JP3335287B2 - Hexokinase gene - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a hydroxyl group at the 6-position of hexose in the presence of at least adenosine diphosphate (hereinafter abbreviated as ADP) without using adenosine triphosphate (hereinafter abbreviated as ATP). Is replaced with a phosphate group, and hexose 6-phosphate and adenosine monophosphate (hereinafter, AMP)
Gene encoding the amino acid sequence of an ADP-dependent hexokinase that catalyzes the reaction that produces
A and a substantially pure genetically modified microorganism capable of producing said hexokinase.

[0002]

2. Description of the Related Art In recent years, without using ATP, at least A
In the presence of DP, a special hexokinase that catalyzes the reaction of hexose 6-phosphate and AMP by replacing the hydroxyl group at the 6-position of hexose with a phosphate group (hereinafter, this hexokinase is referred to as ADPHK, and the enzyme activity of the hexokinase is referred to as ADPH
K activity) is a hyperthermophilic archaebacterium Pyrococcus furiosus DSM3638 strain (Deutsche).
Sammlung von Mikroorganis
men und Zellkulturen Gmb
H) (J. Bi)
ol. Chem. , 269 (26) 1994, 1753
7-17541).

[0003] This ADPHK uses ADP, and AT
Without using P, at least in the presence of ADP, the characteristic of catalyzing the reaction of generating hexose-6-phosphate and AMP from ADP and hexose indicates that the ADP concentration in a biological fluid such as serum, urine, or saliva as a test solution. In quantification, for example,
Application of various biological components such as glucose, glyceride, creatinine, and urea to the ADP generation and release reaction system to measure these biological components is expected in the field of clinical diagnosis.

[0004]

However, ultra-high thermophilic archaebacteria require a special component in the culture medium or have a very high growth temperature (85 to 100 ° C.). In addition, the amount of ADPHK produced is very small, which hinders practical application. Further, it is difficult to purify ADPHK, which can obtain only a trace amount, and the partial amino acid sequence of the polypeptide constituting ADPHK is analyzed, and the ADPHK gene DN
It was also difficult to separate A.

[0005] Pyrococcus furiosus DSM3
The sequence of 10 amino acids from the N-terminus of ADPHK of strain 638 is S. cerevisiae. Reported by Kengen et al. (J.B.
iol. Chem, 270 (51) 1995, 3045.
3-30457), and this sequence data is obtained from Pyrococcus furiosus DSM3638 identified by the present invention.
Compared to the N-terminal amino acid sequence of the strain ADPHK, the intermediate two amino acids are unknown, and furthermore, the proline after the methionine at the N-terminus is incomplete and inaccurate, and the gene DNA is isolated. Was completely inadequate. Accordingly, an object of the present invention is to provide efficient production of ADPHK.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies on the various problems described above, and as a result, isolated ADPHK from Pyrococcus furiosus DSM3638 strain.
After purification, the complete sequence of 39 amino acids from the N-terminus of the constituent polypeptide was successfully determined. Based on this N-terminal amino acid sequence, the DNA encoding the amino acid sequence of ADPHK was separated, and the DNA of the ADPHK gene DNA was isolated. The nucleotide sequence was determined.

[0007] The nucleotide sequence of ADPHK gene DNA and the amino acid sequence of ADPHK revealed from the nucleotide sequence are obviously novel, and are described in European Bioi.
nformatics Institute's nucleic acid sequence database, National Biomedica
No other genes and proteins with similar sequences were found in the Protein Database of l Research Foundation.

The ADP of Pyrococcus furiosus
By a hybridization operation using a probe prepared based on DNA containing a part of HK gene DNA,
An ultra-high thermophilic archae bacterium Thermococcus litoralis ATCC 51850 strain (Americic) which is different from Pyrococcus
an Type Culture Collection
The DNA of the ADPHK gene was also isolated from n), its nucleotide sequence was determined, and the entire amino acid sequence encoded by the DNA was determined. Hereinafter, ADPHK derived from Pyrococcus furiosus is abbreviated as PfuHK, and ADPHK derived from Thermococcus litoralis is abbreviated as TliHK.

Further, these ADPHK gene DNAs are introduced into an arbitrary vector, a host microorganism is used as a transformant in a preferable host-vector system, and the transformant is cultured to express the ADPHK gene DNA information. ADPHK was confirmed from the culture, an excellent industrial production method was established, and the present invention was completed.

[0010] The present invention has been made based on the above-mentioned findings, and does not utilize ATP, and at least in the presence of ADP, in the presence of ADPHK, which catalyzes a reaction for producing hexose 6-phosphate and AMP from ADP and hexose. DNA encoding the amino acid sequence, one of the amino acid sequences in Sequence Listing 1.
Encoding the amino acid sequence represented by 455
A, a DNA having a nucleotide sequence represented by 1 to 1365 of the nucleotide sequence of Sequence Listing 1, and a DNA encoding an amino acid sequence represented by 1 to 467 of the amino acid sequence of Sequence Listing 2.

[0011] The present invention also relates to a DNA which is positive by a hybridization operation using a DNA having a nucleotide sequence encoding an amino acid sequence represented by amino acids 1 to 455 of the amino acid sequence shown in SEQ ID NO: 1 to prepare a probe;
DNA that is positive by a hybridization operation using a DNA having a base sequence of 1 to 1365 of the base sequence of SEQ ID NO: 1 to produce a probe, and a DNA having a base sequence of 1 to 1401 of the base sequence in Sequence Listing 2. ,
It is a DNA that becomes positive by a hybridization operation using a DNA having a base sequence encoding an amino acid sequence represented by any one of amino acids 1 to 467 in Sequence Listing 2 for preparing a probe.

Further, in the present invention, a DNA which is positive by a hybridization operation using a DNA having a nucleotide sequence represented by any one of nucleotide sequences from 1 to 1401 in SEQ ID NO: 2 in the nucleotide sequence of SEQ ID NO: 1 to 1 from the amino acid sequence in SEQ ID NO: 1 455
A DN encoding an amino acid sequence obtained by deleting, adding, or substituting the amino acid sequence represented by
A, DNA encoding an amino acid sequence obtained by deleting, adding, or substituting the amino acid sequence represented by 1 to 467 of the amino acid sequence in Sequence Listing 2.

Furthermore, the present invention relates to a microorganism belonging to Escherichia coli, which is substantially exogenous to a host from a nucleotide sequence encoding an amino acid sequence represented by 1 to 455 of the amino acid sequence of Sequence Listing 1. A
Holding a DNA encoding the amino acid sequence of DPHK,
A substantially pure microorganism having the ability to produce ADPHK, the microorganism Escherichia coli (Esc
herichia coli) JM109 / pcHK
1. The microorganism Escherichia coli
a coli) JM109 · pcHK2.

The DNA encoding the amino acid sequence represented by 1 to 455 of the amino acid sequence of Sequence Listing 1 and the amino acid sequence of 1 to 467 of the amino acid sequence of Sequence Listing 2 according to the present invention comprises the N-terminal and C-terminal amino acid residues. Any DNA may be used as long as the DNA consists of any one of a series of codons corresponding to each amino acid of the amino acid sequence including the group or the polypeptide residue.

Also, a DNA encoding the amino acid sequence of PfuHK represented by 1 to 455 of the amino acid sequence of Sequence Listing 1 of the present invention, and the amino acid sequence of TliHK represented by 1 to 467 of the amino acid sequence of Sequence Listing 2 May have a DNA having a base sequence encoding one or more amino acid residues or polypeptide residues at the 5 ′ end and the 3 ′ end. In addition, the DN
At the 5 ′ end of A, initiation codons such as ATG and GTG
Usually, termination codons such as TAA, TAG, and TGA are present at the 3 'end.

In the natural world, mutations in the base sequence of DNA frequently occur, which may result in a mutant DNA encoding an amino acid sequence in which one or more amino acids have been deleted, added or substituted. Furthermore, if a so-called genetic engineering technique is used, it is possible to prepare a mutant DNA encoding an amino acid sequence in which amino acids are arbitrarily deleted, added or substituted. Thus A
Among the mutant DNAs generated from the DPHK gene DNA, peptides having the amino acid sequence encoded by
Those having K activity are defined as mutant ADPHK gene DNA. Mutant ADPHK generated from DNA encoding the amino acid sequence represented by 1 to 455 of the amino acid sequence of Sequence Listing 1 and 1 to 467 of the amino acid sequence of Sequence Listing 2
All gene DNAs are included in the scope of the present invention.

Furthermore, among the DNAs encoding the amino acid sequence of the peptide having ADPHK activity, the DNAs encode the amino acid sequences represented by 1 to 455 of the amino acid sequence of Sequence Listing 1 or 1 to 467 of the amino acid sequence of Sequence Listing 2. All DNAs that are positive by a hybridization operation using a part of the DNA for preparing a probe are included in the scope of the present invention. The DNA that becomes positive by the hybridization operation is at least one of the positive strand and the complementary strand of the double-stranded DNA fragment by having homology to the DNA sequence used as a probe in the hybridization operation. Is capable of causing complementary binding of a base to the DNA strand used as a probe.
Represents NA.

