JPH0797986B2 - Heat-resistant acetic acid kinase and method for producing the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a thermostable acetate kinase and a method for producing the same, and more specifically to a novel thermostable acetate kinase having various properties described below and production of various enzymes. The present invention relates to a method for efficiently producing the enzyme from a bacterium belonging to the genus Thermoanaerobium having the ability.

(Prior Art) Acetate kinase (EC 2.7.2.1) is an enzyme whose presence is found only in microorganisms and catalyzes the following reaction.

Adenosine triphosphate (ATP) + Acetate Adenosine diphosphate (ADP) + Acetyl phosphate This enzyme is used for the quantification of acetic acid in foods, etc., and its application to the ATP regeneration system in bioreactors has also been tried.

Conventionally, regarding acetate kinases, Escherichia coli, Clostridium acidi-urici, Clostridium thermoaceticum, Desulfovibrio desulfuric
ans), Propionibacterium shermani (Propion
ibacterium shermannii), Propionibacterium freudenreichii, Bacillus
Bacillus stearothermophilu
s), Veillonella alc
alescens), Acceleplasma Raideravi (Acheleplas)
Ma laidlavii) was attempted, and the Bayronella arcalessense [Archives of Biochemi and Biophysics (Archives of Biochemi
stry and Biophysics) 189 424,1978] and Bacillus stearothermophiles [Journal of Biochemistry 84 193,1978]
Is highly refined.

Among these, the use of Bacillus stearothermophilus-produced acetate kinase as an ATP-regenerating enzyme in constructing a bioreactor involving ATP by utilizing its thermostability has been investigated, but ( Japanese Patent Publication 59-3335
5), Acetate kinase as an enzyme for ATP regeneration in a better bioreactor is required to have properties such as higher temperature and pH stability.

(Object of the Invention) The inventors of the present invention have conducted various studies to obtain an acetate kinase having excellent stability and high enzymatic activity, which can be used for an ATP regeneration system in a bioreactor. The characteristics of acetic acid kinase, which has different properties in terms of temperature stability and pH stability and is highly enzymatically active, are the obligate anaerobic thermophile Thermoan Aerobium Blocky (Thermoanaero
bium brockii) found that it was produced, and completed the present invention.

(Structure of the invention) That is, the present invention is a thermostable acetate kinase having the following physicochemical properties a. Action Positive reaction produces adenosine diphosphate and acetyl phosphate from adenosine triphosphate and acetic acid, the reverse reaction is , Produces adenosine triphosphate and acetic acid from adenosine diphosphate and acetyl phosphate.

b. Substrate specificity Acetate is the best organic acid that can serve as a phosphate acceptor, and it also has a slight activity with propionic acid and n-butyric acid, and guanosine triphosphate (GTP) is a nucleotide that can serve as a phosphate donor. ) Is the most effective, followed by uridine triphosphate (UTP), cytidine triphosphate (CTP), and adenosine triphosphate in this order.

c.Km value Km value for acetyl phosphate: 0.025 mM, Km for ADP
Value: 1.25 mM d. Requirement of metal ion: Magnesium ion or manganese ion is required for the activity expression of this enzyme.

e. No decline in activity was observed even after treatment in the stable pH range of pH 4 to 11 at 30 ° C for 16 hours.

f. Optimum temperature of action: 55-60 ℃ g. Heat resistance: 75 ℃ Stable against heating for 15 minutes. 75 ° C 2
70% or more of the activity remains after heating for an hour.

h. Molecular weight: about 50,000 (gel filtration method using Sephadex G-100) and a novel heat-resistant acetic acid belonging to the genus Thermoan Aerobium and having the above-mentioned physical properties (a) to (h). A thermostable acetate kinase characterized in that a novel thermostable acetate kinase having the above properties (a) to (h) is collected from the culture by culturing a strain having a kinase-producing ability in a medium. It is a manufacturing method.

