JPH0648986B2 - Acetate kinase gene - Google Patents

Description

TECHNICAL FIELD The present invention relates to a gene encoding acetate kinase (hereinafter referred to as an acetate kinase gene) useful for producing acetate kinase.

Acetate kinase is an enzyme that catalyzes the reaction represented by the following reaction formula.

And, acetate kinase is extremely useful for production of ATP and the like.

[Conventional technology]

Conventionally, acetate kinase has been produced, for example, by culturing Escherichia coli and separating and purifying the acetate kinase from bacterial cells [J. Biol. Chem. (J. Biol. Chem.),
261, No. 29, 13487-13497 (1986)].

[Problems to be Solved by the Invention]

However, when the above-mentioned method for producing acetate kines is used, there are drawbacks such as a markedly low yield of the enzyme.

[Means for Solving the Problems]

Therefore, the inventors of the present invention have conducted various studies to solve the above-mentioned problems, and as a result, D containing an acetate kinase gene has been investigated.
Recombinant DNA in which NA was inserted into vector DNA was obtained, and this recombinant was contained in a strain belonging to the genus Escherichia.
We obtained the knowledge that kinase is produced, and filed a patent application (Japanese Patent Application Nos. 62-273146 and 62-273147).

After that, the present inventors found that Escheri
As a result of further study of the acetate kinase gene derived from chiacoli), we succeeded in isolating and determining the structure of the acetate kinase gene derived from Escherichia coli for the first time, and completed the present invention.

That is, the present invention is an acetate kinase gene encoding the amino acid sequence shown in the claims.

Hereinafter, the present invention will be described in detail.

First, DNA containing the acetate kinase gene
The preparation will be described.

For example, E. coli, preferably E. coli 1100 (Max-Pla
nk-Institut West Germany, obtained from Heidelberg) to obtain a culture.

In order to cultivate the above-mentioned microorganism, it may be cultivated by a usual solid culturing method, but it is preferable to employ a liquid culturing method as much as possible.

The medium for culturing the above-mentioned microorganisms includes, for example, yeast extract, peptone, meat extract, corn sweep liquor, soybean or wheat bran leachate, and the like, at least one nitrogen source, potassium potassium phosphate, and phosphoric acid. Second potassium,
One or more kinds of inorganic salts such as magnesium sulfate, sodium chloride, magnesium chloride, ferric chloride, ferric sulfate or manganese sulfate are added, and if necessary, sugar raw materials, vitamins and the like are appropriately added. .

The initial pH of the medium is appropriately adjusted to 7-9. Culturing is performed at 30 to 42 ° C, preferably around 37 ° C for 4 to
It is preferably carried out for 24 hours, preferably for 6 to 24 hours, by aeration and agitation deep culture, shaking culture, static culture and the like.

The culture thus obtained is, for example, 3,000 rpm
As described above, centrifugation at 8,000 to 10,000 rpm for 5 minutes or more, preferably 10 to 15 minutes is performed to obtain Escherichia coli 1100 cells.

From this cell, for example, the method of Saito and Miura [Biochem.
Biofizz. Actor. (Biochem.Biophys.Acta.), No. 7
Volume 2, page 619 (1963)], KS Kirby (K.
S. Kirby) [Biochem. Jay. (Biochem.J.),
64, 405 (1956)] and the like.
NA can be obtained.

Then, a restriction enzyme, such as Sau3AI (manufactured by Toyobo Co., Ltd.), which produces a protruding end is added to the chromosomal DNA at a temperature of 30 ° C. or higher, preferably 37 ° C., and an enzyme concentration of 1 to 10 units / ml.
The mixture is allowed to stand for at least 30 minutes, preferably 30 minutes to 2 hours, and digested to obtain a mixture of various stained DNA fragments.

And, the acetate kinase gene is linked to a linkage map [Microbiological Reviews, Vol. 47, Vol.
No. 180-230 (1983)], purF (glutamine.
Glutamine Phosphoribosyl-pyrophosph
ate amidotransferase) gene, it is located in the vicinity of the chromosomal DNA fragment mixture, so that the method of chromosomal walking (Chromosomal Walking) [Nature, Vol. 300, No. 4, pp. 35-42 (1982).
Year)], the acetates present near the purF gene
A DNA fragment containing the kinase gene can be searched.