D encoding the amino acid sequence of ADPHK
NA is, for example, DNA (t) derived from a microorganism producing ADPHK, which is a donor of ADPHK gene DNA.
total DNA) is separated and purified, then cut with restriction enzymes and the like, and the total DNA fragment prepared is ligated to the linearly cut and linear expression vector using DNA ligase or the like. The thus obtained recombinant DNA vector is introduced into a host microorganism, and a marker for the expression vector and AD
Screening using PHK activity expression or DNA probe as an index, isolating microorganisms holding recombinant DNA vector containing ADPHK gene DNA,
Culturing the genetically modified microorganism, separating and purifying the recombinant DNA vector from the cultured cells,
It is obtained by obtaining ADPHK gene DNA from NA vector.

Microorganisms that are DNA donors include A
Any microorganism can be used as long as it is a microorganism producing DPHK, but it is preferably, but not limited to, Pyrococcus furiosus DSM3638 or Thermococcus litoralis A.
TCC51850. In order to obtain a DNA encoding the amino acid sequence of ADPHK, specifically, the following method may be used. Among the manipulation methods, a common method is, for example, the method of Maniatis et al.
is, T .; , Et al. MolecularClon
ing. Cold Spring Harbor La
1982, 1989) or various commercially available enzymes and procedures attached to kits.

The microorganism selected as a DNA donor
To collect talDNA, for example, a microorganism that produces ADPHK is cultured by culturing a microorganism that produces ADPHK, collecting cells from the resulting culture, and lysing the lysate to prepare a lysate containing the ADPHK gene DNA. As a lysis method, for example, treatment with a cell wall lysing enzyme such as lysozyme is performed, and if necessary, another enzyme such as a protease or a surfactant such as sodium lauryl sulfate is used in combination,
Furthermore, freeze-thaw (see JP-A-63-185371), which is a method for physically destroying the cell wall, or French press treatment may be performed in combination with the lysis method described above.

From the lysate thus obtained, tota
The lDNA can be separated and purified according to a conventional method, for example, by appropriately combining methods such as protein removal treatment by phenol extraction, protease treatment, ribonuclease treatment, alcohol precipitation, and centrifugation.
The method of cutting the separated and purified total DNA may be performed by a restriction enzyme treatment according to a conventional method, and particularly, in order to facilitate the binding of the obtained total DNA fragment to the vector, restriction enzymes, particularly those acting on a specific nucleotide sequence, for example, , SalI, BglII, BamH
Type II restriction enzymes such as I, XhoI, MluI are suitable.

As the vector into which the total DNA fragment is incorporated, a phage or a plasmid capable of autonomously growing in a host microorganism, or a vector constructed for gene recombination from a cosmid is suitable. As the phage vector, for example, Escherichia coli Λgt.λC, λgt.λB, etc. can be used when the microorganism belonging to

As a plasmid vector, for example, when Escherichia coli is used as a host microorganism,
Plasmids pBR322, pBR325, pACYC1
84, pUC12, pUC13, pUC18, pUC1
9, pUC118, pUC119, pINI, Blu
E.script KS ⁺ , p.
UB110, pKH300PLK, pIJ680 and pIJ702 when using actinomycetes as hosts, and YRp when using yeast, especially Saccharomyces cerevisiae as hosts.
7, pYC1, pYES2, etc. can be used. As a cosmid vector, for example, pWE15 or the like using Escherichia coli as a host microorganism can be used.

[0024] Such a vector is converted into total DNA.
DNA fragments produced by the restriction enzyme used to cleave the DNA and a restriction enzyme producing the same ends to produce a vector fragment, and the total DNA fragment and the vector fragment are
What is necessary is just to couple | bond with an A ligase enzyme according to a conventional method.
The host microorganism into which the vector to which the total DNA fragment is ligated may be any one as long as the recombinant DNA can be stably and autonomously propagated. For example, when the host microorganism is a microorganism belonging to Escherichia coli, Escherichia coli DH1, Escherichia coli Coli JM109, Escherichia coli W3110, Escherichia coli C600 and the like can be used. Further, when the microorganism host is a microorganism belonging to Bacillus subtilis, Bacillus subtilis ISW1214 or the like, a microorganism belonging to actinomycetes, Streptomyces lividans TK24, or a microorganism belonging to Saccharomyces cerevisiae such as Saccharomyces cerevisiae IN
VSC1 or the like can be used.

As a method for transferring the recombinant DNA to the host microorganism, for example, when the host microorganism is a microorganism belonging to Escherichia coli, Saccharomyces cerevisiae, or Streptomyces lividans, the host cell is transformed into a competent cell according to a conventional method. The recombinant DNA may be transferred to the strain, and depending on the strain, electroporation may be used.

The selection as to whether or not the desired recombinant DNA has been transferred to the host microorganism can be made by screening from a gene library prepared by the host microorganism into which the recombinant DNA has been transferred by the above method, using the expression of ADPHK activity as an index. It may, was labeled with like ³² P or phosphor,
A positive strain may be selected by colony hybridization or plaque hybridization using a DNA probe designed and synthesized in advance based on the partial amino acid sequence of ADPHK, and this strain may be used as a target transformant.

As a method for preparing a DNA probe,
Separated from the culture of a microorganism producing ADPHK selected as a DNA donor, by analyzing a partial amino acid sequence of the purified ADPHK, designed oligonucleotides coding for the appropriate site of the amino acid sequence, synthesized, ³² according to a conventional method What is necessary is just to label with P or a fluorescent substance. The DNA containing the ADPHK gene DNA may be isolated from the transformant according to a conventional method. AD obtained by the above method
The base sequence of the PHK gene DNA was determined by the dideoxy method (Sa
ngar, F .; (1981) Science, 214,
1205-1210) and ADPH
The entire amino acid sequence of the polypeptide constituting K can be predicted and determined from the nucleotide sequence.

The ADPHK gene DNA of the present invention also comprises
It is also possible to cause a mutation within a range in which the encoded peptide does not impair ADPHK activity. As a method for producing this mutation, a natural mutation of the base sequence of DNA may be used, or an artificial genetic engineering technique may be used. Specific examples of mutations by genetic engineering techniques include site-specific mutagenesis by the method of Zoller (Zoller, MJ and Smith, M.S.
(1983) Methods in Enzymolo
gy, 154, 367), isolation from a group of random mutants by screening, replacement of a specific DNA fragment in a target gene with synthetic DNA, and a so-called evolutionary engineering method for artificially promoting gene evolution in an organism (Oshima Tairo, et al., Miami-Biotechnol.
t-Rep. , (1995) 6, 3).

In this way, once the ADPHK gene D
If NA is isolated, the isolated ADPHK gene DN
It is also possible to produce a probe using a part of A and to isolate new ADPHK gene DNA from other organisms. As an example of newly separating ADPHK gene DNA from another organism from ADPHK gene DNA that has already been separated, an example of the ADPHK gene of Thermococcus litoralis using the ADPHK gene DNA of Pyrococcus furiosus
Separation of K gene DNA. The reverse is naturally also possible.

Specifically, the obtained ADPHK gene D
A DNA fragment containing 80% or more of NA was prepared by a conventional method, and was subjected to a random primer method (Feinberg, A .;
P. and Vogelstein, B.A. (1983)
Anal. Biochem. 132, 6-13), a DNA probe labeled with a radioisotope or a fluorescent substance is prepared, and a hybridization operation using the probe is carried out from chromosomal fragments or cDNA of an organism whose ADPHK gene DNA is to be newly separated. This may be performed on the prepared gene library, and a clone showing a positive reaction may be selected. By the above operation, at least 55
% Of the gene DNA of ADPHK derived from different organisms and the gene DNA of mutant ADPHK having homology of not less than%.

The probe prepared by the random primer method is a collection of DNA fragments having a part of the base sequence or the complementary base sequence of the DNA fragment used as the template. A DNA fragment consisting of 80% or more of the DNA having the nucleotide sequence encoding the amino acid sequence represented by 1 to 455 of the amino acid sequence shown in Table 1 and a specific nucleotide sequence encoding the amino acid sequence are listed in Sequence Listing 1. 1 to 125 of the nucleotide sequence
A DNA fragment comprising at least 80% of the DNA having the nucleotide sequence encoding the amino acid sequence represented by 1 to 467 of the DNA fragment of SEQ ID NO: 5
Specific examples of the nucleotide sequence encoding the amino acid sequence include a DNA fragment comprising 80% or more of the DNA having the nucleotide sequence represented by 1 to 1401 of the nucleotide sequence in Sequence Listing 2.

