JPS6016232B2 - Method for producing enzyme for reproducing adenosine triphosphate - Google Patents

Description

[Detailed description of the invention] The present invention relates to adenosine triphosphate (hereinafter referred to as ATP).

) This relates to a method for producing enzymes for reproduction. Recently, the excellent properties of enzymes as catalysts, such as their high reaction, substrate and optical specificity, and mild reaction conditions, have attracted attention, and their use in the chemical industry, food, medicine, and many other fields has attracted attention. It is being considered and implemented.

However, these are currently mostly limited in their use as hydrolytic enzymes. However, in vivo,
Biosynthesis is carried out by many synthetic enzymes, mainly using ATP as an energy source. At this time, ATP used as an energy source was adenosine diphosphate (
Hereinafter referred to as ADP. ) will be converted to Therefore, in order to break away from the conventional use of hydrolytic enzymes and expand the industrial use of enzymes, we developed a completely new production system that aims to achieve biosynthesis in vitro similar to that carried out in vivo. Attempts have been made to create systems commonly referred to as bioreactors. In order to complete and implement such a bioreactor, a large amount of ATP is required as an energy source, and it is necessary to regenerate the used ATP from ADP. In line with this purpose, as a method for regenerating ATP from ADP, it is possible to use enzymes collectively called phosphotransferases that catalyze the reaction of the following formula. (R~■; High energy phosphoric acid compound, RH:R~■
represents a hydrolyzate of

) Among the many phosphotransferases, ■ the equilibrium reaction is sufficiently biased toward ATP synthesis, ■ the phosphoric acid compounds R ~ ■ can be obtained industrially easily and inexpensively, and ■ the enzyme preparations are It is important that it is easily made sufficiently pure, or even if it is impure, that it does not contain contaminating ATP-degrading enzymes or inhibitors.

An example of an enzyme that satisfies these conditions is acetate kinase (enzyme number 2.7.2.1). In the case of acetate kinase, the equilibrium reaction is shifted approximately 123 times toward the ATP synthesis side.Furthermore, acetyl phosphate, which is a phosphoric acid compound R~■, can be easily obtained industrially at low cost, and under the above conditions (■ and ③ is fully satisfied. However, the previously known acetate kinase is
Acetate kinase is obtained from bacteria such as Coli or Bacillus stearothermophilus, but since acetate kinase is an intracellular enzyme, it is extracted from the supernatant obtained by destroying the bacterial cells. This method is common, and as a result, the supernatant contains various substances derived from the bacterial cells, making it extremely difficult to separate and purify the desired enzyme.

For example, when collecting acetate kinase from Escherichia coli, a crude fractionated extract was obtained as described in Journal of Biological Chemistry, Volume 211, Page 73, page 195. Later, ion exchange chromatography, adsorption chromatography,
Even with complicated purification methods such as gel chromatography and dew point electrophoresis, it was usually possible to purify the product to a purity of only about 30%. In particular, the isokame spot electrophoresis method is difficult to scale up, which has been a major obstacle to purifying and producing acetate kinase in large quantities. In addition, when collecting acetate kinase from Bacillus stearothermophilus, please refer to Mochiseki No. 52-25088 and Journal of
As described in Biochemistry, Tsurumaki, p. 198, 1978, after the bacterial cells were disrupted, nucleic acid was removed with streptomycin, and the resulting supernatant was salted out with ammonium sulfate to obtain a crude enzyme precipitate. DEAE cell. Purification is carried out through a complicated process including acetic acid kinase column chromatography, hydroxyl acetate kinase column chromatography, Ultrogel column chromatography, and DEAE Sephadex column chromatography. The production cost per unit enzyme is high, which is a significant hindrance to the industrial development of bioreactors. Therefore, since it is absolutely necessary to efficiently obtain acetate kinase from these points of view, the present inventors have conducted intensive research with the aim of efficiently obtaining highly pure acetate kinase through simple purification operations. As a result, when purified using a specific water-insoluble carrier,
It has been found and devised that all of the above objectives are achieved, and the present invention has been arrived at.

