JPH06319542A - Novel thermostable phosphoenolpyruvate carboxylase and production thereof - Google Patents

Abstract

PURPOSE:To obtain the novel enzyme stable at a pH during the use of a reagent for measuring CO2 without being inhibited with ADP by culturing a microorganism, belonging to the genus Acetobacter and having the ability to produce a specific thermostable phosphoenolpyruvate carboxylase. CONSTITUTION:A strain, belonging to the genus Acetobacter and having the ability to produce a thermostable phosphoenolpyruvate carboxylase (e.g. Acetobacter pasteurianus IFO14814) is cultured in a nutrient culture medium to collect the resultant product from the culture. Thereby, the objective thermostable phosphoenolpyruvate carboxylase, capable of catalyzing the reaction expressed by the formula and having the following physico-chemical properties is obtained: optimum action pH: 7.0; pH stability: pH6.0-8.5 (treatment at 25 deg.C for 24hr); optimum action temperature: 60 deg.C; thermostability: stable to 55 deg.C (treatment at pH7.0 for 15min) and molecular weight: 390000+ or -10000 (measured by a gel filtration method) and 100000+ or -5000 (measured by an SDS- PAGE method).

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a thermostable phosphoenolpyruvate carboxylase (hereinafter abbreviated as PEPC) useful for measuring bicarbonate ion content in body fluids and a method for producing the same.

[0002]

2. Description of the Related Art PEPC acts on phosphoenolpyruvate and bicarbonate ions to catalyze the reaction to produce oxaloacetate and phosphate ions, and is therefore used for the measurement of bicarbonate ions in body fluids. Measurements of carbon dioxide content in body fluids, especially serum and plasma, are useful in the medical diagnosis of diseases related to acid-base balance, e.g. a high bicarbonate content indicates metabolic alkalosis or respiratory acidosis,
Low levels are seen in respiratory metabolic acidosis and respiratory alkalosis. It has been reported that PEPC is widely present in plant tissues and most bacteria. P currently on the market
Most EPCs are derived from plants such as corn and wheat. The pH of the bicarbonate ion measuring reagent is adjusted to pH 8.0 so that bicarbonate ions in the sample are not in the form of carbon dioxide gas.
Although the above-mentioned materials are widely used, it has been known that PEPCs of plant origin have poor stability at the pH used (8.0 or more) and have problems in practical use. Further, as a highly stable PEPC, one originating from a thermophilic bacterium is considered, but most of PEPCs of the bacterium are expensive acetyl coenzyme A and fructose-1,6 for their activity expression.
Since it requires an activator of diphosphoric acid and glucose-6-phosphoric acid, it has been known that it is not practical.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a PEPC which is stable in the use pH (8.0) of a reagent for measuring carbon dioxide and which does not require an expensive activator.

[0004]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to obtain a PEPC excellent in practicability that overcomes the above-mentioned problems, and as a result, Acetobacter pasteurianus (Acet
PEPC for the purpose of bacterium pasteurianus) IFO14814
The present invention has been completed and the present invention has been completed.

That is, the present invention is a thermostable phosphoenolpyruvate carboxylase having the following physicochemical properties. (1) It catalyzes the following reaction.

[0006]

[Chemical 3] (2) Optimum action pH: 7.0 (3) pH stability: pH 6.0 to 8.5 (25 ° C, 2
(4 hours treatment) (4) Optimum working temperature: 60 ° C (5) Thermal stability: 55 ° C or less (pH 7.0, 15 minutes treatment) (6) Molecular weight: 390,000 ± 10,000 (gel filtration method) 100,000 ± 5,000 (SDS-PAGE)

The present invention also belongs to the genus Acetobacter and is capable of producing a thermostable phosphoenolpyruvate carboxylase having the above-mentioned properties. The strain is cultured in a nutrient medium, and the thermostable phosphoenolpyruvate carboxylase is extracted from the culture. A method for producing a thermostable phosphoenolpyruvate carboxylase, which is characterized by collecting.

As the origin of the enzyme of the present invention, any origin such as plant or microorganism may be used as long as it can produce phosphoenolpyruvate carboxylase having the above-mentioned properties. Preferably, a bacterium belonging to the genus Acetobacter capable of producing phosphoenolpyruvate carboxylase having the above-mentioned properties, and a suitable example thereof is Acetobacter pasteurianus IFO1.
4814 is mentioned.