Further, if a plurality of ADPHK gene DNAs are isolated, hybridization with DNA probes designed based on the highly conserved gene region will result in other ADPHK gene DNAs and mutations having lower homology. It is also possible to isolate ADPHK gene DNA. The recombinant DN once selected in this way
A can be easily removed from a transformed microorganism carrying the recombinant DNA and transferred to another host microorganism.

Further, another DNA encoding the amino acid sequence of the polypeptide constituting ADPHK is cleaved from the recombinant DNA by restriction enzymes or the like, and the resulting DNA is cleaved by the same method as described above. DN having novel characteristics by binding to the terminal of DNA
A can be easily prepared and transferred to other host microorganisms.

As a preferred example of the ADPHK gene DNA obtainable by the above-mentioned method, a hybridization using a DNA having a nucleotide sequence encoding an amino acid sequence represented by 1 to 455 of the amino acid sequence of Sequence Listing 1 was used. DNA that is positive by the operation of
As a nucleotide sequence encoding a specific amino acid sequence, a probe was prepared using a DNA having a nucleotide sequence encoding the amino acid sequence represented by 1 to 1365 of the nucleotide sequence of Sequence Listing 1 and 1 to 467 of the amino acid sequence of Sequence Listing 2. Which becomes positive by the hybridization operation used for
A, which is a specific base sequence encoding an amino acid sequence, includes a DNA having a base sequence represented by 1 to 1401 of the base sequence in Sequence Listing 2.

The microorganism as a transformant thus obtained can produce ADPHK by being cultured in a nutrient medium, but depending on the host microorganism, the ADPHK gene DN.
There is a possibility that ADPHK productivity may not be obtained only by transferring A. In such a case, the obtained DNA fragment containing the ADPHK gene DNA is used to prepare a restriction enzyme recognition site by site-directed mutagenesis according to the above-mentioned Zoller method,
A new transformant was prepared using a vector into which a DNA having the ADPHK gene DNA ligated downstream of a gene promoter compatible with the host microorganism was isolated by excision in an appropriate form such as by excision with a restriction enzyme.
K may be produced.

For example, when the host microorganism is Escherichia
In the case of microorganisms belonging to the genus E. coli, the gene promoter for connecting the ADPHK gene DNA includes lacZ promoter, tac promoter, T7 promoter, pyruvate oxidase gene promoter (Japanese Patent Laid-Open No. 1-1).
No. 44976) and an ADPHK gene DNA having an initiation codon ATG or GTG is connected downstream of these promoters via a ribosome binding site, and incorporated into a vector using Escherichia coli as a host. Escherichia coli may be transformed with the vector created in this manner.

The quantitative relationships commonly used in the above genetic manipulations include DNA from donor microorganisms and vector DNA.
About 0.1 to 10 μg, about 1 to 10 u of the restriction enzyme,
About 300 u of ligase and about 1 to 10 u of other enzymes are exemplified. Contains ADPHK gene DNA, ADP
As a specific example of a transformed microorganism capable of producing HK, a plasmid pcHK1 (restriction map is shown in FIG. 1) containing a microorganism belonging to Escherichia coli as a host microorganism and containing ADPHK gene DNA therein is used. Escherichia coli (Esch)
erichia coli) JM109 / pcHK1
(FERM BP-5453), and plasmid pc
A transformed microorganism carrying HK2 (restriction map is shown in FIG. 2), Escherichia coli
ia coli) JM109 / pcHK2 (FERM)
BP-5851).

In producing the ADPHK by the transformed microorganism, the transformed microorganism is cultured in a nutrient medium to produce the ADPHK in the cells or in a culture solution.
After completion of the culture, the obtained culture is collected by filtration or centrifugation to collect cells, and then the cells are destroyed by a mechanical method or an enzymatic method such as lysozyme. And / or adding an appropriate surfactant or the like to concentrate the aqueous solution of ADPHK, or, without concentration, treating the solution with adsorption chromatography such as ammonium sulfate fractionation, gel filtration, affinity chromatography, etc., or ion exchange chromatography. ADPH with good purity
K can be obtained.

The culture conditions of the transformed microorganisms may be selected in consideration of their nutritional and physiological properties. Usually, in many cases, liquid culture is used, but industrially, deep aeration and stirring culture is performed. Is advantageous. As the nutrient source of the medium, those commonly used for culturing microorganisms can be widely used.
The carbon source may be any assimilable carbon compound,
For example, glucose, saccharose, lactose, maltose, fructose, molasses and the like are used. Any available nitrogen compound may be used as the nitrogen source. For example, peptone, meat extract, yeast extract, casein hydrolyzate and the like are used. In addition, salts such as phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and the like are used as necessary.

The cultivation temperature can be appropriately changed within a range in which the microorganism grows and produces ADPHK. In the case of Escherichia coli, it is preferably about 20 to 42 ° C. The cultivation conditions are slightly different depending on the conditions, but the cultivation may be terminated at an appropriate time in anticipation of the timing when ADPHK reaches the maximum yield. In the case of Escherichia coli, it is usually 12 to 48.
About an hour. The pH of the medium can be appropriately changed within a range in which the bacteria grow and produce ADPHK. In the case of Escherichia coli, the pH is preferably about 6 to 8.

ADPHK in the culture can be used by directly collecting and using the culture solution containing the cells. However, in general, if ADPHK is present in the culture solution, filtration and centrifugation are performed. It is used after separating the ADPHK-containing solution from the microbial cells by, for example, the above method. When ADPHK is present in the cells, the obtained culture is collected by filtration or centrifugation, and then the cells are collected. Then, if necessary, the cells are subjected to a mechanical method or an enzymatic method such as lysozyme. ADPHK is solubilized by adding a chelating agent such as EDTA and / or a surfactant, and separated and collected as an aqueous solution. ADPH obtained in this way
The K-containing solution may be precipitated by a fractional precipitation method, for example, by concentration under reduced pressure, membrane concentration, and salting-out treatment with ammonium sulfate, sodium sulfate, or the like.

The precipitate is then dissolved in water and dialyzed through a semipermeable membrane to remove lower molecular weight impurities. Further, ADP obtained by purification by gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography such as affinity chromatography, ion exchange chromatography, etc., and using these means is used.
From the HK-containing solution, ADPHK purified by a treatment such as freeze-drying under reduced pressure is obtained.

The measurement of the activity of ADPHK is as follows. Measurement reagent 50 mM Tris-HCl buffer (pH 7.5) 20 mM glucose 2 mM ADP 2 mM MgCl ₂ 5 U / ml G6PDH (glucose-6-phosphate dehydrogenase: manufactured by Toyobo Co., Ltd.) 0.025% NBT (nitrotetrazonium blue) 1 mM NADP 1% Triton X-100 5 U / ml DIP (Diaphorase: manufactured by Asahi Kasei Corporation )

After preliminarily heating 1 ml of the measuring reagent at 37 ° C. for 1 minute, 0.02 ml of the enzyme solution is added and reacted for 10 minutes. After the reaction, the reaction was stopped by adding 2 ml of 0.1 N hydrochloric acid, and within 5 minutes using a cell having a layer length of 1.0 cm,
The absorbance at a wavelength of 550 nm is measured (As). As a blind test, absorbance is measured by performing the same operation using 0.02 ml of distilled water instead of the enzyme solution (Ab),
Absorbance (As) using this enzyme and absorbance (Ab) of blind test
The enzyme activity is determined from the absorbance difference (As-Ab). One unit of enzyme activity is 1 μmol of reduced NAD per minute at 37 ° C.
The amount of the enzyme that generates P is calculated by the following formula. Enzyme activity (U / ml) = (As-Ab) × 0.795 ×
Enzyme dilution factor

As the ADPHK obtained by the above production method, for example, ADPHK having the following various physical properties is exemplified. Physicochemical properties <Derived from Escherichia coli JM109 / pcHK1> (1) Enzymatic action: The enzymatic action using glucose as a substrate is shown below. Hexose + ADP → hexose-6-phosphate + AMP (2) Molecular weight: 100,000 ± 5,000 However, TSK-G3000SW _XL (0.
(75 × 30 cm) by gel filtration. (3) Optimum pH: pH 6.0 to 7.0 (phosphate buffer) (4) Optimum temperature: 80 to 100 ° C

<Escherichia coli JM109.pcH
Derived from K2> (1) Enzymatic action: Enzymatic action using glucose as a substrate is shown below. Hexose + ADP → hexose-6-phosphate + AMP (2) Molecular weight: 50,000 ± 5,000 provided that TSK-G3000SW _XL (0.
(75 × 30 cm) by gel filtration. (3) Optimum pH: pH 7.0 to 7.5 (phosphate buffer) (4) Optimum temperature: 80 to 100 ° C

[0047]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples. In the Examples, the operation described as being in accordance with a conventional method can be carried out, for example, according to the above-mentioned method of Maniatis et al., Or the procedure attached to various commercially available enzymes and kits. In addition, recombinant DNA experimental enzyme reagents (restriction enzymes, etc.) used in the experiment, vector DN
A, kits were purchased from Takara Shuzo Co., Ltd. unless otherwise noted.

