JPH10215878A - New hexokinase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the new subject enzyme capable of producing D-hexose-6- phosphate and ADP by acting on D-hexose in the presence of ATP and magnesium, excellent in stability and useful for measurement, etc., of creatine kinase, glucose, etc. SOLUTION: The subject enzyme is a new hexokinase having activities for producing D-hexose-6-phosphate and ADP by acting on D-hexose in the presence of ATP and magnesium, and stability in a measuring solution of creatine kinase, capable of maintaining 60% activity for at least one week at 40 deg.C, being an index for clinical diagnosis for muscular disease, nervous disease, myocardial infarction, etc., and used for measurement of creatine kinase in body fluid and glucose as the diagnosis of disease causing hyperglycemia and hypoglycemia, etc. The enzyme is obtained by culturing Saccharomyces pastorianus ATCC 2366 to produce the hexokinase and collecting the hexokinase from the culturing material.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel enzyme having hexokinase activity which can be used for measuring creatine kinase, glucose, etc., a gene encoding the enzyme, and a method for producing the enzyme by genetic engineering techniques. And a reagent for measuring creatine kinase.

[0002]

2. Description of the Related Art Conventionally, hexokinase (EC 2.7.1.
1) is clinically used as a diagnostic index for muscular disease, neurological disease, myocardial infarction, etc., as a measurement of creatine kinase in body fluids, or as a diagnosis of various diseases and conditions that cause hyperglycemia and hypoglycemia. It is used together with glucose-6-phosphate dehydrogenase for measuring glucose and the like. Such hexokinases are enzymes that are ubiquitous from higher animals to microorganisms, but for clinical testing applications, glucokinase (EC 2.
7.1.2), bacteria of the genus Saccharomyces, bacteria of the genus Bacillus (JP-B-63-26991), and bacteria of the genus Sirmas (JP-B-63)
-26990).

[0003] Saccharomyces cerevisiae (Sacch
aromyces serevisiae), the gene encoding the hexokinase has been cloned, and the amino acid sequence has been reported (Nucleic Acid Research, vol. 14).
No. 2, 1986).

[0004]

However, the conventional hexokinase is not sufficient in its stability.
Particularly, in the creatine kinase measurement solution, the residual activity after long-term storage is low. Therefore, a hexokinase having excellent stability when stored for a long period of time is desired. An object of the present invention is to provide a novel hexokinase having excellent stability in a creatine kinase measurement solution.

[0005]

Means for Solving the Problems The present inventors have made intensive studies to achieve the above object, and as a result, have found that Saccharomyces
Pasteurian (Saccharomyces pastorianus) ATCC
Hexokinase produced by 2366 was found to be stable in a creatine kinase measurement solution,
The gene encoding hexokinase was isolated from the chromosomal DNA extracted from the cells, and the enzyme was successfully mass-produced from the gene, thereby achieving the present invention.

That is, the present invention is a novel hexokinase having the following physicochemical properties. Action: Acts on D-hexose in the presence of ATP and magnesium to produce D-hexose-6-phosphate and ADP. Stability in creatine kinase assay solution: at least 6
0% (Store at 40 ° C for 1 week)

Further, the present invention is a novel hexokinase which is a protein of the following (a) or (b): (A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing; (b) an amino acid sequence (a) consisting of an amino acid sequence in which one or more amino acids have been added, deleted or substituted, and a hexokinase Active protein

[0008] The present invention is a gene encoding hexokinase, which is a protein of the following (a) or (b): (A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing; (b) an amino acid sequence (a) consisting of an amino acid sequence in which one or more amino acids have been added, deleted or substituted, and a hexokinase Active protein

The present invention provides the following (c), (d) or (e)
Is a gene encoding hexokinase consisting of the following DNA: (C) DNA consisting of the nucleotide sequence of SEQ ID NO: 2 in the Sequence Listing (d) In the nucleotide sequence of (c), one or more bases are added, deleted or substituted, and the hexokinase activity is Encoding an amino acid sequence having
A: a bacterial DN which hybridizes with a DNA consisting of the nucleotide sequence of (e) and (c) under stringent conditions and encodes a protein having hexokinase activity
A

[0010] The present invention is a recombinant vector containing the gene encoding the above hexokinase.

The present invention is a transformant obtained by transforming a host cell with the above recombinant vector.

The present invention provides a method for producing hexokinase, which comprises culturing the above transformant, producing hexokinase, and collecting the hexokinase.

The present invention provides a method for producing hexokinase, which comprises culturing Saccharomyces pastorianus ATCC2366, producing hexokinase, and collecting the hexokinase.

Further, the present invention is a reagent for measuring creatine kinase, comprising the above-mentioned hexokinase, buffer solution, glucose, magnesium, ADP, NAD or NADP, and glucose-6-phosphate dehydrogenase solution.