Examples of the bacterium used in the present invention include Thermoan Aerobium Blocky DSM 1457 which belongs to the genus Thermoan Aerobium mentioned above, but in addition to this bacterium, it belongs to the genus Thermoan Aerobium, and this novel heat-resistant acetic acid is used. All kinase-producing bacteria can be used in the present invention.

According to the present invention, the novel thermostable acetate kinase is accumulated in the bacterial cells by anaerobically culturing the thermostable acetate kinase belonging to the genus Thermoan Aerobium in a nutrient medium. The enzyme powder can be obtained by extracting, purifying, and drying with.

More specifically, the Thermoan Aerobium Blocky DSM 1457 belonging to the Thermoan Aerobium genus
By adding an appropriate carbon source, nitrogen source, inorganic salts and, if necessary, other nutrients and trace elements to the culture medium in an anaerobic atmosphere. Can be accumulated in bacterial cells.

As the carbon source, glucose, cellobiose, lactose, sucrose, maltose, pyruvic acid, starch, and other carbon compounds that can be assimilated and those containing them can be used.

As the nitrogen source, ammonium sulfate, ammonium chloride, ammonium phosphate, urea, ammonium nitrate, sodium nitrate and the like which are commonly used for culturing microorganisms are used.

As inorganic salts, potassium phosphate, magnesium chloride,
In addition to sodium chloride, ferrous sulfate and ferric chloride, addition of metal salts such as manganese, cobalt, calcium, zinc, copper, boric acid, molybdenum and selenium is effective.

Biotin, folic acid, vitamins B ₁ , B ₂ , B ₆ , B as trace elements
₁₂ , vitamins such as nicotinic acid, pantothenic acid, p-aminobenzoic acid and lipoic acid are used, and corn steve liquor, yeast extract, peptone and the like can be added as natural nutrition sources.

In the present invention, the culture needs to be performed anaerobically. To perform the culture anaerobically, a reducing agent such as sodium sulfide, L-cystine, and titanium citrate is added to the medium,
It is desirable to ventilate oxygen-free nitrogen gas, carbon dioxide gas or a mixed gas thereof into the medium so that oxygen gas is not mixed in the gas phase of the culture vessel.

The culture temperature can be 35 to 85 ° C, preferably 60 to 75 ° C, and more preferably 65 to 70 ° C. The pH can be adjusted to 6-9, but 7-8 is preferable. Further, it is preferable to stir the cells during the culture so that the cells do not precipitate. Culturing is usually completed in 6 to 15 hours, but it can be changed appropriately according to changes in other culturing conditions.

To isolate and purify a novel thermostable acetate kinase from the thus obtained culture, first, from the obtained culture,
For example, the cells are collected by centrifugation or filtration, and the obtained cells are disrupted by a conventional method such as ultrasonic treatment or French press treatment and then centrifuged to obtain a cell-free extract. Furthermore, from this extract, a precipitation method in which salts such as ammonium sulfate and a hydrophilic organic solvent such as acetone or methanol are added, an ion exchange chromatography method such as DEAE-cellulose, a gel perchromatography method such as Sephadex, hydroxyapatite, etc. Adsorption chromatography method, other isoelectric focusing method,
The novel thermostable acetate kinase can be isolated and purified by applying an appropriate combination of separation methods such as electrophoresis.

Next, the physicochemical properties of the novel thermostable acetate kinase obtained by the present invention are shown.

a. Action The positive reaction produces adenosine diphosphate and acetylphosphate from adenosine triphosphate and acetic acid, and the reverse reaction produces adenosine triphosphate and acetic acid from adenosine diphosphate and acetylphosphate.

b. Substrate specificity Acetic acid was the best organic acid that could serve as a phosphate acceptor, followed by slight activity with propionic acid and n-butyric acid. On the other hand, guanosine triphosphate was the most effective nucleotide as a phosphate donor, followed by uridine triphosphate, cytidine triphosphate, and adenosine triphosphate in this order.