Example 1 <Culture of Pyrococcus furiosus> Thermophilic medium (0.
1% yeast extract, 0.5% tryptone, 0.72% maltose, 2.39% sodium chloride, 0.4% sodium sulfate, 0.07% potassium chloride, 0.02% sodium bicarbonate, 0.01% Potassium bromide, 0.03% boric acid, 1.08% magnesium chloride, 0.15% calcium chloride, 25ppm strontium chloride, 0.025%
Ammonium chloride, 0.014% dipotassium hydrogen phosphate, 0.1% sodium acetate, 15 ppm nitrilotriacetic acid, 5 ppm manganese sulfate, 14 ppm ferrous sulfate,
ppm nickel chloride, 1 ppm cobalt sulfate, 1 ppm
Zinc sulfate, 0.1 ppm copper sulfate, 0.01 ppm sodium tungstate, 0.01 ppm sodium molybdate, 0.1% cysteine hydrochloride, pH 7) 500 m
was dispensed into 10 500 ml Erlenmeyer flasks at 50 ml each and heat-sterilized at 120 ° C. for 20 minutes, and then Pyrococcus furiosus DSM3638 strain (Deuts)
che Sammlung von Mikroorg
anismen und Zellkulturen
(Sustained from GmbH) was transplanted and cultured at 95 ° C. for 20 hours with stirring to obtain a seed culture. Next, 200 l of the thermophilic bacterium medium was prepared in a 300 l tank, sterilized, transplanted with a seed culture, cultured at 95 ° C. for 15 hours while stirring, and cultured at 5 mU / ml with a culture medium of 2 mU / ml.
00l was obtained.

Example 2 <Purification of PfuHK> 200 l of the obtained culture was centrifuged, and the obtained cells were washed with 20% NaCl containing 0.9% NaCl.
Washed once with mM Tris-HCl buffer (pH 7.5). In addition, the culture solution 50
Cells equivalent to 0 ml were separately frozen and stored. 2 washed cells
Suspended in 0 mM Tris-HCl buffer (pH 7.5), adjusted to 2 liters, and an ultrasonic crusher manufactured by Kubota (INSON)
(ATOR 201M) at 180 W for 30 minutes to obtain a disrupted cell suspension.

The crushed liquid was centrifuged at 8,000 rpm for 30 minutes to obtain 1.8 l (enzyme activity: 980 U) of a supernatant. The supernatant was dialyzed overnight at 5 ° C. against 8 liters of 10 mM Tris-HCl buffer (pH 7.5) using a dialysis tube, and then 10 mM Tris-HCl buffer (pH 7.0).
DEAE-Sepharose FF buffered in 5)
(Pharmacia) 200ml (2.6 × 38cm)
And elution was performed with a linear gradient of 0 to 1 mol of NaCl. As a result, an active fraction (950 U) was eluted at a NaCl concentration of 0.08 to 0.1 mol. NaCl was added to the obtained active fraction so as to be 4M.
And Phenyl buffered with 4M NaCl
-Sepharose FF (Pharmacia) 20
Pass through a 0 ml (2.6 × 38 cm) column, 4-0 M
Was eluted with a linear gradient of NaCl.

As a result, 0.02 to 0.07 mol of N
An active fraction (900 U) was obtained at an aCl concentration. The obtained active fraction was subjected to 10 mM Tris-HCl (pH 7.5).
After dialysis against 8 l at 5 ° C. overnight, 100 ml (2.6 × 19 cm) of hydroxyapatite (Pentax) buffered with 10 mM Tris-HCl buffer (pH 7.5).
Through a 0 to 0.5 M phosphate buffer (pH
Elution was performed using the linear gradient of 7.5). As a result, an active fraction (850 U) was eluted at a phosphate buffer concentration of 0.02 to 0.03 M. This enzyme solution was freeze-dried to obtain 5 mg of enzyme powder (170 U / mg).

Example 3 <Analysis of N-terminal amino acid sequence of PfuHK> The N-terminal amino acid sequence of purified PfuHK obtained in Example 2 was
Determined by the Edman decomposition method. The determined N-terminal amino acid sequence is represented by amino acids 1 to 39 in Sequence Listing 1.

Example 4 <Preparation of radioactive DNA probe for cloning PfuHK gene> Pf encoding the identified partial amino acid sequence
The nucleotide sequence of the uHK gene DNA was predicted. The DNA probe designed based on this sequence has a myriad of shapes. Among them, in the present invention, an oligonucleotide named HK6 having the base sequence shown in Sequence Listing 3 was used.

This oligonucleotide was prepared by outsourcing to an external organization (Vex), and 200 ng of the completed oligonucleotide was ligated to a T4 polynucleotide kinase buffer (50 mM Tris-HCl buffer (pH 8.
0) 37 ° C. with 8.5 u of T4 polynucleotide kinase in the presence of 10 mM magnesium chloride, 10 mM 2-mercaptoethanol) and 740 kBq (kilo becquerel) [γ- ³² P] ATP (available from Daiichi Kagaku). ,
The reaction was carried out for 30 minutes, and radioisotope ³² P was incorporated to obtain a radioactive DNA probe.

Example 5 <Extraction of DNA from Pyrococcus furiosus> Pyrococcus furiosus DS cryopreserved in Example 2
M3638 strain cells were thawed, 20 mM sodium acetate (pH 7.0), 1 mM EDTA, 0.5% SD
After suspending in 20 ml of a solution consisting of S and adding an equal amount of water-saturated phenol, stirring, keeping the mixture at 65 ° C. for 5 minutes,
The aqueous layer was collected by centrifugation at 0 rpm for 15 minutes.
After performing the same phenol treatment again on the collected aqueous layer,
After adding 20 ml of a 24: 1 mixture of chloroform / isoamyl alcohol and stirring for 5 minutes, the mixture was stirred at 12,000 rpm for 1 hour.
After centrifugation for 5 minutes, the aqueous layer was collected again.

A 1/10 amount of 3M sodium acetate (pH 5.5) was mixed with the recovered aqueous layer, and then gently overlaid with a double amount of ethanol. The chromosomal DNA was wound around a glass rod and separated. The separated chromosomal DNA was combined with 10 mM Tris-HCl (pH 8.0), 1 mM EDTA aqueous solution (T
E buffer) dissolved in 20 ml, and 20 mg / ml R
Add 200 μl of NaseA and incubate at 37 ° C. for 1 hour,
Mixed RNA was degraded. Next, an equal amount of a phenol / chloroform mixed solution was added, the mixture was treated in the same manner as above, and the aqueous layer was separated. 1/10 amount of 3 in the separated aqueous layer
M sodium acetate (pH 5.5) and twice the amount of ethanol were added, and the chromosomal DNA was separated once again by the method described above.

This chromosome was placed in 50 ml of TE (10 mM Tris-HCl buffer (pH 8.0), 1 mM EDTA).
(PH 8.0)), 20 ml of a 1: 1 mixture of TE-saturated phenol and chloroform was added, and the whole was suspended. The same centrifugation was repeated, and the upper layer was transferred to another container again. 2 ml of 3M sodium acetate buffer (pH 5.5) and 50 ml of ethanol were added to 20 ml of the separated upper layer.
After stirring at −70 ° C. for 5 minutes, the mixture was centrifuged (2,000 G, 4 ° C., 15 minutes), and the precipitated chromosome was washed with 75% ethanol and dried under reduced pressure. By the above operation, 1 mg of a chromosome standard of Pyrococcus furiosus DSM3638 strain was obtained.

Example 6 <Test of PfuHK gene-containing DNA fragment> Pyrococcus furiosus DS obtained by the procedure of Example 5
In order to prepare a gene library from the chromosomal DNA of the M3638 strain, the present chromosome was cut with various restriction enzymes, and an operation for testing the chain length of the DNA fragment containing the target gene was performed. That is, Pyrococcus furiosas DS
M3638 strain chromosomal DNA (10 μg) was digested with various restriction enzymes, and 1.5% agarose gel (H1 available from Takara Shuzo Co., Ltd.).
4, 40 mM Tris-acetate buffer (pH 7.4),
Electrophoresis was performed at 150 V for 1.5 hours with 2 mM EDTA, and Southern blotting was performed according to a conventional method to transfer DNA from the agarose gel to a nylon membrane (manufactured by PALL: Biodyne A).