The enzyme of the present invention can be produced by culturing the phosphoenolpyruvate carboxylase-producing bacterium in a nutrient medium and collecting phosphoenolpyruvate carboxylase from the culture. As the medium used for culturing the phosphoenolpyruvate carboxylase-producing bacterium, a synthetic medium and a natural medium may be used as long as they contain an appropriate amount of a carbon source, a nitrogen source, an inorganic substance and other necessary nutrients that can be assimilated by the strain used. Can be used. Examples of carbon sources include peptones,
Nitrogen-containing natural products such as meat extract and yeast extract, and inorganic nitrogen-containing compounds such as ammonium chloride and ammonium citrate are used. As the inorganic substance, potassium phosphate, sodium phosphate, magnesium sulfate or the like is used. The culture medium is usually shake culture or aeration-agitation culture. The culture temperature is 20 to 40 ° C., preferably 25 to 30 ° C., and the culture pH is in the range of 5 to 9, preferably 7 to 8. It can be carried out under conditions other than these as long as the strain to be used grows. The cultivation period is usually 1 to 10 days, and phosphoenolpyruvate carboxylase is produced and accumulated in the cells.

As a method for purifying the enzyme of the present invention, a generally used purification method may be used. For example, for the extraction method, any of ultrasonic crushing, mechanical crushing using glass beads, French press, surfactant and the like may be used. Further, regarding the extract, salting-out method such as ammonium sulfate and sodium sulfate, metal coagulation method such as magnesium chloride and calcium chloride, coagulation method such as protamine and polyethyleneimine, and further DEAE
It can be purified by an ion exchange chromatography method such as (diethylaminoethyl) -sepharose, CM (carboxymethyl) -sepharose and the like. The crude enzyme solution and the purified enzyme solution obtained by these methods can be powdered by, for example, spray drying or freeze drying. Furthermore, it can be used as an immobilized enzyme by immobilizing it on a suitable carrier.

The activity of phosphoenolpyruvate carboxylase can be measured by the following method. 50m
M Tris-HCl (pH 8.0), 10 mM Na
₂ CO ₃ , 3.2 mM phosphoenolpyruvate, 1
00 mM MgSO ₄ , 0.14 mM NADH, 50U
/ Ml reaction mixture containing malate dehydrogenase 2.9
Prepare ml in a cuvette (d = 1 cm) and preheat at 30 ° C. for about 5 minutes. Next, 0.1 ml of enzyme solution was added, and after gently mixing, the change in absorbance at 340 nm was recorded for 2 to 3 minutes by a spectrophotometer controlled at 30 ° C. with water as a control, and from the initial linear portion, per minute The change in absorbance of is determined (ΔODtest). Blinding is 5 instead of enzyme solution
Add 0 mM phosphate buffer (pH 7.0) and perform the same operation to determine the change in absorbance per minute (ΔOD
blank). The activity of phosphoenolpyruvate carboxylase is 1 micromol N / min under the above conditions.
The amount of enzyme that consumes ADH is 1 unit (U).

[0012]

INDUSTRIAL APPLICABILITY The present invention does not require an expensive effector, has good stability at pH 8.0, and further has a temperature of 55 ° C. and 15 ° C.
A phosphoenolpyruvate carboxylase that is stable and has excellent heat resistance can be obtained even after treatment for a minute. By using the enzyme of the present invention, a stable carbon dioxide measuring reagent can be obtained at low cost.

[0013]

EXAMPLES The present invention will be specifically described below with reference to examples. Example 1 100 ml of a medium (pH 7.0) containing 0.5% of polypeptone, 0.5% of yeast extract, 0.5% of glucose and 0.05% of magnesium sulfate was transferred to a 500 ml Sakaguchi flask and the temperature was 121 ° C. for 15 minutes. It was autoclaved.
As an inoculum, Acetob
acter pasteurianus) IFO14814 inoculated with 1 platinum loop
Culturing was carried out for 72 hours at 0 ° C. to obtain a seed culture. Next, 6 liters of the same medium
Transfer to a 10 liter jar fermenter and stand at 121 ° C for 15
After autoclaving for 1 minute and allowing to cool, seed culture 100m
l, transfer at 300 rpm, aeration rate 21 / min, 7 at 30 ° C
It was cultured for 2 hours. The culture solution was collected by centrifugation to obtain 50m
It was suspended in M phosphate buffer (pH 7.0). This solution was treated with a French press and centrifuged to obtain a supernatant.
The resulting crude enzyme solution was purified to a specific activity of 51 U / mg by ammonium sulfate fractionation, DEAE-Sepharose chromatography, hydroxyapatite chromatography, and gel filtration with Sephadex G-200.