From the DNA fragment mixture thus obtained, a DNA fragment mixture is obtained by, for example, an ordinary agarose gel electrophoresis method, further purified by a purification means such as phenol extraction, and further concentrated by a concentration means such as ethanol precipitation method. Then, a purified DNA fragment mixture (which contains a DNA fragment containing the acetate kinase gene) is obtained.

On the other hand, the vector DN that can be used in the present invention
Any A may be used, and examples thereof include plasmid vector DNA and bacteriophage vector DNA. Specifically, for example, plasmid pUC19D
NA (Takara Shuzo) and the like are preferable.

A restriction enzyme, such as EcoRI or BamHI (both manufactured by Takara Shuzo Co., Ltd.), which produces protruding ends is added to the vector DNA
A digested vector DNA is obtained by digesting at a temperature of 30 ° C or higher, preferably 37 ° C, at an enzyme concentration of 10 to 1000 units / ml for 1 hour or longer, preferably 1 to 3 hours.

Then, a mixture of the DNA fragments containing the acetate kinase gene derived from Escherichia coli 1100 obtained as described above and the cleaved vector DNA are mixed, and this is mixed with, for example, Escherichia coli DNA ligase (New England Biolabs). , T4 DNA ligase (manufactured by Boehringa Mannheim), etc., preferably T4 DNA ligase at a temperature of 4 to 37 ° C., preferably 4 to 16 ° C., and an enzyme concentration of 1 to 1.
Recombinant DNA is obtained by reacting with 100 units for 1 hour or more, preferably 6 to 24 hours.

Using this recombinant DNA, for example, E. coli K-12,
Preferably Escherichia coli JM101 (ATCC33876), Escherichia coli HB101
(ATCC33694), E. coli DHI (ATCC33849), E. coli x-17
76 (ATCC31244) or the like is transformed or transduced to obtain each strain. This transformation is DM
DM Morrison [Methods in Enzymology, Vol. 68, Vol. 326
~ 331 (1979)]. The method of transduction is B. Hohn [Methods in Enzymology, Vol. 68, 299-309 (1979)].
Can be done by.

Then, by screening a strain having the ability to produce acetate / kinase from the above strains, DNA containing the acetate / kinase gene is used as a vector DN.
It is possible to obtain a strain belonging to the genus Escherichia that contains the recombinant DNA inserted in A and has the ability to produce acetate kinase.

To obtain a purified novel recombinant DNA from the strain thus obtained, for example, P. Gue (P. Gue
rry) etc. [J. Bacteriology (J. Bacteriol
ogy) 116, 1064-1066 (1973)], and the method of DB Clewell [J. Bacteriology 110, 667-676 (197)]. it can.

Then, to the above purified novel recombinant DNA,
For example, restriction enzymes MluI and PstI (both manufactured by Takara Shuzo)
Is digested at a temperature of 30 ° C or higher, preferably 37 ° C, at an enzyme concentration of 5 to 20 units / ml for 0.5 to 2 hours, preferably about 1 hour to obtain a DNA fragment mixture.

To isolate a DNA containing an acetate kinase gene from the above DNA fragment mixture, a method such as T. Maniatis (Molecular Cloning, Cold Spring Harbor Laboratory ( Cold Spring Harbof Laborator
y), pages 173 to 178 (1982)].

Using the DNA containing the above-mentioned acetate kinase gene, by the method as shown in item (7) of the Example,
The entire nucleotide sequence of only the acetate kinase gene is analyzed (see FIG. 4), and then the amino acid sequence of the polypeptide translated by the gene having the nucleotide sequence is determined (see FIG. 5).

The gene encoding the amino acid sequence thus determined is the acetate kinase gene of the present invention.