After the membrane was air-dried, pre-hybridization was carried out according to the manual attached to the nylon membrane, and hybridization using the HK6 radioactive probe prepared in Example 4 was carried out at 45 ° C. for 1 hour.
I went there in the evening. After hybridization, 5 membranes
5 ° C. washing solution (6 × SSC, 0.05% sodium pyrophosphate [1 × SSC: 0.15 M sodium chloride, 15
mM sodium citrate]: 100 square cm of membrane
(About 50 ml per 10 minutes), and the membrane was air-dried. The dried membrane was overlaid on an X-ray film (New RXO-H, manufactured by Fuji Photo Film Co., Ltd.), and subjected to autoradiography at -70 ° C for 24 hours under light shielding.

After completion of the autoradiography, the film was developed, and the size of the positive band indicated by the chromosome cut by each restriction enzyme was observed. As a result, about 5 kb (kilobase: a unit of DNA chain length,
PfuH on a DNA fragment (1,000 base pairs)
It was revealed that the K gene was contained.
A gene library was prepared from the 5 kb fragment of the chromosomal DNA cleaved in the above.

Example 7 <Preparation of Gene Library> 10 μg of the chromosomal DNA of Pyrococcus furiosus DSM3638 strain obtained by the procedure of Example 5 was digested with a restriction enzyme BglII, and a DNA fragment of about 5 kb was separated according to a conventional method. This D
The NA fragment is digested with a restriction enzyme BglII and alkaline phosphatase (hereinafter abbreviated as BAP) 1u
1 μg of pUC118 dephosphorylated at the cleaved end with
The DNA was ligated with DNA Ligation Kit (Takara Shuzo). Using this, Escherichia coli JM109 (ATCC53) was prepared as competent cells according to a conventional method.
323, sold by Toyobo Co., Ltd.), and 3.7% B containing 50 μg / ml ampicillin.
The cells were cultured overnight on an HI agar medium (manufactured by DIFCO) to obtain about 1,000 ampicillin-resistant colonies, which were used as a gene library.

Example 8 <Screening of PfuHK Gene-Containing Clones> The gene library obtained in Example 7 was replicated on a nylon membrane (manufactured by PALL: Biodyne A), and bacterial cells were collected according to the manual attached to this filter. The DNA was fixed. This filter was subjected to pre-hybridization and Example 4 according to the attached manual.
Hybridization using the HK6 probe prepared in the above. After hybridization, the membrane was washed with the washing solution at 55 ° C. shown in Example 6 for 10 minutes, and then the membrane was air-dried. This dried membrane was overlaid on an X-ray film, and subjected to autoradiography at -70 ° C for 24 hours under light shielding. After completion of the autoradiography, the film was developed, and two colonies showing a positive signal were confirmed.

Example 9 <Extraction of Recombinant Plasmid> A colony showing a positive signal selected in Example 8 was inoculated into 1.5 ml of LB liquid medium containing 50 μg / ml of ampicillin and cultured with shaking at 37 ° C. for 16 hours. The plasmid was extracted according to a conventional method. As a result, the plasmids extracted from the two colonies contained the same chromosomal DNA fragment. One of the plasmids was named pcHK1, and the Escherichia coli JM109 carrying the plasmid was identified as Escherichia coli JM109. It was named pcHK1. The structure of plasmid pcHK1 is shown in FIG. In the figures, "pfuhk" represents the PfuHK structural gene, "ap" represents the ampicillin resistance structural gene, and "ori" represents the replication origin region of the E. coli plasmid.

Example 10 <Determination of DNA Sequence of PfuHK Gene> From the plasmid pcHK1 obtained in Example 9, the nucleotide sequence of the PfuHK structural gene was determined by the dideoxy method. The determined base sequence and the encoded amino acid sequence are shown in Sequence Listing 1.

Example 11 <Preparation of radioactive DNA probe for cloning of TliHK gene> Next, similar to Pyrococcus fuliosus, it has a primary structural homology with PfuHK in Thermococcus litoralis, which is an extremely hyperthermophilic archaeon. And AD
Focusing on the presence or absence of the enzyme TliHK having PHK activity, in order to clone this TliHK gene, Pf
Using a DNA fragment containing a part of the uHK gene,
It was planned to prepare a DNA probe to be used for isolating the iHK gene, and the following operation was performed.

1 μg of plasmid p prepared in Example 9
cHK1 was digested with the restriction enzyme EcoRV, and a DNA fragment of about 1.3 kb containing a part of the PfuHK gene was separated by a conventional method. To 50 ng of this DNA fragment, a random primer labeling kit (Takara Shuzo Co., Ltd .: BcaBEST)
Labeling Kit) and 370 kBq of [α-
^{Using [32} P] dCTP (available from Daiichi Kagaku), the reaction was carried out in accordance with the attached manual to prepare a ³² P-labeled radioactive DNA probe.

Example 12 <Culture of Thermococcus litoralis> Thermophilic medium 50
0 ml was prepared in a 1000 ml Erlenmeyer flask,
At 20 ° C. for 20 minutes, and then added to Thermococcus litoralis ATCC 51850 strain (American).
(Distributed from Type Culture Collection) was transplanted, and cultured at 85 ° C. for 20 hours while stirring. The culture was centrifuged at 6,000 rpm for 30 minutes to collect the bacteria.

Example 13 <Extraction of DNA from Thermococcus litoralis> The cells of Thermococcus litoralis ATCC 51850 cultured and collected in Example 12 were isolated from 20 mM sodium acetate (pH 7.0) and 1 mM EDTA. , 0.5% S
The suspension was suspended in 20 ml of a solution containing DS, an equal amount of water-saturated phenol was added, and the mixture was stirred and kept at 65 ° C. for 5 minutes.
The aqueous layer was collected by centrifugation at 00 rpm for 15 minutes. The collected aqueous layer was again subjected to the same phenol treatment, and then 20 ml of chloroform / isoamyl alcohol 2
Add a 4: 1 mixture and stir for 5 minutes, then 12,000 rpm
For 15 minutes, and the aqueous layer was collected again.

The recovered aqueous layer was mixed with 1/10 volume of 3M sodium acetate (pH 5.5), gently overlaid with 2 volumes of ethanol, and the chromosomal DNA was wound around a glass rod and separated. . The separated chromosomal DNA is dissolved in 20 ml of TE buffer (10 mM Tris-hydrochloric acid (pH 8.0), 1 mM aqueous EDTA solution), 200 μl of 20 mg / ml RNaseA is added, and the mixture is kept at 37 ° C. for 1 hour. RNA was degraded. Next, an equal amount of a phenol / chloroform mixed solution was added, the mixture was treated in the same manner as above, and the aqueous layer was separated. One-tenth amount of 3M sodium acetate (pH 5.5) and two-fold amount of ethanol were added to the separated aqueous layer, and the chromosomal DNA was separated again by the above method.

The chromosome was dissolved in 50 ml of TE buffer, 20 ml of a 1: 1 mixture of phenol and chloroform saturated with TE buffer was added, and the whole was suspended.
The same centrifugation was repeated, and the upper layer was transferred again to another container. To the separated upper layer (20 ml) were added 3 M sodium acetate buffer (pH 5.5) (2 ml) and ethanol (50 ml). After stirring, the mixture was cooled at -70 ° C for 5 minutes and centrifuged (2,000 G, 4 ° C, 15 minutes). The precipitated chromosome was washed with 75% ethanol and dried under reduced pressure. By the above operation, Thermococcus litoralis ATCC 51850
1 mg of a chromosome sample of the strain was obtained.

Example 14 <Assay for PfuHK Gene Homologous DNA Fragment> In order to prepare a gene library from the chromosomal DNA of Thermococcus litoralis ATCC 51850 obtained by the procedure of Example 13, this chromosome was cut with various restriction enzymes. Then, an operation for testing the chain length of the DNA fragment containing the target gene was performed. That is, Thermococcus
Litoralis ATCC 51850 chromosomal DNA (10
μg) was cleaved with various restriction enzymes, and 150 V was applied to a 1.5% agarose gel (Takara Shuzo H14, 40 mM Tris-acetate buffer (pH 7.4), 2 mM EDTA).
After 1.5 hours of electrophoresis, Southern blotting was performed according to a conventional method, and DNA was transferred from the agarose gel to a nylon membrane (manufactured by PALL: Biodyne A).

After air drying the membrane, 0.05 ml of a hybridization solution (0.1 ml / cm 2) was used.
% Ficoll, 0.1% polyvinylpyrrolidone, 0.1
% Fetal bovine serum albumin (hereinafter abbreviated as BSA)
(Manufactured by Sigma), 0.75 M sodium chloride, 75 mM
Sodium citrate, 50 mM trisodium phosphate,
0.1% sodium dodecyl sulfate, 250 μg / ml salmon sperm DNA (Boehringer Mannheim), 30
% Formamide) and prehybridized at 42 ° C for 2 hours.