The resulting phosphoenolpyruvate carboxylase had the following characteristics. (1) The following reaction was catalyzed.

[0015]

[Chemical 4]

(2) Km value The Km value for phosphoenolpyruvate is 2.79 m.
It was M. (3) Optimum pH 50 mM MES buffer (pH 6.0 to 7.5), 50
The enzyme activity was measured in mM Tris-HCl buffer (pH 7.5-9.0). The result is as shown in FIG. 1, and the optimum pH was 7.0. (4) Stable pH Britton-Robinson's buffer (pH 3 to 12) and Tris-HCl buffer (pH 7.5 to 9.0) were stored at 25 ° C for 24 hours, and the residual activity was measured. As a result, stable p
H had a pH of 6.0 to 8.5 (Fig. 2). (5) Optimum temperature The enzyme activity at each temperature was measured. The result is as shown in FIG. 3, and the optimum temperature was 60 ° C. (6) Thermostability The enzyme of the present invention was kept in 50 mM K-phosphate (pH 7.0) for 15 minutes, and the residual enzyme activity was measured. The results are shown in Fig. 4, and were stable up to 55 ° C. In addition, after keeping the temperature in 50 mM K-phosphate (pH 8.0) for 15 minutes, the residual enzyme activity was measured. As a result, it was stable up to 50 ° C. (7) Optimum magnesium concentration The enzyme activity was measured while changing magnesium sulfate to 20 to 200 mM. The result is as shown in FIG.
00 mM was optimal. (8) Molecular weight 390,000 ± 10,000 (TSK-gel G-
Gel filtration method using 3000SW) 100,000 ± 5,000 (SDS-PAGE) (9) Isoelectric point The isoelectric point was 5.8 ± 0.1. (10)
The effects of acetyl CoA and ADP were as follows.

[0017]

[Table 1]

(11) Comparison with known enzyme The following was a comparison between the enzyme of the present invention and the known enzyme derived from acetic acid bacterium.

[Table 2]

Further, it was compared with known enzymes from other sources.

[Table 3]

As is clear from Tables 1 to 3 above, the enzyme of the present invention was not activated by acetyl coenzyme A, and AD
Not inhibited by P and stable at pH 8.0,
It is stable below 55 ° C.

[Brief description of drawings]

FIG. 1 shows the optimum pH of the enzyme of the present invention.

FIG. 2 shows the stable pH of the enzyme of the present invention.

FIG. 3 shows the optimum temperature of the enzyme of the present invention.

FIG. 4 shows the thermostability of the enzyme of the present invention.

FIG. 5 shows the optimum magnesium concentration of the enzyme of the present invention.

Claims (2)

[Claims] 1. A thermostable phosphoenolpyruvate carboxylase having the following physicochemical properties. (1) It catalyzes the following reaction. [Chemical 1] (2) Optimum action pH: 7.0 (3) pH stability: pH 6.0 to 8.5 (25 ° C, 2
(4 hours treatment) (4) Optimum working temperature: 60 ° C (5) Thermal stability: Stable up to 55 ° C (pH 7.0, 15)
Treatment for minutes) (6) Molecular weight: 390,000 ± 10,000 (gel filtration method) 100,000 ± 5,000 (SDS-PAGE) 2. A strain belonging to the genus Acetobacter and having the ability to produce thermostable phosphoenolpyruvate carboxylase having the following physicochemical properties is cultivated in a nutrient medium, and thermostable phosphoenolpyruvate carboxylase is produced from the culture. A method for producing a thermostable phosphoenolpyruvate carboxylase, which comprises collecting. (1) It catalyzes the following reaction. [Chemical 2] (2) Optimum action pH: 7.0 (3) pH stability: pH 6.0 to 8.5 (25 ° C, 2
(4 hours treatment) (4) Optimum working temperature: 60 ° C (5) Thermal stability: 55 ° C or less (pH 7.0, 15 minutes treatment) (6) Molecular weight: 390,000 ± 10,000 (gel filtration method) 100,000 ± 5,000 (SDS-PAGE)