Next, using a strain belonging to the genus Escherichia containing the recombinant DNA in which the DNA containing the acetate kinase gene obtained as described above is inserted into the vector DNA, acetate kinase is obtained. For production, the culture is obtained by culturing in exactly the same manner as the culturing method of Escherichia coli 1100.

After the completion of the culture, to collect the acetate kinase from the culture, it can be obtained by a usual enzyme collecting means.

For example, the cells are subjected to ultrasonic disruption treatment, grinding treatment, etc. by a conventional method, or the enzyme is extracted using a lysing enzyme such as lysozyme, or shaken or left in the presence of toluene or the like. The enzyme is self-digested and the enzyme is excreted outside the cells. The solution is filtered, the solid portion is removed by centrifugation, etc., and if necessary, nucleic acid is removed with streptomycin sulfate, protamine sulfate or manganese sulfate, and then ammonium sulfate, alcohol, acetone or the like is added to fractionate the solution. The precipitate is collected, dialyzed against water, and dried under vacuum to obtain a crude enzyme preparation.

Further, in order to obtain a purified product of acetate kines, for example, DEAE-cellulose (diethylamineethylcellulose, manufactured by U.S. Brown Co.), DEAE-Sephadex (diethylamine ethyl sephadex, Purified by an adsorption elution method using an ion-exchange substance such as Pharmacia of Sweden) or QAE-Sephadex (Pharmacia of Sweden),
Also, a gel filtration method using Sephadex G-200 (Pharmacia, Sweden), Sepharose 6B (Pharmacia, Sweden), etc., Hydroxyl apatite (Bio-Gel HT, Bio-Rad, USA)
A highly purified acetate-kinase standard product can be obtained by appropriately selecting and combining the adsorption and elution method using and the electrophoresis using polyacrylamide gel.

The physicochemical properties of the purified acetate kinase obtained by the above purification means are described in [J. Biol. Chem. (JB
iol.Chem.), Vol. 261, No. 29, No. 13487-13497 (198
6 years)] and the physicochemical properties of the described acetate kines are exactly the same.

〔The invention's effect〕

As is clear from the above, the acetate of the present invention
By culturing in a medium a strain belonging to the genus Escherichia containing a recombinant DNA in which a DNA containing a kinase gene is inserted into a vector DNA, acetate kinase can be obtained in a high yield. It is useful.

〔Example〕

Hereinafter, the present invention will be described more specifically with reference to examples.

Example (1) Preparation of Escherichia coli 1100 strain DNA Escherichia coli 1100 (obtained from Max-Plank-Institut West Germany, Heidelberg) strain was mixed with TY medium [1% (W / V) Bacto-trypton [Difco]. (Manufactured by (Difco)), 0.5
% (W / V) Bacto-yeast extract
extract) (manufactured by Difco) and 0.5% (W / V) NaCl
(PH 7.2)] 100 ml was inoculated and shake-cultured at a temperature of 37 ° C. for 8 hours to obtain a culture.

This culture was subjected to centrifugation at 10,000 rpm for 15 minutes by a conventional method to obtain 0.5 g of wet microbial cells, and then Saito from the microbial cells,
Miura's method [Biochem. Biofizz. Actor. (Bio
chrm.Biophys.Acta.), 72, 619 (1963)].

Next, 60 μg of this chromosomal DNA and the restriction enzyme Sau 3AI
3 units (manufactured by Toyobo Co., Ltd.) were mixed with 10 mM Tris-hydrochloric acid buffer solution (containing 50 mM NaCl, 10 mM MgSO ₄ and 1 mM dithiothreitol) (pH 7.4) and reacted at a temperature of 37 ° C. for 30 minutes. After completion of the reaction, the reaction solution is subjected to phenol extraction treatment and ethanol precipitation treatment by a conventional method, and in order to prevent the DNA fragment digested with Sau3AI from recombining, molecular cloning (Molecular Cloning), No. 133-
Treatment with bacterial alkaline phosphatase (Bacterial Alkaline Phosphatase) according to the method described on page 134
The A fragment was dephosphorylated, phenol-extracted and ethanol-precipitated by a conventional method to obtain 50 μg of a chromosomal DNA fragment of Escherichia coli 1100 strain digested with Sau3AI.