After completion of the treatment, the hybridization solution was replaced with a new one.
An NA probe was added at 74 kBq, and a hybridization treatment was performed at 42 ° C. overnight. After hybridization, 50 ml of membrane per 100 square cm
Wash solution (75 mM sodium chloride, 7.5 mM sodium citrate, 0.1% SDS) at 50 ° C.
After washing for 0 minutes, the membrane was air-dried. An X-ray film (Fuji Photo Film Co., Ltd.)
New RXO-H).
Timed autoradiography was performed.

After completion of the autoradiography, the film was developed, and the size of the positive band indicated by the chromosome cut by each restriction enzyme was observed. As a result, EcoT22
By digestion with I, a positive band was confirmed on a DNA fragment of about 2.5 kb, suggesting that a base sequence having homology to the PfuHK gene DNA was contained.
A gene library was created from the b fragment.

Example 15 <Preparation of Gene Library> 10 μg of the chromosomal DNA of Thermococcus litoralis ATCC 51850 obtained by the procedure of Example 13 was digested with a restriction enzyme EcoT22I, and a DNA fragment of about 2.5 kb was digested according to a conventional method. separated. This DNA fragment is used as the restriction enzyme Pst
I and 1 μg of p-phosphorylated end-phosphorylated with 1u of alkaline phosphatase (hereinafter abbreviated as BAP)
UC119 was ligated with a DNA ligation kit (Takara Shuzo: DNA Ligation Kit). Using this, Escherichia coli JM109 transformed into competent cells was transformed according to a conventional method, and 3.7 containing 50 μg / ml ampicillin was used.
% Of BHI agar medium overnight to obtain about 2,500 ampicillin-resistant colonies, which was used as a gene library.

Example 16 <Screening of clone containing PfuHK gene homologous DNA> The gene library obtained in Example 15 was
Nylon membrane (Pall: Biodyne A)
And the DNA of the bacterial cells was fixed according to the manual attached to the membrane. The membrane on which the DNA was immobilized was immersed in the hybridization solution described in Example 14 (20 μl per square cm of the membrane).
Prehybridization treatment was performed at 42 ° C for 2 hours. After the completion of the treatment, the hybridization solution was replaced with a new one, the radioactive DNA probe prepared in Example 11 was added at 74 kBq, and the hybridization treatment was performed at 42 ° C. overnight. After the hybridization, the membrane was washed with a washing solution at 50 ° C. shown in Example 14 (50 ml per 100 cm 2 of the membrane) for 10 minutes, and then naturally dried. This dried membrane is converted to an X-ray film (New RXO- manufactured by Fuji Photo Film Co., Ltd.).
H), and autoradiography was performed at -70 ° C for 24 hours under light shielding. After autoradiography,
The film was developed, and three colonies showing a positive signal were confirmed.

Example 17 <Extraction of Recombinant Plasmid> A colony showing a positive signal selected in Example 16 was inoculated into 1.5 ml of a 50 μg / ml ampicillin-containing LB liquid medium and incubated at 37 ° C.
After shaking culture for 16 hours, the plasmid was extracted according to a conventional method. As a result, plasmids extracted from the three colonies contained the same chromosomal DNA fragment, and one of the plasmids was named pcHKT.

Example 18 <Determination of DNA Base Sequence of TliHK Gene> Example 17
The base sequence of the 2.5 kb DNA derived from Thermococcus litoralis contained in the plasmid pcHKT obtained in the above was determined by the dideoxy method. As a result, D
A structural gene having a length of about 1.4 kb in NA and having a homology of 64.8% with the base sequence of the PfuHK structural gene (according to GENETYX ver. 9.0, manufactured by Software Development Corporation) was confirmed. Was identified as the TliHK gene. This indicates that it is possible to obtain ADPHK gene DNA and mutant ADPHK gene DNA derived from different organisms having at least 60% or more nucleotide sequence homology. The amino acid sequence encoded by the TliHK structural gene is 58.9% the amino acid sequence of PfuHK.
(Id.). This indicates that it is possible to obtain ADPHK gene DNA and mutant ADPHK gene DNA from different organisms having at least 55% or more amino acid homology. Tli decided
The nucleotide sequence of the HK gene DNA and the amino acid sequence encoded thereby are shown in Sequence Listing 2.

Example 19 <Preparation of TliHK Expression Plasmid> 1 μg of pcH
Using KT, restriction enzymes SalI and SphI were 5
Cut in units of 2 hours at 37 ° C. according to the attached manual, and perform about 2.0 kb by 0.7% agarose gel electrophoresis.
The DNA fragment containing the TliHK gene was separated and recovered.
This DNA fragment was ligated with 1 μg of pUC118 cut with a restriction enzyme as described above using a DNA ligation kit. Using this, the cells were transformed into competent Escherichia coli JM109 according to a conventional method, and cultured overnight on a 3.7% BHI agar medium containing 50 μg / ml ampicillin.

Thus, the SalI of pUC118
And about 2.0k including the TliHK gene at the SphI site
Escherichia coli JM109 carrying the plasmid into which the DNA fragment b was inserted was obtained, the transformed strain was named Escherichia coli JM109 · pcHK2, and the plasmid carried by this strain was named pcHK2. Further pcH
K2 was extracted by a conventional method. The structure of the plasmid pcHK2 is shown in FIG. “Tlihk” in the figure is TliH
The K structural gene, “ap” represents the ampicillin resistance structural gene, and “ori” represents the replication origin region of the E. coli plasmid.

Example 20 <Culture of E. coli carrying pcHK1 and pcHK2 and preparation of cell extract thereof> Escherichia coli JM109 · p
cHK1 and Escherichia coli JM109 / pcHK2
Was 3.7% containing 50 μg / ml ampicillin.
37 ml with 1.5 ml of BHI (DIFCO) liquid medium
At 16 ° C. for 16 hours, and 1 ml thereof was centrifuged (1
5,000 G, 1 minute, 4 ° C.), 200 μl
Of 10 mM Tris-HCl buffer (pH 8.0)
After disrupting the cells using an ultrasonic disrupter, centrifugation (1
4,000 G, 5 minutes, 4 ° C.), and the supernatant was obtained as a cell extract. Similarly, an extract of Escherichia coli JM109 / pUC118 transformed with the cloning vector pUC118 containing no ADPHK gene was also prepared.

Example 21 <Confirmation of ADPHK Enzyme Activity in Cell Extract> Example 2
0 Escherichia coli JM109.pcHK
1. Escherichia coli JM109 / pcHK2, and
And Escherichia coli JM109 / pUC118 cells
The ADPHK enzyme activity in the extract was determined by the following ADPHK enzyme:
It was measured by elementary activity measurement method. Measurement reagent 1 50 mM Tris-HCl buffer (pH 7.5) 20 mM glucose 2 mM ADP 2 mM MgCl_Two Measurement reagent 2  50 U / ml G6PDH 0.25% NBT (nitrotetrazonium blue) 10 mM NADP 10% Triton X-100 5 U / ml DIP

After preliminarily heating 1 ml of the measuring reagent 1 at 80 ° C. for 5 minutes, 0.02 ml of the enzyme solution is added and reacted for 10 minutes. After the reaction, the reaction solution is preliminarily heated at 37 ° C. for 5 minutes, and then 0.1 ml of measurement reagent 2 is added and reacted for 10 minutes. After the reaction, the reaction was stopped by adding 2 ml of 0.1 N hydrochloric acid, and within 5 minutes using a cell having a layer length of 1.0 cm,
The absorbance at a wavelength of 550 nm is measured (As). As a blind test, the same operation is performed using 0.02 ml of distilled water instead of the enzyme solution, and the absorbance is measured (Ab). The absorbance difference between the absorbance of the enzyme (As) and the absorbance of the blind test (Ab) The enzyme activity is determined from (As-Ab). One unit of enzyme activity is 1 μmol of reduced NADP per minute at 37 ° C.
And the calculation formula is as follows. Enzyme activity (U / ml) = (As-Ab) × 0.795 ×
Enzyme dilution factor

The result of measuring the activity by the above method is as follows.
For Escherichia coli JM109 / pcHK1, 0.11 unit per 1 ml of culture solution, Escherichia coli JM109
0.13 / ml of culture solution for M109 / pcHK2
The activity of the unit (both at the time of reaction at 80 ° C.) was detected, and no activity was detected in Escherichia coli JM109 / pUC118. With this, Escherichia Kori JM
109 PCHK1 and Escherichia coli JM10
9. Expression of ADPHK activity in pcHK2 was confirmed.

[0085]

EFFECT OF THE INVENTION Chromosome DN derived from a PfuHK-producing strain was obtained using an oligonucleotide synthesized based on the partial amino acid sequence of PfuHK having ADPHK activity.
A library obtained by isolating a novel PfuHK gene whose entire nucleotide sequence of the PfuHK gene DNA was clarified from the A library, and isolating another DNA TliHK gene having ADPHK activity based on the PfuHK gene. Highly efficient AD using DNA
PHK production is now possible.