The relative activity of each substrate is shown in Tables 1 and 2.

c. Km values for various substrates are as follows. (Table 3) d. The requirements for metal ions are as follows.

Divalent metal ions of manganese and magnesium were essential for the activity of this enzyme, and the addition effect of manganese ion was particularly large.

When the relative activity is 100 (%) magnesium ion,
Manganese ion was 191%.

e. pH stability The pH stability of this enzyme was investigated using a Briton-Robinson broad range buffer (pH 2-14). Each pH
After maintaining at 30 ° C. for 16 hours, the residual activity of each was measured. As shown in FIG. 1, no decrease in activity was observed in the range of pH 4 to pH 11.

f. Optimum temperature of action As a result of examining the influence of the reaction temperature on the enzyme activity using the enzyme activity measuring method (II), the maximum activity was shown between 55 ° C and 60 ° C (Fig. 2).

g. Heat-resistant enzyme solution containing 0.2M phosphate buffer (pH 7.0) at 15
After keeping for a minute, the remaining activity was measured (FIG. 3). As a result, this enzyme was quite stable up to 75 ° C. When the same enzyme solution was kept at 75 ° C. and the residual activity was measured with time (FIG. 4), 70% or more of the activity was observed even after 2 hours of treatment.

h. Molecular weight As a result of measuring the molecular weight of this enzyme by the gel filtration method using Sephadex G-100, as shown in FIG.
I got the value.

In the present invention, the acetate kinase activity was measured by either one of the following two methods I and II, or by using both methods in combination, depending on the experimental conditions.

Enzyme activity measurement method (I) ATP amount produced from acetyl phosphate and ADP was quantified by coupling hexose kinase and glucose-6-phosphate dehydrogenase, and the amount of enzyme producing 1 micromol ATP per minute. Was defined as 1 unit. That is, 6.25 μmol of acetyl phosphate, 5 μmol of magnesium chloride, ADP
1.9 μmol, glucose 25 μmol, NADP 0.4 μmol, hexose kinase (Sigma) 2.5 units, glucose-6-phosphate dehydrogenase (Sigma) 1 unit, 0.1 M Tris / HCl buffer (pH 8.3), succinate To the reaction mixture containing 10 mM sodium and 50 mM calcium chloride, add an appropriate amount of enzyme sample solution to 1 ml, let the reaction proceed at 40 ° C, measure the absorbance at 340 nm, and increase the absorbance to generate NADPH per minute. The amount of ATP produced is calculated based on the obtained amount, and the enzyme titer in the sample is calculated.

Enzyme activity measurement method (II) Measure the amount of acetyl phosphate produced from acetic acid and ATP, and
The amount of enzyme that produces 1 μmol of acetyl phosphate per minute was defined as 1 unit. That is, sodium acetate 10 mM, AT
P10 mM, magnesium chloride 10 mM, hydroxylamine 500
Add an appropriate amount of enzyme sample solution to the reaction solution containing mM, sodium succinate 10 mM, 50 mM Tris / HCl buffer (pH 8.3)
The volume was adjusted to 0.5 ml and the reaction was carried out at 40 ° C. 10% after reacting for 30 minutes
A solution containing ferric chloride, 3% trichloroacetic acid and 0.7N hydrochloric acid 0.7
After adding 5 ml and shaking vigorously, the amount of ferric chloride-hydroxamic acid complex produced was calculated from the absorbance at 535 nm, and based on this, the amount of acetyl phosphate produced was calculated and the enzyme titer in the sample was calculated. To do.

Among the acetate kinases that have been reported so far, the one having the highest heat resistance is acetate kinase purified from Bacillus stearothermophilus. This enzyme retains 100% activity after treatment at 65 ° C for 15 minutes, and decreases to about 20% after treatment at 70 ° C for 15 minutes. [Journal of Biochemistry 84 193 (1978)] However, the thermostable acetate kinase obtained by the present invention does not inactivate at 75 ° C for 15 minutes, and retains 70% or more activity even at 75 ° C for 2 hours. Was. Also in terms of pH stability, the stable pH range of Bacillus stearothermophilus acetate kinase is 7-9.
(Treated at 4 ° C. for 24 hours), the acetate kinase of the present invention was stable at pH 4 to 11 (treated at 30 ° C. for 16 hours).

