JP3125955B2 - Thermostable phosphoenolpyruvate carboxylase and method for producing the same - Google Patents

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 a body fluid, and a method for producing the same.

[0002]

2. Description of the Related Art PEPC is used for measuring bicarbonate ions in body fluids because it acts on phosphoenolpyruvate and bicarbonate ions to catalyze a reaction that produces oxalacetic acid and phosphate ions. Measurements of carbon dioxide content in body fluids, especially serum and plasma, are useful for medical diagnosis of diseases related to acid-base equilibrium, for example, elevated bicarbonate content can indicate metabolic alkalosis, respiratory acidosis,
Low levels are seen in respiratory metabolic acidosis and respiratory alkalosis. PEPC has been reported to be widely present in plant tissues and most bacteria. P currently on the market
Many EPCs are made from plants such as corn and wheat. The pH of the bicarbonate ion measurement reagent was adjusted to pH 8.0 so that bicarbonate ions in the sample did not form carbon dioxide gas.
These are widely used, but it has been known that plant-derived PEPC has poor stability at the used pH (8.0 or more) and has a problem in practicality. Although PEPC having excellent stability may be derived from a thermophilic bacterium, most of the bacterial PEPC is acetyl coenzyme A, fructose-1,6 which is expensive for expressing its activity.
It was also known that it was not practical because it required an activator of -diphosphate or glucose-6-phosphate.

[0003]

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

[0004]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to obtain a practically useful PEPC which overcomes the above-mentioned problems, and as a result, Acetobacter pasturianus (Acetobacter) has been studied.
Bacteria pasteurianus) PEPC targeted by IFO14814
And found that the present invention was completed.

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

[0006]

Embedded image (2) Optimum action pH: 7.0 (3) pH stability: pH 6.0 to 8.5 (25 ° C., 2
(4 hours treatment) (4) Optimum action 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)

[0007] The present invention also relates to a bacterium belonging to the genus Acetobacter and having the above-mentioned properties and capable of producing a thermostable phosphoenolpyruvate carboxylase, which is cultured in a nutrient medium. A method for producing thermostable phosphoenolpyruvate carboxylase, which comprises collecting.

[0008] The enzyme of the present invention may be of any origin, such as plants and microorganisms, as long as it can produce phosphoenolpyruvate carboxylase having the above properties. Preferably, it is an Acetobacter bacterium capable of producing phosphoenolpyruvate carboxylase having the above properties, and a preferred example is Acetobacter pasteurianus IFO1.
4814.

The enzyme of the present invention can be produced by culturing the above-mentioned phosphoenolpyruvate carboxylase-producing bacterium in a nutrient medium and collecting phosphoenolpyruvate carboxylase from the culture. The culture medium used for culturing phosphoenolpyruvate carboxylase-producing bacteria may be any of a synthetic medium and a natural medium as long as the used strain can utilize carbon sources, nitrogen sources, inorganic substances, and other necessary nutrients in appropriate amounts. Can be used. As the carbon source, for example, 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 is usually performed by shaking culture or aeration and stirring culture. The cultivation temperature is controlled in the range of 20 to 40 ° C, preferably 25 to 30 ° C, and the culture pH is 5 to 9, preferably 7 to 8. It can be carried out under conditions other than these provided that 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.

The enzyme of the present invention may be purified by a commonly used purification method. 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 methods such as ammonium sulfate and sodium sulfate, metal aggregation methods such as magnesium chloride and calcium chloride, aggregation methods such as protamine and polyethyleneimine, and DEAE
It can be purified by an ion-exchange chromatography method such as (diethylaminoethyl) -sepharose and CM (carboxymethyl) -sepharose. 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 immobilized on a suitable carrier and used as an immobilized enzyme.

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, 50 U
Reaction mixture containing 2.9 / ml malate dehydrogenase 2.9
Prepare a ml in a cuvette (d = 1 cm) and preheat at 30 ° C. for about 5 minutes. Next, 0.1 ml of the enzyme solution was added, and after gentle mixing, the change in absorbance at 340 nm was recorded for 2 to 3 minutes with a spectrophotometer controlled at 30 ° C. using water as a control. The change in absorbance is determined (△ ODtest). Blind is 5 instead of enzyme solution
0 mM phosphate buffer (pH 7.0) was added, and the same operation was performed to determine the change in absorbance per minute (ΔOD
blank). The activity of the phosphoenol pyruvate carboxylase was determined to be 1 μmol N
The amount of enzyme that consumes ADH is defined as one unit (U).

