenase is most produced, and it is usually about 24 to 100 hours. 3-hydroxybutyrate dehydrogenase is mainly contained in the cells,
It is accumulated and may be extracted from the cells. 3-
To illustrate the extraction method of hydroxybutyrate dehydrogenase, first the culture is subjected to solid-liquid separation, the obtained wet cells are dispersed in a solution such as phosphate buffer or Tris-hydrochloric acid buffer, lysozyme treatment, ultrasonication. A crude 3-hydroxybutyrate dehydrogenase-containing solution is obtained by appropriately selecting and combining various types of bacterial cell treatment means such as treatment, French press treatment, and dynomyl treatment.

Purified 3-hydroxybutyrate dehydrogenase is obtained from the crude liquid containing 3-hydroxybutyrate dehydrogenase by isolation and purification means of known proteins and enzymes. For example, the crude 3-hydroxybutyrate dehydrogenase-containing solution is subjected to fractional precipitation with an organic solvent such as acetone, methanol, or ethanol, or salting-out with ammonium sulfate or sodium chloride to precipitate 3-hydroxybutyrate dehydrogenase. Let it collect.
Furthermore, after subjecting this precipitate to dialysis and isoelectric point precipitation as needed, column chromatography using an ion exchanger, gel filtration agent, adsorbent, etc. until a single band is shown by electrophoresis etc. Purify by. Further, when the purification degree of 3-hydroxybutyrate dehydrogenase is improved by appropriately combining these methods, they can be appropriately combined.

The enzyme obtained by these methods is used as a stabilizer in a liquid form by a method of ultrafiltration concentration and lyophilization without or with addition of various salts, sugars, proteins, lipids, surfactants and the like. Solid 3-hydroxybutyrate dehydrogenase can be obtained, and may be appropriately lyophilized. In this case, saccharose, mannitol, sodium chloride, albumin, etc. are used as stabilizers in an amount of 0.5.
You may add about 10%.

Next, the physicochemical properties of the 3-hydroxybutyrate dehydrogenase obtained in the present invention and the method for measuring the enzyme activity will be described. Method for measuring activity of 3-hydroxybutyrate dehydrogenase 0.2 M Tris-hydrochloric acid buffer solution (pH 8.5) 0.2 m
1, 50 mM 3-hydroxybutyric acid 0.1 ml, 10 m
MNAD 0.1 ml, 100 U / ml diaphorase 0.05 ml, 0.25% NTB (nitrotetrazonium blue) 0.02 ml, 10% Triton X-10.
0 ml of 0.01 ml and 1 ml of a reaction liquid consisting of 0.52 ml of distilled water were preheated at 37 ° C. for 1 minute, and then 20 μm
l of enzyme solution is added and reacted for 10 minutes. After the reaction,
The reaction is stopped by adding 0.1N hydrochloric acid, and the absorbance at a wavelength of 550 nm is measured using a cell having a layer length of 1.0 cm within 5 minutes (As). Further, as a blind test, the same operation was performed using 20 μl of distilled water instead of the enzyme solution to measure the absorbance (Ab). The difference in absorbance between the absorbance (As) using this enzyme and the blind absorbance (Ab) (As) -Determine the enzyme activity from Ab). One unit of enzyme activity is the amount of enzyme that produces 1 μmol of reduced NAD per minute at 37 ° C., and the calculation formula is as follows.

[0015]

[Equation 1]

Physicochemical properties (1) Enzymatic action: The enzymatic action using 3-hydroxybutyric acid as a substrate is shown below.

[0017]

Embedded image

(2) Substrate specificity: Shows substrate specificity for 3-hydroxybutyric acid. The specificity for various substrates is shown in Table 1.

[0019]

[Table 1]

(3) Km value: 1.6 ± 0.5 (mM)
(For 3-hydroxybutyric acid) 0.12 ± 0.02 (mM) (for NAD) 4) Isoelectric point: 5.0 ± 0.2 (by electrophoresis using carrier-ampholine) ) (5) Molecular weight: 60,000 ± 5,000 (TSK G-3
Gel filtration with 000 SW), 30,000 ± 50
00 (by SDS polyacrylamide gel electrophoresis) (6) Optimum pH The optimum pH was determined according to the enzyme activity measuring method described above, and the results are shown in FIG. The pH range of 5.0 to 6.0 is 100 mM acetate buffer, the range of pH 6.0 to 7.5 is 100 mM phosphate buffer, the range of pH 7.5 to 9.0 is 100 mM Tris-HCl buffer, pH 9. 0-11.
The range of 0 indicates the activity value when 100 mM glycine-sodium hydroxide buffer is used, and the optimum pH is 8
It was in ~ 9. (7) pH stability 100 mM various buffer solutions (3-hydroxybutyrate dehydrogenase 1 U / ml) were treated at 37 ° C for 60 minutes, and the residual activity was measured according to the above-mentioned enzyme activity measuring method. The result is shown in FIG. pH 4.0-5.0 is 100 mM citrate buffer, pH 5.0-6.0 is 100 mM acetate buffer,
pH 6.0-7.5 is 100 mM phosphate buffer, pH
7.5-9.0 is 100 mM Tris-HCl buffer, pH
For 9.0 to 11.0, 100 mM glycine-sodium hydroxide buffer was used. The best stability was shown in the pH range of 7.5 to 11. (8) Optimum temperature The optimum temperature of the present enzyme is shown in Fig. 3 when the optimum temperature was determined by changing the temperature in the range of 25 ° C to 60 ° C according to the method for measuring enzyme activity. Was 45-50 ° C. (9) Thermostability 100 mM Tris-HCl buffer (pH 8.5) (3-
Hydroxybutyrate dehydrogenase 1U / ml) at each temperature 10
The residual activity after the heat treatment for 1 minute was measured according to the above-mentioned enzyme activity measuring method. As a result, it was stable up to at least 37 ° C. as shown in FIG. (10) Effect of Metal Ions The results of examining the effects of various metal ions (1 mM) on the activity of this enzyme are shown in Table 2, and strong inhibition by copper ions was observed.