That is, the present invention involves culturing a microorganism that produces acetate kinase, and culturing the resulting culture with the general formula (1) (where R, ,
R2 is selected from an atom or a S03Na group and is different from each other, and R3 represents a water-insoluble polymer.

) A method for producing an enzyme for regenerating adenosine triphosphate is characterized in that acetate kinase is adsorbed onto the carrier by treatment with a water-insoluble carrier, and then the adsorbed acetate kinase is eluted.

As the acetate kinase-producing microorganism used in the present invention, any microorganism can be used as long as it has acetate kinase productivity.

Preferred microorganisms include, for example, Bacillus stearothermophilus.
Examples include arthromophilic vessels) and Escherjchiacoli. In particular, Bacillus stearothermophilus, which produces thermostable acetate kinase, is preferred. In order to culture these microorganisms, for example, as described in Japanese Patent Application Laid-Open No. 52-25088, glucose, sugar, etc. are used. carbon sources such as carbonaceous acid or malic acid and/or veptone, meat extract,
The culture may be carried out under medium conditions commonly used for culturing microorganisms, which contain a nitrogen source such as amino acids, and if necessary, inorganic salts and/or vitamins. In the present invention, the water-impregnable carrier represented by the general formula (1) refers to a water-insoluble compound obtained by reacting a blue dye with a water-impregnable polymer, and as this water-insoluble polymer, For example, cellulose, dextran, agarose, derivatives of polysaccharides such as starch, cellulose polyacetate, derivatives of polyvinyl alcohol, polystyrene, polypropylene, polyethylene, polyvinyl chloride, poly(methyl methacrylate) ester, polybutene, polybentene, polyvinyldene chloride , polyacrylonitrile, polymethacrylic acid, polyacrylic acid, polyaminostyrene, polybutadiene, polyisopresso, polymaleic acid monoester, crosslinked polyacrylamide, polymethacrylamide, polyvinylamine, poly(dialkylaminoethyl methacrylate), poly(dialkylaminomethyl) styrene), poly(vinylpyridine), poly(vinylpyrrolidone), polyacrylic anhydride, polymethacrylic anhydride, polymaleic anhydride, polymethacrylonitrile, poly(trifluoroethylene), poly(tetrafluoroethylene) , poly(divinylbenzene), poly(Q
-methylstyrene), poly(N-vinylamine), poly(tetramethylene glycol divinyl ether), polyvinyl sulfone, polyvinyl sulfoxide, polyacrolein, polymethylpinyl ketone, and other polymers made of monomers containing unsaturated carbon, polyphenylene Polyethers such as nylene oxide, polymethylene oxide, polyethylene oxide, and polytetramethylene oxide, polypeptides such as polyalanine and polyphenylalanine, nylon-13, nylon-4, and nylon.

N-5, Nylon-6, Nylon-7, Nylon-11
, nylon-12, nylon-16,6, nylon-16,
1 Poly(m-phenylene-isophthalamide, Poly(
polyamides such as p-phenylene-terephthalamide),
Polycarboxylic acids such as terephthalic acid, isophthalic acid, adipic acid, maleic acid, fumaric acid, trimellitic acid,
Ethylene glycol, p. Pyrene glycol, butylene glycol, bentaerythritol, bisphenol A
Polyesters derived from polyols such as glycols, lactic acid, polyesters derived from hydroxypyribalic acid, dimethylpolysiloxane, methylphenylpolysiloxane, methylvinylpolysiloxane, cyanoalkylmethylpolysiloxane, fluoroalkyl Silicone rubber such as methylpolysiloxane, polyisocyanate such as toluene diisocyanate, xylene diisocyanate, phenylene diisocyanate, ethylene diisocyanate, diphenylmethane diisocyanate, and toluene diisocyanate, and polyethylene glycol. , polypropylene glycol, O at both ends
Polyurethanes derived from polyols such as polyesters having H groups, formaldehyde resins such as phenol-formaldehyde resins, xylene-formaldehyde resins, urea-formaldehyde resins, and melamine-formaldehyde resins, polyimides, polybenzimidazole, polythiazole, etc. Synthetic polymers such as polymers containing four-membered rings, polycarbonate, polysulfone, and inorganic derivatives such as glass, asbestos, clay, mica, hydroxylapatite activated carbon, silica gel, alumina, and synthetic inorganic polymers such as polyphosphazene etc. A specific example of the water-proof and sexual essence represented by the general formula (1) is, for example, commercially available Blue Sefa.