[0015]

DETAILED DESCRIPTION OF THE INVENTION The hexokinase of the present invention is ATP.
And act on D-hexose in the presence of magnesium,
It is an enzyme that produces D-hexose-6-phosphate and ADP. Examples of D-hexose include D-glucose, D-fructose, D-galactose and the like.

The enzyme of the present invention is characterized by having at least 60% residual activity when stored in a creatine kinase measurement solution at 40 ° C. for one week.
Here, the creatine kinase measurement solution refers to a creatine kinase in a sample that acts on creatine in the presence of ADP, and the generated ATP and glucose reacts with hexokinase in the presence of magnesium ions to produce ADP and glucose-6. Creatine kinase activity is measured by generating phosphate and then allowing glucose-6-phosphate dehydrogenase to act on glucose-6-phosphate and NAD and measuring the absorbance of the change from NAD or NADP to NADH or NADPH A pH buffer, ADP, D-glucose,
A solution containing NAD or NADP, magnesium ion, glucose-6-phosphate dehydrogenase and hexokinase as components and having a pH of about 6.6 at 30 ° C.

More specifically, at least imidazole acetate buffer, magnesium acetate, ADP, D-
Glucose, NAD or NADP, glucose-6
A solution containing phosphate dehydrogenase and hexokinase as components and having a pH at 30 ° C. of about 6.6.

The preferred reagent composition of the solution is determined by the method recommended by the Japanese Society of Clinical Chemistry (Clinical Chemistry, Vol. 19, No. 2, p. 185, June 1990).

The stability in the creatine kinase assay solution refers to the ratio of the enzyme activity after storage of the enzyme in the following solution and storage at 40 ° C. for one week, relative to the enzyme activity before storage of the enzyme. In the present invention, the stability is at least 60%,
Preferably it is 70%. The conventional enzyme derived from Saccharomyces cerevisiae and Bacillus thearothermophilus is about 50% (see FIG. 6).

One embodiment of the present invention is a novel hexokinase having the following physicochemical properties. Action: Acts on D-hexose in the presence of ATP and magnesium to produce D-hexose-6-phosphate and ADP. Stability in creatine kinase assay solution: at least 6
0% (stored at 40 ° C for 1 week) Optimum temperature: about 45 to 50 ° C Optimum pH: about 8.0 to 9.0 Molecular weight: 53,998 (value obtained from amino acid composition) 55,000 ( SDS-PAGE)

Another embodiment further has the following physicochemical properties. Thermal stability: about 40 ° C. or less (pH 7.0, 30 minutes) pH stability: about 5.5 to 7.5 (25 ° C., 20 hours) Km value for glucose: about 0.21 mM Km value for ATP: about 0.074 mM substrate specificity: D-glucose, D-fructose, D-
Acts on mannose, D-galactose, 2-deoxy-D-glucose.

One embodiment of the present invention is a Saccharomyces pastorianus ATCC2.
366 is an enzyme produced.

The present invention also relates to a hexokinase which is a protein of the following (a) or (b): (A) a protein consisting of the amino acid sequence of SEQ ID NO: 1 in the sequence listing; (B) a protein comprising an amino acid sequence in which one or more amino acids have been added, deleted or substituted in the amino acid sequence (a), and which has hexokinase activity.

One embodiment of the present invention is a hexokinase having the amino acid sequence set forth in SEQ ID NO: 1.

The hexokinase of the present invention is a hexokinase-producing microorganism such as Saccharomyces pasturianus.
(Saccharomyces pastorianus) ATCC2366 can be cultured and harvested from the culture. Culture conditions may be selected in consideration of the nutritional and physiological properties of the cells. Usually, liquid culture is generally used, but aeration and agitation culture is advantageous industrially. As the nutrient source of the medium, those commonly used for culturing microorganisms can be widely used. The carbon source may be any carbon compound that can be assimilated, such as glucose, sucrose, lactose, maltose, fructose, molasses, and pyruvic acid. Any available nitrogen compound may be used as the nitrogen source, for example, peptone, meat extract, yeast extract, casein hydrolyzate,
An alkali extract of soybean meal is 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 culture temperature can be appropriately changed within a range in which the bacteria grow and produce hexokinase. Preferably, it is about 25 to 30 ° C. The cultivation time varies somewhat depending on the conditions, but the cultivation may be terminated at an appropriate time in consideration of the time when the hexokinase reaches the maximum yield, and is usually about 24 to 48 hours. The pH of the medium can be appropriately changed within a range in which the bacteria grow and produce hexokinase.
5

The culture solution containing the cells producing hexokinase in the culture can be collected and used as it is. Generally, when hexokinase is present in the culture solution, filtration, centrifugation, etc., are carried out according to a conventional method. Is used after separating the hexokinase-containing solution from the microbial cells. When hexokinase is present in the cells, the cells are collected from the obtained culture by means such as filtration or centrifugation, and then the cells are destroyed by a mechanical method or an enzymatic method such as lysozyme, ED if necessary
A chelating agent such as TA and / or a surfactant is solubilized with hexokinase and separated and collected as an aqueous solution.