[0012]

According to the present invention, an expensive effector is not required, the stability is good at pH 8.0,
A stable phosphoenolpyruvate carboxylase having excellent heat resistance can be obtained even after treatment for minutes. By using the enzyme of the present invention, a highly stable reagent for measuring carbon dioxide gas 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 121 ° C., 15 minutes Autoclave was performed.
Acetobacter pasteurianus (Acetob)
acter pasteurianus) Inoculate a platinum loop of IFO14814, 30
C. for 72 hours to obtain a seed culture solution. Next, 6 l of the same medium
Was transferred to a 10-liter jar fermenter at 121 ° C. for 15 minutes.
After autoclaving for 10 minutes and allowing to cool,
1 at 300 rpm, aeration 21 / min, 30 ° C.
Incubated for 2 hours. The culture is collected by centrifugation, and 50 m
M phosphate buffer (pH 7.0). This solution was treated with a French press and centrifuged to obtain a supernatant.
The obtained 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 using Sephadex G-200.

The obtained phosphoenolpyruvate carboxylase had the following properties. (1) The following reaction was catalyzed.

[0015]

Embedded image

(2) Km value The Km value for phosphoenolpyruvate is 2.79 m
M. (3) Optimum pH 50 mM MES buffer (pH 6.0-7.5), 50
The enzyme activity was measured in a mM Tris-HCl buffer (pH 7.5 to 9.0). The result was as shown in FIG. 1 and the optimum pH was 7.0. (4) Stable pH The solution was stored in a Britton-Robinson's buffer (pH 3 to 12) and Tris-HCl buffer (pH 7.5 to 9.0) at 25 ° C. for 24 hours, and its residual activity was measured. As a result, stable p
H was pH 6.0-8.5 (FIG. 2). (5) Optimum temperature The enzyme activity at each temperature was measured. The result was as shown in FIG. 3, and the optimum temperature was 60 ° C. (6) Thermostability After the enzyme of the present invention was incubated for 15 minutes in 50 mM K-phosphate (pH 7.0), the remaining enzyme activity was measured. The result was as shown in FIG. 4 and was stable up to 55 ° C. Further, after keeping the solution in 50 mM K-phosphate (pH 8.0) for 15 minutes, the remaining enzyme activity was measured, and as a result, it was stable up to 50 ° C. (7) Optimal magnesium concentration Enzyme activity was measured by 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 It was 5.8 ± 0.1 by isoelectric focusing. (10)
The effects of acetyl-CoA and ADP were as follows.

[0017]

[Table 1]

(11) Comparison with known enzymes Comparison between the enzymes of the present invention and known enzymes derived from acetic acid bacteria was as follows.

[Table 2]

In addition, comparison was made with known enzymes from other sources.

[Table 3]

As is apparent from Tables 1 to 3, the enzyme of the present invention is not activated by acetyl coenzyme A,
Not inhibited by P and stable at pH 8.0;
Further, it is stable at 55 ° C. or lower.

[Brief description of the 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.

Continuation of front page (56) References JP-A-4-228074 (JP, A) JP-A-5-111397 (JP, A) Arch. Microbiol. , 122 (1979), p. 109J. Bacteriol. , 98 (1969), p. 1005 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 9/88 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A thermostable phosphoenolpyruvate carboxylase having the following physicochemical properties: (1) catalyze the next reaction. Embedded image (2) Optimum action pH: 7.0 (3) pH stability: pH 6.0 to 8.5 (25 ° C., 2
(4 hours treatment) (4) Optimal operating temperature: 60 ° C (5) Thermal stability: stable up to 55 ° C (pH 7.0, 15
(6 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 capable of producing a thermostable phosphoenolpyruvate carboxylase having the following physicochemical properties is cultured in a nutrient medium, and a thermostable phosphoenolpyruvate carboxylase is obtained from the culture. A method for producing thermostable phosphoenolpyruvate carboxylase, which comprises collecting. (1) catalyze the next reaction. Embedded image (2) Optimum action pH: 7.0 (3) pH stability: pH 6.0 to 8.5 (25 ° C., 2
(4 hours treatment) (4) Optimum action 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)