[0021]

[Table 2]

[0022]

EXAMPLES Next, examples of the present invention will be described in detail.
The present invention is not limited to this.

[0023]

[Example 1] Alcaligenes SP NO. Strain 981 was inoculated into 100 ml of a medium (pH 7.0) consisting of 2% glutamic acid and 5.0% yeast extract, cultured at 28 ° C for 48 hours, and the resulting seed culture was defoamed in the same composition medium as above. To the medium (pH 7.0) containing the agent, and add 28
It was cultured at ° C for 48 hours. After the culture is completed, the culture is
The cells were centrifuged at 0 rpm for 30 minutes to collect 560 g of bacterial cells.

2 l of a 0.1% lysozyme solution containing 10 mM EDTA and 50 mM Tris-hydrochloric acid buffer (pH 7.5) was added to the cells, and the mixture was reacted at 37 ° C for 30 minutes.
Solubilization was performed. Then, this is 4500 rpm for 30
Insoluble matter was removed by centrifugation for 1 minute, and the supernatant 1870
ml (41100 U) was obtained. Then, 18.7 ml of 5% protamine sulfate was added to this supernatant to remove the precipitate by centrifugation, and the supernatant was added to a 20 mM Tris-hydrochloric acid buffer solution (pH 8.
5) It was dialyzed overnight with DEAE-Sepharose (2.7
X30 cm) (Pharmacia) ion exchange chromatography was performed. Elution is performed with a 0 to 1 M linear gradient of potassium chloride, 0.20.3 MKCl
The elution fraction (31700 U) was collected. NaCl was dissolved in this enzyme solution to a concentration of 4 M, and hydrophobic chromatography of phenyl sepharose (2.6 × 19.5 cm) (Pharmacia) was performed. Elution from 4M
Performed by a linear gradient of 0M NaCl, 1M-0.5
The elution fraction of M NaCl (2080 U) was collected.

Then, this enzyme solution was added to 10 mM Tris-
It was dialyzed against a hydrochloric acid buffer solution (pH 8.5) overnight, and Q-Sepharose (2.6 × 19.5 cm) (Pharmacia).
Was subjected to ion exchange chromatography. Elution is 0
Performed with a linear gradient of 0.4 M NaCl,
Elution fraction of 0.2-0.25M NaCl (20,000
U) was recovered. Furthermore, this enzyme solution was added to 10 mM Tris-
It was dialyzed overnight against a hydrochloric acid buffer solution (pH 7.5) and subjected to chromatography on hydroxyapatite (1.6 × 26.5 cm) (manufactured by Pentax). Elution is 0-0.3
Performed by a linear gradient with M phosphate buffer,
Elution fraction of 0.06-0.1M phosphate buffer (160
00U) was collected and lyophilized to obtain purified 3-hydroxybutyrate dehydrogenase (213U / mg, 75mg).

[0026]

According to the present invention, it is possible to provide a novel production method using a microorganism belonging to the genus Alcaligenes,
It was possible to provide an enzyme for measuring 3-hydroxybutyric acid, which is one of the ketone bodies using this enzyme.

[Brief description of drawings]

FIG. 1 shows an optimum pH curve of 3-hydroxybutyrate dehydrogenase of the present invention.

FIG. 2 shows a pH stability curve of the 3-hydroxybutyrate dehydrogenase of the present invention.

FIG. 3 shows an optimum temperature curve of 3-hydroxybutyrate dehydrogenase of the present invention.

FIG. 4 shows a thermostable curve of 3-hydroxybutyrate dehydrogenase of the present invention.

Claims (2)

[Claims] 1. A 3-hydroxybutyrate dehydrogenase comprising culturing a 3-hydroxybutyrate dehydrogenase-producing bacterium belonging to the genus Alcaligenes in a medium and collecting the 3-hydroxybutyrate dehydrogenase from the culture. Manufacturing method. 2. A 3-hydroxybutyrate dehydrogenase-producing bacterium belonging to the genus Alcaligenes belongs to Alcaligenes sp. The method for producing a 3-hydroxybutyrate dehydrogenase according to claim 1, which is strain 981 (Microtechnical Laboratory Article 2570).