(2) Preparation of Escherichia coli chromosomal DNA library using bacteriophage vector DNA (EMBL4) 20 μg of bacteriophage vector DNA (EMBL4) [Promega Biotec] and 200 units of restriction enzyme BamHI (Takara Shuzo) Was mixed with 50 mM Tris-hydrochloric acid buffer (containing 100 mM NaCl and 10 mM MgSO ₄ ) (pH 7.4) and reacted at a temperature of 37 ° C. for 2 hours to obtain a digestive solution, which was extracted with phenol and precipitated with ethanol by a conventional method. Intermediate DNA from bacteriophage vector after treatment
To prevent the fragments from recombining to form bacteriophage, ethanol precipitate 20
μg and 100 units of restriction enzyme SalI were mixed with 50 mM Tris-hydrochloric acid buffer (containing 100 mM NaCl and 10 mM MgSO ₄ ) (pH 7.4) and reacted at a temperature of 37 ° C. for 2 hours to obtain a digestive solution. BamHI-digested bacteriophage EMBL4 after phenol extraction and ethanol precipitation by the method
DNA was obtained.

Then, this BamHI digested bacteriophage E
MBL4DNA 1 μg, Sau3AI obtained in the above item (1)
1 μg of chromosomal DNA fragment of Escherichia coli 1100 strain digested with 2 and 2 units of T4 DNA ligase (manufactured by Boeringer Manheim) were added to 66 mM MgCl ₂ , 10
It was added to 66 mM Tris-HCl buffer (pH 7.5) containing mM dithiothreitol and 10 mM ATP at 16 ° C at a temperature of 16 ° C.
After reacting for a time, DNA was ligated.

Then, the DNA mixture is subjected to an in vitro packaging method [Methods in Enzymology, Volume 68, 281-29].
8 (1979)], bacteriophage particles were prepared by wrapping them with a bacteriophage coat protein.

The bacteriophage particles thus obtained were then transformed into E. coli NM539 [Promega Biote
c)) as an indicator bacterium, tryptone agar medium [tryptone [Difco (manufactured by)] 1%, NaCl 0.25%, agar 1.2%, sterilized under pressure, and 30 ml each is divided into a petri dish with a diameter of 9 cm. It is a note. ] Sprinkle on, at a temperature of 37 ℃
After stationary culture for 16 hours, about 5,000 lysates were obtained and used as a library.

(3) Preparation of DNA containing purF gene Gene ac involved in expression of Escherichia coli acetate kinase
kA is located near the purF gene, as described above. The nucleotide sequence of the purF gene is reported in J. Yun Tso et al. [Journal of Biological Chemstr.
y), 257, pp. 3525-3351, 1982]. 5'GTCGG which is a part of the base sequence of this purF gene
TATCGCCGGTG3 '16-base oligonucleotide was DNA
The synthesis was performed using a synthesizer (Beckmann). Less than 5'of this 20 ng oligonucleotide [ ³²
P] ATP (manufactured by Amersham), Molecular Cloning, pages 122-126,
Cold Spring Harbor Laboratory (Cold
Labeling was performed according to the method described in Spring Harbor Laboratory (1982).

Recombinant bacteriophage EMB prepared in item (2) using as an probe an oligonucleotide that is a part of the purF gene labeled with ³² P prepared by the above method.
Escherichia coli 1100 strain chromosomal DNA using L4 DNA as a vector
The library was cloned using the plaque hybridization method [(Molecular Cloning
g), pages 312-328, Cold Spring Harbor
Laboratory (Cold Spring Harbor Laboratory) (198
2)], a plaque having the purF gene was obtained. The plaque was labeled with Molecular Cloning.
Ning), pp. 371-372, Cold Spring Harbor Laboratory (19
According to the method described in 82), the bacteriophage DNA containing the purF gene was purified, and this recombinant bacteriophage DNA was named hy102.