As described above, the acetate kinase obtained in the present invention is an enzyme that is more excellent in thermostability and pH stability than conventionally reported acetate kinase.

The acetate kinase obtained in the present invention is excellent in temperature and pH stability, and has a very small Km value of 0.025 mM against acetyl phosphate.
It has been shown that it can be used particularly effectively as an enzyme for ATP regeneration in the use of a reaction for synthesizing ATP, particularly when constructing a bioreactor involving ATP.

This enzyme can be used in the form of an aqueous solution or immobilized.

(Effects of the invention) As described above, the thermostable acetate kinase of the present invention has a temperature and temperature higher than those of known acetate kinases.
It has excellent pH stability and high affinity with acetyl phosphate. From this, this enzyme
It can be particularly effectively used as an enzyme for ATP regeneration when constructing a bioreactor involving ATP.

Also, since it is stable even at high temperatures, impure proteins can be denatured and removed by heat treatment without impairing the activity of acetate kinase, and a highly pure acetate kinase can be easily obtained in high yield. You can

(Examples) Next, the present invention will be described with reference to Examples, but the present invention is not limited thereto.

After sterilizing the medium 7 shown in Table 4 for 15 minutes at 121 ° C., a culture of Thermoan aerobium blocky DSM 1457 was pre-cultured for 6 hours in a medium of the same composition separately sterilized similarly.
Was inoculated into the medium. After making the gas phase of the culture vessel an atmosphere of 95% nitrogen and 5% carbon dioxide in an anaerobic state, 65 to 70 ° C
The cells were statically cultivated for 6 hours.

After culturing, the culture was centrifuged to collect the bacterial cells, which were washed three times with 0.1 M phosphate buffer (pH 7.0) to obtain about 12 g of wet bacterial cells.

3 g of wet cells were suspended in 40 ml of 1M phosphate buffer (pH 7.0),
The cells were crushed by treating with an ultrasonic crusher (200 W) for 5 minutes. This disrupted solution was centrifuged (15000 rpm for 30 minutes) to remove cell debris, and a cell-free extract containing acetate kinase was obtained. Ammonium sulfate fractionation is performed on this extract,
A precipitate with 75% saturated ammonium sulfate was obtained. The precipitate containing this acetate kinase was dissolved in 3 mM phosphate buffer (pH 7.0), and dialyzed against the same buffer overnight at 4 ° C. Pass the dialyzed solution through a DEAE-cellulose column equilibrated with 3 mM phosphate buffer (pH 7.0) and elute with a solution containing sodium chloride added to the above buffer to elute the target acetate kinase at around 0.15 M. did. Further, it was passed through a Sephadex G-100 column equilibrated with 3 mM phosphate buffer (pH 7.0) and eluted with the same buffer.

The eluted active fraction was passed through a hydroxylapatite column equilibrated with 3 mM phosphate buffer (pH 7.0), and a linear curve from 3 mM phosphate buffer (pH 7.0) to 500 mM phosphate buffer (pH 7.0) was obtained. When elution was performed with a gradient, the target acetate kinase was eluted at around 200 mM concentration. The thermostable acetate kinase obtained in this example gave a single band by acrylamide disk electrophoresis, and further gave a single peak at a molecular weight of about 50,000 in Sephadex G-100 column chromatography.

The yield of acetate kinase obtained in this example is about 20 mg.
Shows a titer of about 155 units per mg of enzyme protein (measured by the method of II), and its purification degree is about 1 when the cell-free extract is 1.
It was 31 times.

The Km value of the obtained acetate kinase was 0.025 mM for acetyl phosphate (measured by the method of II) and 1.25 mM for ADP (I
It was measured by the method of.