Examples of these include ``Full-A'' and Matrex Flue A. In the present invention, it is necessary to adsorb acetate kinase onto the above-mentioned carrier. To do this, collect bacteria from the culture,
The collected bacterial cells may be crushed using a French press, Manton Gaulin, ultrasound, freeze-thaw, Dynomyl, etc. to obtain a crude extract, or the nucleic acid removed by treatment with streptomycin sulfate or protamine sulfate, etc. extract,
A crudely fractionated extract obtained by fractionation using ammonia sulfate, acetone, etc. or a pre-fractionated extract obtained by pretreatment by ion exchange chromatography, adsorption chromatography, etc. may be used. Acetate kinase can be adsorbed by simply mixing this extract and the above-mentioned carrier, or by passing the solution through the above-mentioned carrier packed in a column. Next, in the present invention, it is necessary to elute the adsorbed acetate kinase.

To this end, it is desirable to first wash the carrier on which acetate kinase has been adsorbed with a buffer solution to remove extraneous proteins in the extract that do not adsorb to the raccoon body. At this time, it is desirable to select conditions that do not cause the adsorbed acetate kinase to elute. For this purpose, the ionic strength of the buffer solution must be between 0.001 and o. In the range of 15 degrees, preferably from 0.01 to 0.10 and even 0.0
A range of 5 to 0.10 is optimal. After washing in this manner, acetate kinase may then be eluted using, for example, the eluent described below. Any eluent may be used as long as the activity of acetate kinase is not impaired and acetate kinase can be eluted from the carrier, but among these, the eluent contains 10 M ATP, which specifically elutes acetate kinase. buffer, 10 M ATP and 10
Buffers containing 0 M potassium chloride, low M ATP and 100 mM sodium chloride are preferred, and when Bacillus stearothermophilus is used, 2 M ATP and 1 A buffer solution containing fructose-1,6-diphosphate is preferred. The pH of this buffer is preferably 5 to lu, preferably 6 to 9, particularly preferably 7 to 9. Further, the elution method may be a so-called batch elution method in which a buffer solution is added at once, or a so-called concentration gradient elution method in which elution is performed by continuously increasing the concentration of a buffer solution. According to the present invention, highly pure acetate kinase can be obtained efficiently without using isomerical point electrophoresis and by omitting some of the related chromatography, and the time required for purification is reduced. It can be shortened to large.

Next, the present invention will be specifically explained using examples.

Example 1 Bacillus stearothermophile NCA15
500 wet bacterial cells of No. 03 were suspended in twice the amount of 100 M Tris-HCl buffer (pH 8.0), the cells were sufficiently disrupted using Dynomil, cell debris was removed by centrifugation, and acetic acid was added. A crude extract containing the kinase was obtained.

This crude extract was passed through a column filled with Blue Sepharose (manufactured by Pharmacia) that had been equilibrated with 50 mM Tris-HCl buffer (pH 80) containing 5 mM magnesium chloride and 100 mM potassium chloride. After passing the crude extract, unadsorbed hybrid proteins and the like were washed with the above buffer. Then add 2WHATP to the above buffer solution and the above buffer solution.
When linear gradient elution is performed with a buffer containing 2 M fructose-1.6-diphosphate, the ATP concentration is approximately 0.5
The target acetate kinase was eluted near the enemy M. The fractions were collected and passed through a DEAE-Sephadex column pre-equilibrated with 30mM phosphate buffer (pH 7.0) and 30mM phosphate buffer (pH 7.0).
Elution was carried out by a linear concentration gradient with a solution supplemented with 0.00 M potassium chloride. Potassium chloride concentration approximately 150m
The target acetate kinase was eluted near M. Acetate kinase obtained in this way gave a single band in acrylamide disc electrophoresis, and also gave a single peak at a molecular weight of about 16 in Ultrogel ACA I chromatography, indicating almost 100% purity. have.