D obtained by single and double digestion of the recombinant bacteriophage hy102 DNA with restriction enzymes HindIII, EcoRI, BamHI, BglII and SalI (all manufactured by Takara Shuzo)
The NA fragment was analyzed for mobility pattern by agarose electrophoresis, and the obtained mobility pattern was compared with the standard mobility pattern of the DNA fragment obtained by digesting bacteriophage DNA (Takara Shuzo) with HindIII. The restriction enzyme map obtained by the above was as shown in FIG.

(4) Search for Acetate Kinase Gene--Preparation of probe DNA 5 μg of recombinant bacteriophage hy102 DNA was
5 μl TE buffer [10 mM Tris containing 1 mM EDTA-
Hydrochloric acid buffer solution (pH 7.5)] and add 2 μl of Hig
h buffer [50 mM Tris-HCl buffer (pH 7.5) / 1000 mM
NaCl / 100mM MgCl ₂ / 10mM dithiothreitol] and 30
A unit of EcoRI was added, and the mixture was cut at a temperature of 37 ° C. for 2 hours.

The total amount of this DNA was separated by electrophoresis using a 0.7% (W / V) agarose gel. Agarose gel electrophoresis
Methods such as T. Maniatis [Molecular Cloning, pp. 156-161, Cold Spring Harbor Laboratory (Cold Spri
ng Harbor Laboratory) (1982)]. 3.45 Kbp EcoRI / EcoRI in bacteriophage hy102 DNA
The gel portion containing the DNA fragment was cut out from the gel, placed in a transparent tube, 2 ml of TE buffer was added, the transparent tube was sealed, and the DNA was eluted from the gel into the buffer by electrophoresis. After adding an equal volume of water-saturated phenol to this solution and stirring, the aqueous layer was recovered, and DNA was recovered by ethanol precipitation according to a conventional method.

The obtained 3.45 Kbp DNA fragment was transformed with [ ³² P] dCTP.
(Manufactured by Amersham) was labeled by the nick translation method to obtain a probe. Nick translation, using a kit manufactured by Takara Shuzo Co., Ltd., directed by Takara Shuzo Co., Ltd., J. Mol. Biol. Volume 113,
Pages 237-251 (1977) and molecular cloning
(Molecular Cloning), pp. 109-112, Cold Spring Habor L.
aboratory) (1982).

(5) Search for acetate kinase gene --- Search for acetate kinase gene by chromosomal walking method 3.45 Kbp DNA labeled with ³² P prepared by the above method
Using the fragment as a probe, the recombinant Escherichia coli 1100 chromosomal DNA library prepared in item (2) using the recombinant bacteriophage EMBL4 DNA as a vector,
A plaque having a 3.45 Kbp DNA fragment was obtained by searching by the plaque hybridization method as in (3). Each of the plaques was molecularly cloned (Molecul
ar Cloning), pp. 371-372, Cold Spring
Harbor Laboratory (Cold Spring Habor Laborator
y) The bacteriophage DNA was purified by treatment according to the method described in (1982). The recombinant bacteriophage DNA containing the 3,45 Kbp DNA fragment was designated hy122.

The recombinant bacteriophage hy122 DNA
According to the method described in (3), using the various restriction enzymes,
After digestion, a restriction enzyme map was obtained as shown in FIG.

10 μg of the recombinant bacteriophage hy122 DNA
Was dissolved in 15 μl TE buffer and 2 μl Hi
A gh buffer solution, 30 units of restriction enzyme EcoRI and 30 units of restriction enzyme BamHI were added, and the reaction was carried out at a temperature of 37 ° C. for 2 hours to cut the DNA. From the digested recombinant bacteriophage hy122 DNA, 4.5 Kbp EcoRI / BamHI
The DNA fragment was isolated according to the method using the agarose gel electrophoresis method described above to obtain 6 μg of EcoRI / BamHI DNA fragment.

On the other hand, 1 μg of plasmid pUC19DNA (Takara Shuzo)
Was dissolved in 18 μl TE buffer and 3 μl Hi
A gh buffer, 5 units of BamHI and 5 units of EcoRI were added and digested at a temperature of 37 ° C. for 1 hour, followed by phenol extraction and ethanol precipitation treatment by a conventional method to obtain a precipitate.