The pH stability of this enzyme was investigated using Briton-Robinson wide-range buffer (pH 2-14), and the residual activity of each enzyme was measured after keeping it at 30 ° C for 16 hours at each pH. As shown in Figure 1. No decrease in activity was observed in the range of pH 4 to pH 11.

In addition, as a result of examining the influence of the reaction temperature on this enzyme activity using the enzyme activity measuring method (II), the maximum activity was shown between 55 ° C and 60 ° C (Fig. 2).

Next, the enzyme solution containing 0.2 M phosphate buffer (pH 7.0) was kept at each temperature for 15 minutes, and the remaining activity was measured (FIG. 3). As a result, this enzyme was quite stable up to 75 ° C.
When the same enzyme solution was kept at 75 ° C and the residual activity was measured over time (Fig. 4), 70% or more of the activity was observed even after 2 hours of treatment.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing pH stability of the present enzyme. FIG. 2 is a diagram showing the optimum action temperature of the present enzyme. 3 and 4 are diagrams showing the thermostability of this enzyme. FIG. 5 is a diagram showing the results of molecular weight determination of this enzyme by Sephadex G-100 gel permeation method.

Claims (2)

[Claims] 1. At least the following physicochemical properties (a) to
Thermostable acetate kinase with (h) a. Action The positive reaction produces adenosine diphosphate and acetylphosphate from adenosine triphosphate and acetic acid, and the reverse reaction produces adenosine diphosphate and acetylphosphate from adenosine diphosphate. It produces triphosphoric acid and acetic acid. b. Substrate specificity Acetic acid is the best organic acid that can serve as a phosphate acceptor, followed by propionate and n-butyric acid that have a slight activity, and guanosine triphosphate is the most likely nucleotide as a phosphate donor. It is effective, and then acts in the order of uridine triphosphate, cytidine triphosphate, and adenosine triphosphate. c.Km value Km value for acetyl phosphate: 0.025 mM, Km for ADP
Value: 1.25 mM. d. Requirement of metal ion: Magnesium ion or manganese ion is required for the activity expression of this enzyme. e. No decline in activity was observed even after treatment in the stable pH range of pH 4 to 11 at 30 ° C for 16 hours. f. Optimum temperature of action: 55-60 ℃ g. Heat resistance: 75 ℃ Stable against heating for 15 minutes. After heating at 75 ° C for 2 hours, 70% or more of the remaining activity remains. h. Molecular weight: about 50,000 (gel filtration method using Sephadex G-100) 2. A thermoan aerobium.
um) belonging to the genus and culturing in a medium, a bacterium that produces a novel thermostable acetate kinase having the following properties (a) to (h):
A method for producing an acetate kinase, which comprises collecting a novel thermostable acetate kinase having the following properties (a) to (h) from the culture. a. Action The positive reaction produces adenosine diphosphate and acetyl phosphate from adenosine triphosphate and acetic acid, and the reverse reaction produces adenosine triphosphate and acetic acid from adenosine diphosphate and acetyl phosphate. b. Substrate specificity Acetic acid is the best organic acid that can serve as a phosphate acceptor, followed by propionate and n-butyric acid that have a slight activity, and guanosine triphosphate is the most likely nucleotide as a phosphate donor. It is effective, and then acts in the order of uridine triphosphate, cytidine triphosphate, and adenosine triphosphate. c.Km value Km value for acetyl phosphate: 0.025 mM, Km for ADP
Value: 1.25 mM. d. Requirement of metal ion: Magnesium ion or manganese ion is required for the activity expression of this enzyme. e. No decline in activity was observed even after treatment in the stable pH range of pH 4 to 11 at 30 ° C for 16 hours. f. Optimum temperature of action: 55-60 ℃ g. Heat resistance: 75 ℃ Stable against heating for 15 minutes. After heating at 75 ° C for 2 hours, 70% or more of the remaining activity remains. h. Molecular weight: about 50,000 (gel filtration method using Sephadex G-100)