[0086]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1365 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: genomic DNA Origin Organism: Pyrococcus furiosas
strain name: DSM3638 Sequence characteristics PCDS sequence ATG CCC ACT TGG GAG GAG CTT TAT AAA AAT GCA ATA GAG AAG GCC ATA 48 Met Pro Thr Trp Glu Glu Leu Tyr Lys Asn Ala Ile Glu Lys Ala Ile 1 5 10 15 AAA TCA GTG CCA AAG GTT AAA GGG GTT CTG CTT GGA TAT AAC ACA AAC 96 Lys Ser Val Pro Lys Val Lys Gly Val Leu Leu Gly Tyr Asn Thr Asn 20 25 30 ATC GAC GCT ATA AAA TAC TTG GAC AGC AAG GAC CTT GAG GAA AGA ATA 144 Ile Asp Ala Ile Lys Tyr Leu Asp Ser Lys Asp Leu Glu Glu Arg Ile 35 40 45 ATA AAA GCT GGA AAA GAG GAA GTG ATA AAG TAT TCA GAA GAG CTC CCA 192 Ile Lys Ala Gly Lys Glu Glu Val Ile Lys Tyr Ser Glu Glu Leu Pro 50 55 60 GAT AAA ATC AAC ACT GTC TCT CAA CTC CTT GGT TCA ATA CTT TGG AGC 240 Asp Lys Ile Asn Thr Val Ser Gln Leu Leu Gly Ser Ile Leu Trp Ser 65 70 75 80 ATA AGA AGG GGA AAA GCT GCA GAA CTG TTC GTC GAA AGT TGC CCA GTG 288 Ile Arg Arg Gly Lys Ala Ala Glu Leu Phe Val Glu Ser Cys Pro Val 85 90 95 AGA TTT TAC ATG AAG AGA TGG GGC TGG AAT GAG CTC A GA ATG GGA GGC 336 Arg Phe Tyr Met Lys Arg Trp Gly Trp Asn Glu Leu Arg Met Gly Gly 100 105 110 CAA GCT GGA ATA ATG GCA AAT CTC TTG GGA GGA GTT TAT GGG GTT CCT 384 Gln Ala Gly Ile Met Ala Asn Leu Leu Gly Gly Val Tyr Gly Val Pro 115 120 125 GTA ATT GTT CAC GTT CCC CAG CTT TCA AGA CTC CAA GCT AAT CTA TTC 432 Val Ile Val His Val Pro Gln Leu Ser Arg Leu Gln Ala Asn Leu Phe 130 135 140 TTG GAC GGC CCG ATC TAC GTT CCA ACT TTG GAG AAT GGA GAA GTA AAA 480 Leu Asp Gly Pro Ile Tyr Val Pro Thr Leu Glu Asn Gly Glu Val Lys 145 150 155 160 TTG ATC CAT CCA AAG GAG TTT AGT GGA GAC GAA GAG AAC TGT ATC CAC 528 Leu Ile His Pro Lys Glu Phe Ser Gly Asp Glu Glu Asn Cys Ile His 165 170 175 TAC ATT TAT GAA TTC CCC AGG GGA TTC AGA GTT TTT GAG TTT GAA GCA 576 Tyr Ile Tyr Glu Phe Pro Arg Gly Phe Arg Val Phe Glu Phe Glu Ala 180 185 190 CCT AGA GAG AAT AGA TTC ATA GGC TCC GCC GAT GAT TAC AAC ACA ACT 624 Pro Arg Glu Asn Arg Phe Ile Gly Ser Ala Asp Asp Tyr Asn Thr Thr 195 200 205 CTC TTC ATA AGA GAG GAG TTT AGA GAA A GC TTT AGC GAA GTA ATA AAG 672 Leu Phe Ile Arg Glu Glu Phe Arg Glu Ser Phe Ser Glu Val Ile Lys 210 215 220 AAC GTC CAG TTA GCA ATA CTG AGT GGA CTG CAG GCT TTA ACA AAA GAG 720 Asn Val Gln Leu Ala Ile Leu Ser Gly Leu Gln Ala Leu Thr Lys Glu 225 230 235 240 AAC TAC AAG GAG CCT TTT GAG ATT GTC AAG TCG AAC TTG GAG GTT CTG 768 Asn Tyr Lys Glu Pro Phe Glu Ile Val Lys Ser Asn Leu Glu Val Leu 245 250 255 AAC GAG AGG GAA ATC CCA GTT CAC TTG GAA TTT GCA TTT ACG CCT GAC 816 Asn Glu Arg Glu Ile Pro Val His Leu Glu Phe Ala Phe Thr Pro Asp 260 265 270 GAA AAA GTT AGA GAA GAG ATA TTG AAC GTT CTT GGA ATG TTC TAC AGT 864 Glu Lys Val Arg Glu Glu Ile Leu Asn Val Leu Gly Met Phe Tyr Ser 275 280 285 GTA GGG CTT AAC GAG GTA GAG CTG GCA TCA ATA ATG GAA ATC TTG GGA 912 Val Gly Leu Asn Glu Val Glu Leu Ala Ser Ile Met Glu Ile Leu Gly 290 295 300 GAG AAG AAG CTC GCA AAA GAA CTA CTG GCC CAC GAT CCC GTA GAT CCA 960 Glu Lys Lys Leu Ala Lys Glu Leu Leu Ala His Asp Pro Val Asp Pro 305 310 315 320 ATA GCT GTG ACT GAA G CA ATG TTA AAG CTT GCC AAG AAG ACT GGG GTT 1008 Ile Ala Val Thr Glu Ala Met Leu Lys Leu Ala Lys Lys Thr Gly Val 325 330 335 AAG AGG ATA CAC TTC CAC ACG TAT GGT TAT TAT CTC GCA CTA ACC GAA 1056 Lys Arg Ile His Phe His Thr Tyr Gly Tyr Tyr Leu Ala Leu Thr Glu 340 345 350 TAC AAG GGA GAG CAC GTG AGG GAT GCT CTC CTA TTT GCA GCC CTA GCA 1104 Tyr Lys Gly Glu His Val Arg Asp Ala Leu Leu Phe Ala Ala Leu Ala 355 360 365 GCC GCT GCC AAG GCC ATG AAA GGA AAC ATA ACA AGC CTT GAA GAA ATA 1152 Ala Ala Ala Lys Ala Met Lys Gly Asn Ile Thr Ser Leu Glu Glu Ile 370 375 380 AGA GAA GCC ACA AGT GTT CCA GTC AAT GAA AAA GCC ACC CAG GTC GAA 1200 Arg Glu Ala Thr Ser Val Pro Val Asn Glu Lys Ala Thr Gln Val Glu 385 390 395 400 GAG AAA CTA AGA GCA GAG TAT GGA ATT AAG GAA GGA ATA GGA GAA GTG 1248 Glu Lys Leu Arg Ala Glu Tyr Gly Ile Lys Glu Gly Ile Gly Glu Val 405 410 415 GAG GGA TAT CAA ATA GCT TTC ATT CCA ACT AAA ATA GTG GCA AAA CCA 1296 Glu Gly Tyr Gln Ile Ala Phe Ile Pro Thr Lys Ile Val Ala Lys Pro 420 425 430 AA G AGC ACT GTC GGA ATT GGG GAC ACC ATC TCA AGC TCG GCA TTT ATT 1344 Lys Ser Thr Val Gly Ile Gly Asp Thr Ile Ser Ser Ser Ala Phe Ile 435 440 445 445 GGT GAG TTT TCA TTC ACT CTC 1365 Gly Glu Plu Ser Phe Thr Leu 450 455