Plasmid pUC19D digested with 0.5 μg EcoRI and BamHI
NA and 0.5 μg of 4.5 Kbp Eco derived from the above hy122 DNA
Each RI / BamHI DNA fragment was dissolved in 7 μl of water and
l mixed solution [77 mM Tris-HCl buffer (pH 7.4) / 15 mM
MgCl _2/15 mM dithiothreitol /0.15MM ATP] and 1
Unit of T4 DNA ligase is added, and the temperature is 8 ℃.
A time ligation reaction was performed. Using this reaction mixture, the Journal of Bac-teriolog
y), Vol. 119, pp. 1072-1074 (1974), Escherichia coli JM101 strain (ATCC 33876) was transformed, and drug resistance (ampicillin resistance) and β-galactosidase activity were examined. A transformant strain was obtained, and the recombinant plasmid DNA containing the acetate kinase gene contained in the strain was named pAK122. Escherichia coli JM101 (pAK122) thus obtained has been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Micro Engineering Research Article No. 1534 (FERM BP-1534).

(6) Plasmid p containing the acetate kinase gene
Preparation of AK122 DNA Escherichia coli 101 strain (FERM BP-1534) containing the plasmid pAK122 DNA obtained above was treated with tryptone 1% (W / W), yeast extract 0.5 (W / W) and NaCl 0.5% (W / V). ) Consisting of medium 1
20 ml of a culture solution of Escherichia coli JM101 (pAK122) obtained by preculturing at 37 ° C. for 24 hours using the medium was inoculated into
After shaking culture at 7 ° C. for 3 hours, 0.2 g of chloramphenicol was added, and the same culture was performed at the same temperature for 20 hours to obtain a culture solution.

Then, this culture solution was centrifuged at 6000 rpm for 10 minutes by a conventional method to obtain 2 g of wet cells, which were added to 20 ml of 25%.
(W / V) 350 mM Tris-HCl buffer containing sucrose (pH
8.0), and then 10 mg of lysozyme (manufactured by Sigma), 8 ml of 0.25M EDTA solution (pH 8.0), and
8% of 20% (W / V) sodium dodecylsulfate solution was added, and the mixture was kept warm at a temperature of 60 ° C. for 30 minutes to lyse the cells to obtain a lysate.
To this lysate, 13 ml of 5M NaCl solution was added and the temperature was 4 ° C.
Processed for 16 hours at 15000r.pm for 30
After centrifuging, the extract was subjected to a phenol extraction treatment and an ethanol precipitation treatment by a conventional method to obtain a precipitate.

Then, this precipitate was subjected to ordinary vacuum drying treatment,
10 mM Tris-HCl buffer containing 1 mM EDTA (pH
7.5) Dissolve in 6 ml, and then add 6 g of cesium chloride
And ethidium bromide (19 mg / ml) 0.2 ml were added, and the recombinant plasmid pAK122 DNA was isolated by performing an equilibrium density gradient centrifugation treatment using an ultracentrifuge at 39000 rpm for 42 hours by a conventional method. Further, ethidium bromide was removed using n-butanol, and then dialyzed against 10 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA to obtain 500 μg of purified recombinant plasmid pAK122DNA.

The recombinant plasmid pAK122 DNA was digested with various restriction enzymes according to the method described in item (3) to obtain a restriction enzyme map as shown in FIG.

(7) DNA containing acetate / kinase gene
Nucleotide sequence analysis of recombinant plasmid pAK122DNA1 obtained in item (6)
5 μg was digested with restriction enzymes MluI and PstI (both manufactured by Takara Shuzo) to obtain 5 μg of 2.9 Kb DNA fragment containing an acetate kinase gene, and this DNA fragment was used as plasmids pUC18 and pUC19DNA (both manufactured by Takara Shuzo).
E. coli JM101 (ACTT33876) was cloned into the SmaI and PstI sites and the resulting recombinant plasmid DNA was used.
Of E. coli JM101 (pAK124-8)
And JM101 (pAK124-9) were obtained respectively.