In addition, the yield is about 30:9, about 1,50 per enzyme Imp.
The titer per mountain is shown. In this way, higher yields were obtained than in the secondary method, and the number of purification operations was reduced. Example 2 5,00 units of a commercially available crude acetate kinase preparation obtained from Escherichia coli (purity was approximately 30% as determined by acrylamide disc electrophoresis) were
Dialysis was performed against a buffer consisting of 0mM imidazole-hydrochloric acid, 5mM magnesium chloride, and 2mM mercaptoethanol.

This solution was passed through a column filled with Matrex Fluor A (manufactured by Amicon) that had been equilibrated with the above buffer solution and was composed of 100mM sodium chloride, 20mM imidazole-hydrochloric acid, 5M magnesium chloride, and 2mM captoethanol. Washed with buffer. Then, elution was carried out using a linear concentration gradient using the washed buffer solution so that the final salt concentration was 500 mM, and the target acetate kinase was eluted near a salt concentration of about 300 mM. The thus obtained acetate kinase was assayed by acrylamide disc electrophoresis and found to have a purity of 91%, and the amount of acetate kinase recovered was 4800 units.

Example 3/Atylus stearothermophilus ATC
C7954 wet cell lk9 was suspended in twice the volume of 100mM phosphate buffer (pH 7.4), the cells were disrupted using a French press, and insoluble matter was removed by centrifugation, and crude extraction including acetate kinase was performed. I got the liquid.

After adding 300 μl of 10% streptomycin sulfate solution per 600 μl of this extract, the resulting particles were removed by centrifugal D separation to obtain a nucleic acid-free extract. This was subjected to ammonium sulfate separation, and from 30% saturation (4℃) to 45% saturation (4℃).
℃) were obtained. This divided portion was dissolved in 20 mM phosphate buffer (pH 7.0). Next, 20
DEAE equilibrated with mM phosphate buffer (pH 7.0)
- After mixing the cellulose resin and the above solution, a precipitate was passed for 80 minutes. This precipitate was washed with the above buffer containing 150mM sodium chloride. 250m further along Meden
The fruit was washed with 20mM phosphate buffer containing M sodium chloride, and the fruit was harvested. This monkey fluid was dialyzed against a 50 mM imidazole-hydrochloric acid buffer containing 100 mM sodium chloride and 5 M magnesium chloride, and then mixed with blue sepharose for 30 minutes. This mixture was filtered, and the makuraden was washed with the above buffer solution, and then with the above buffer solution containing 10 M ATP. Collecting the latter wash yielded 70,00 units of the desired acetate kinase. Next, Ultrogel ACA34.

Claims (1)

[Scope of Claims] 1. A microorganism that produces acetate kinase is cultivated, and the resulting culture is expressed by the general formula I▲There are mathematical formulas, chemical formulas, tables, etc.▼ (However, R_1 and R_2 are selected from H atoms or SO_3Na groups) , which are different from each other, and R_3 represents a water-insoluble polymer. A method for producing an enzyme for regenerating adenosine triphosphate. 2. The production method according to claim 1, wherein the microorganism that produces acetate kinase is Bacillus stearothermophilus. 3. The production method according to claim 1, wherein elution is performed with a buffer containing 10 mM adenosine triphosphate. 4. The production method according to claim 1, wherein elution is performed with a buffer containing 10 mM adenosine triphosphate and 100 mM potassium chloride. 5. The production method according to claim 1, wherein elution is performed with a buffer containing 10 mM adenosine triphosphate and 100 mM sodium chloride. 6. The production method according to claim 1 or 2, wherein elution is performed with a buffer containing 2mM adenosine triphosphate and 1mM fructose-1,6-diphosphate.