SEQ ID NO: 2 Sequence length: 1401 Sequence type: number of nucleic acid strands: double-stranded Topology: linear Sequence type: genomic DNA Origin Organism: Thermococcus litoralis (Thermoc)
Strain name: ATCC51850 Sequence features PCDS sequence ATG AAG GAA AGC CTT AAA GAT AGG ATA AGA TTG TGG AAA AGG CTG TAT 48 Met Lys Glu Ser Leu Lys Asp Arg Ile Arg Leu Trp Lys Arg Leu Tyr 1 5 15 GTA AAC GCC TTT GAG AAT GCC CTA AAC GCT ATC CCA AAC GTT AAG GGT 96 Val Asn Ala Phe Glu Asn Ala Leu Asn Ala Ile Pro Asn Val Lys Gly 20 25 30 GTT CTT CTG GCA TAT AAC ACC AAC ATT GAT GCC ATA AAA TAC TTA GAC 144 Val Leu Leu Ala Tyr Asn Thr Asn Ile Asp Ala Ile Lys Tyr Leu Asp 35 40 45 AAG GAC GAT CTA GAA AAG AGA GTA ACC GAA ATA GGG AAA GAG AAG GTT 192 Lys Asp Asp Leu Glu Lys Arg Val Thr Glu Ile Gly Lys Glu Lys Val 50 55 60 TTT GAA ATT ATT GAA AAT CCA CCT GAA AAG ATC TCC TCC ATT GAA GAG 240 Phe Glu Ile Ile Glu Asn Pro Pro Glu Lys Ile Ser Ser Ile Glu Glu 65 70 75 80 CTT CTT GGT GGA ATA TTG AGG AGC ATA AAA CTT GGC AAG GCT ATG GAG 288 Leu Leu Gly Gly Ile Leu Arg Ser Ile Lys Leu Gly Lys Ala Met Glu 85 90 95 TGG TTT GTA GAG AGT GAG GAA GTG AGG AGA TAC TTA AGG GAA TGG GGA 336 Trp Phe Val Glu Ser Glu Glu Val Arg Arg Tyr Leu Arg Glu Trp Gly 100 105 110 TGG GAT GAG CTG AGA ATA GGA GGG CAG GCA GGG ATA ATG GCG AAC CTT 384 Trp Asp Glu Leu Arg Ile Gly Gly Gln Ala Gly Ile Met Ala Asn Leu 115 120 125 CTT GGG GGA GTG TAT AGA ATT CCA ACG ATT GTT CAT GTT CCT CAG AAT 432 Leu Gly Gly Val Tyr Arg Ile Pro Thrle Le Val His Val Pro Gln Asn 130 135 140 CCA AAG CTT CAG GCG GAG TTG TTT GTA GAT GGT CCT ATT TAT GTC CCC 480 Pro Lys Leu Gln Ala Glu Leu Phe Val Asp Gly Pro Ile Tyr Val Pro 145 150 155 160 GTC TTT GAA GGA AAT AAG TTG AAG CTA GTT CAC CCT AAA GAT GCC ATT 528 Val Phe Glu Gly Asn Lys Leu Lys Leu Val His Pro Lys Asp Ala Ile 165 170 175 GCA GAG GAA GAA GAG TTA ATC CAC TAC ATA TAT GAA TTT CCC AGA GGT 576 Ala Glu Glu Glu Glu Leu Ile His Tyr Ile Tyr Glu Phe Pro Arg Gly 180 185 190 TTT CAG GTT TTT GAT GTT CAA GCA CCA AGA GAG AAT AGA TTC ATA GCC 624 Phe Gln Val Phe Asp Val Gln Ala Pro Arg Glu Asn Arg Phe Ile Ala 195 200 205 AAT GCC GAT GAC TAC AAC GCG AGG GTC TAC ATG AGA AGG GAA TTC AGA 672 Asn Ala Asp Asp Tyr Asn Ala Arg Val Tyr Met Arg Arg Glu Phe Arg 210 215 220 GAA GGC TTT GAG GAA ATT ACC AGA AAT GTT GAG CTA GCG ATA ATA AGC 720 Glu Gly Phe Glu Glu Ile Thr Arg Asn Val Glu Leu Ala Ile Ile Ser 225 230 235 240 GGG CTG CAG GTT TTA AAG GAA TAC TAT CCC GAC GGA ACA ACA TAC AGA 768 Gly Leu Gln Val Leu Lys Glu Tyr Tyr Pro Asp Gly Thr Thr Tyr Arg 245 250 255 GAT GTT CTT GAT AGA GTC GAA AGT CAT TTG AAT ATC CTC AAC CGC TAC 816 Asp Val Leu Asp Arg Val Glu Ser His Leu Asn Ile Leu Asn Arg Tyr 260 265 270 AAC GTG AAG AGC CAT TTT GAA TTT GCA TAT ACT GCA AAC AGA AGG GTT 864 Asn Val Lys Ser His Phe Glu Phe Ala Tyr Thr Ala Asn Arg Arg Val 275 280 285 AGG GAG GCA CTT GTA GAG CTT TTA CCG AAG TTC ACA AGC GTC GGC CTT 912 Arg Glu Ala Leu Val Glu Leu Leu Pro Lys Phe Thr Ser Val Gly Leu 290 295 300 AAT GAA GTA GAA CTT GCC TCA ATA ATG GAG ATA ATA GGA GAC GAG GAG 960 Asn Glu Val Glu Leu Ala Ser Ile Met Glu Ile Ile Gly Asp Glu Glu 305 310 315 320 CTG GCA AAA GAA GTT CTG GAG GGA CAT ATC TTC TCA GTC ATA GAT GCG 1008 Leu Ala Lys Glu Val Leu Glu Gly His Ile Phe Ser Val Ile Asp Ala 325 330 335 ATG AAT GTT TTA ATG GAC GAG ACA GGA ATT GAG AGA ATT CAC TTC CAC 1056 Met Asn Val Leu Met Asp Glu Thr Gly Ile Glu Arg Ile His Phe His 340 345 350 ACC TAC GGC TAC TAC CTC GCC CTA ACT CAA TAC AGA GGA GAA GAG GTA 1104 Thr Tyr Gly Tyr Tyr Leu Ala Leu Thr Gln Tyr Arg Gly Glu Glu Val 355 360 365 AGA GAT GCT CTG CTC TTT GCA TCC CTC GCT GCA GCT GCC AAA GCC ATG 1152 Arg Asp Ala Leu Leu Phe Ala Ser Leu Ala Ala Ala Ala Lys Ala Met 370 375 380 AAA GGA AAC CTT GAA AGA ATA GAG CAG ATT AGA GAT GCC CTA AGT GTA 1200 Lys Gly Asn Leu Glu Arg Ile Glu Gln Ile Arg Asp Ala Leu Ser Val 385 390 395 400 CCA ACA AAT GAA AGG GCA ATA GTG CTT GAA GAA GAA CTT GAA AAG GAG 1248 Pro Thr Asn Glu Arg Ala Ile Val Leu Glu Glu Glu Leu Glu Lys Glu 405 410 415 TTC ACA GAA TTT GAG AAC GGC CTT ATT GAT ATG GTG GAC AGA CAA CTT 1296 Phe Thr Glu Phe Glu Asn Gly Leu Ile Asp Met Val Asp Arg Gln Leu 420 425 430 GCT TTT GTG CCA ACA AAG ATT GTA GCA TCT CCC AAG AGC ACC GTT GGA 1344 Ala Phe Val Pro Thr Lys Ile Val Ala Ser Pro Lys Ser Thr Val Gly 435 440 445 ATT GGA GAT ACC ATT TCA AGC TCT GCT TTT GTC AGT GAA TTT GGC ATG 1392 Ile Gly Asp Thr Ile Ser Ser Ser Ala Phe Val Ser Glu Phe Gly Met 450 455 460 AGG AAA AGG 1401 Arg Lys Arg 465

SEQ ID NO: 3 Sequence length: 32 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Other nucleic acid
(Synthetic DNA) Sequence characteristics S CDS sequence GGW TAY AAC ACW AAC ATW GAY GCW ATW AAG TA 32 Gly Tyr Asn Thr Asn Ile Asp Ala Ile Lys Tyr 1 5 10

[Brief description of the drawings]

FIG. 1 shows a restriction map of plasmid pcHK1 according to the present invention.

FIG. 2 shows a restriction map of plasmid pcHK2 according to the present invention.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12N 9/12 C12R 1:19) (C12N 15/09 ZNA C12R 1:01) (56) Reference The Journal of Biological Chemistry y, December 22, 1995, Vol. 270, No. 51, p. 30453-30457 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/09 C12N 15/31 C12N 15/54 C12N 9/12 C12N 1/21 SwissProt / PIR / GeneSeq GenBank / EMBL / DDBJ / GeneSeq BIOSIS / WPI (DIALOG) PubMed

Claims (4)

(57) [Claims] 1. An adenosine triphosphate and hexose to a hexose hexaphosphate and an adenosine monophosphate without using adenosine triphosphate and at least in the presence of adenosine diphosphate.
Sequence of hexokinase catalyzing the reaction that produces phosphate
A DNA encoding the sequence represented by any one of amino acid sequences 1 to 467 in Table 2 . 2. The method according to claim 1, wherein the DNA is selected from one of the nucleotide sequences in Sequence Listing 2.
Claims: 1. A DNA having a base sequence represented by 1401.
Item 6. The DNA according to Item 1 . 3. The DNA is one of the amino acid sequences shown in Sequence Listing 2.
To the amino acid sequence represented by 467
Is the hexokinase activity obtained by substitution
DNA encoding an amino acid sequence having the formula:
2. The DNA according to 1 . 4. An adenosine which is exogenous to a host.
Does not use triphosphate and contains at least adenosine diphosphate
Hexa from this adenosine diphosphate and hexose
The reaction that produces ose-6-phosphate and adenosine monophosphate is
Characterized by having the ability to produce mediated hexokinase
Escherichi, a substantially pure genetically modified microorganism
Escherichia coli JM1
09 · pcHK2 “Accession number, FERM BP-585
1 " .