The method of digesting DNA with a restriction enzyme, the ligation with T4 DNA ligase and the transformation method is described in the above item (5), and the isolation of a DNA fragment by agarose gel electrophoresis is described in the above item (4). According to the described method.
The transformant thus obtained was infected with Belperphage M13K07 (manufactured by Takara Shuzo Co., Ltd.) and subjected to meshing (Messi).
ng) [Methods in Enzymology (Methods
in Enzymology), 101, 20-78 (1983)].

Sequencing with the obtained single-stranded DNA was performed using 7-
A DEAZA sequence kit (manufactured by Takara Shuzo) was used according to the above meshing method. The primer was synthesized using System 1 Plus DNA synthesizer (manufactured by Beckman).

In addition, gel electrophoresis for analysis of the nucleotide sequence is 8% (W
/ V) Polyacrylamide gel (Fuji Photo Film Co., Ltd.)
Was performed using.

The entire nucleotide sequence of the obtained acetate kinase gene is shown in FIG. 4, and the amino acid sequence of the polypeptide translated from the gene is shown in FIG.

(8) Culture of Escherichia coli JM101 (pAK122) and Preparation of Crude Enzyme Solution Escherichia coli JM101 (pAK122) (FERM BP-1534) was mixed with LB-amp medium [Bactotryptone 1% (W / W), yeast extract 0.5% ( W /
W), NaCl 0.5% (W / W) and ampicillin 50 μg / ml] 3 ml
Culture was carried out at 37 ° C for 18 hours with shaking. This culture
0.5 ml was inoculated into 10 ml of the above LB-amp medium, and the temperature was 37 ° C.
After 6-hour shaking culture at 37 ° C., centrifugation was performed at 3500 rpm for 10 minutes to obtain moist bacterial cells, which were 10 mM MgCl ₂ and 1 mM.
2 ml of 10 mM phosphate buffer (pH 7.5) containing EDTA
And suspended in ultrasonic waves and subjected to ultrasonic destruction by a conventional method to obtain a crude enzyme solution. Acetate in the crude enzyme solution thus obtained
Kinase activity was measured by the following method, and the results are shown in the table below.

The acetate kinase activity in the obtained crude enzyme solution was measured by a method such as Thomas (Journal of.
Bacteriology, No. 144
Volume 672-682 (1980))] and generate N
This was done by calculating the number of moles of ADPH.

That is, 5 mM MgCl ₂ , 10 mM glucose, 0.5 m
5 units / ml glucose 6-phosphate dehydrogenase (Boehringer Mannheim) and 21 units hexokinase (Boehringer) were added to 0.1 mM HEPES buffer (pH 7.0) containing MNADP, 5 mM ADP and 5 mM acetyl phosphate. (Mannheim) was added, and 0.1 ml of a 50-fold diluted crude enzyme solution was mixed with 2.4 ml of this solution, and the amount of NADPH produced was calculated from the change in absorption at 340 nm. .

For comparison, the acetate kinase activity of Escherichia coli JM101 strain [Escherichia coli 101 (pUC19)] containing the plasmid pUC19 DNA was also measured. The results are shown in the table below.

As is clear from the above table, Escherichia coli JM101 (pAK122) transformed with the acetate kinase gene of the present invention
In the case of using E. coli, the amount of NADPH produced was remarkably increased as compared with the case of using Escherichia coli JM101 (pUC19) as a control, and it can be seen that acetate kinase was expressed.

[Brief description of drawings]

FIG. 1 shows recombinant bacteriophage hy102DNA.
2 is a restriction enzyme map of the recombinant bacteriophage hy122 DNA, FIG. 3 is a restriction map of the recombinant plasmid pAK122DNA, and FIG. FIG. 5 is a diagram showing the entire nucleotide sequence of only the kinase gene, and FIG. 5 is a diagram showing the amino acid sequence of a polypeptide translated from the gene having the nucleotide sequence shown in FIG.

Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location (C12N 9/12 C12R 1:19)

Claims (1)

[Claims] 1. An acetate kinase gene encoding the amino acid sequence shown below.