The thus obtained hexokinase-containing solution is precipitated by, for example, concentration under reduced pressure, membrane concentration, salting-out treatment with ammonium sulfate, sodium sulfate or the like, or fractional precipitation with an organic solvent such as methanol, ethanol, acetone or the like. I'll let you go. Heat treatment and isoelectric point treatment are also effective purification means. After that, purified creatinine amidohydrolase can be obtained by performing gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography, and affinity chromatography. For example, gel filtration using Sephadex G-25 (Pharmacia Biotech), DEAE Sepharose CL-6B (Pharmacia Biotech) is added, and separation / separation is performed by phenyl Sepharose CL6-B (Pharmacia Biotech) column chromatography.
After purification, a purified enzyme preparation can be obtained. This purified enzyme preparation has been purified to the extent that it shows a substantially single band on electrophoresis (SDS-PAGE).

In the present invention, a gene encoding hexokinase is isolated, and the resulting enzyme is transformed into another host cell using an appropriate vector, and the expressed hexokinase is isolated. It can be obtained easily and efficiently.

The gene encoding hexokinase of the present invention includes a gene encoding hexokinase which is a protein of the following (a) or (b): (A) a protein consisting of the amino acid sequence of SEQ ID NO: 1 in the sequence listing; (B) a protein comprising an amino acid sequence in which one or more amino acids have been added, deleted or substituted in the amino acid sequence (a), and which has hexokinase activity. As a method of deletion, substitution or addition of an amino acid, a method of mutating a gene encoding the amino acid, for example,
A method using an ordinary nuclease, a polymerase chain reaction (PCR) method, and a commercially available mutagenesis kit can be used. The degree of the deletion, substitution or addition is such that the basic property is not changed or the property is improved.

The gene encoding the hexokinase of the present invention includes the following DNA (c), (d) or (e):
There is a gene encoding a hexokinase consisting of (C) DNA consisting of the nucleotide sequence of SEQ ID NO: 2 in the Sequence Listing (d) In the nucleotide sequence of (c), one or more bases are added, deleted or substituted and have hexokinase activity As a method for deleting, substituting or adding DNA encoding the amino acid sequence, for example, a method using a usual nuclease, a polymerase chain reaction (PCR) method, and a commercially available mutation introduction kit can be used. The degree of the deletion, substitution or addition is such that the basic property is not changed or the property is improved. (E) DN derived from XX which hybridizes with the DNA comprising the nucleotide sequence of (c) under stringent conditions and encodes a protein having hexokinase activity
A Here, the stringent condition is, for example, × 2 SSC
(300 mM NaCl, 30 mM citric acid), 65
° C for 16 hours.

The gene encoding the hexokinase of the present invention is, for example, Saccharomyces pasturianus (Sacchar
omyces pastorianus) ATCC2366 or can be chemically synthesized. By using the polymerase chain reaction (PCR), it is also possible to obtain a DNA fragment containing the hexokinase gene. As a method for obtaining the gene of the present invention, for example, Saccharomyces pasto
rianus) The chromosomal DNA of ATCC2366 was separated and purified, and Saccharomyces p.
Based on the nucleotide sequence encoding a hexokinase of Saccharomyces cerevisiae (Saccharomyces cerevisiae), which is related to ATCC2366 and a gene having a high homology to the gene, encodes a hexokinase of Saccharomyces pastorianus ATCC2366. A primer is prepared to amplify the gene, and a PCR reaction is performed using the prepared chromosomal DNA as a template.

The obtained PCR amplified fragment is combined with a linear expression vector at the blunt end or cohesive end of both DNAs by DNA ligase or the like to construct a recombinant vector. The thus obtained recombinant vector is transferred to a replicable host microorganism, and then screened using the expression of the marker and the enzyme activity of the vector as an index to obtain a microorganism carrying the recombinant vector containing the gene encoding hexokinase. . Next, the microorganism is cultured, the recombinant vector is separated and purified from the cultured cells, and the hexokinase gene may be collected from the recombinant vector.

That is, a culture obtained by stirring and culturing the donor microorganism for, for example, 1 to 3 days is collected by centrifugation, and then lysed to prepare a lysate containing the hexokinase gene. it can. As a method of lysis, for example, treatment with a lytic enzyme such as lysozyme or β-glucanase is performed, and if necessary, a protease or another enzyme or a surfactant such as sodium lauryl sulfate (SDS) is used in combination. Or a physical crushing method such as a French press treatment.

From the lysate thus obtained, DNA
In order to separate and purify, according to a conventional method, for example, a method such as deproteinization by phenol treatment or protease treatment, ribonuclease treatment, alcohol precipitation treatment, etc.
It can be performed by combining them.

As a vector, a vector constructed for gene recombination from a phage or a plasmid capable of autonomous propagation in a host microorganism is suitable. As the phage, for example, Escherichia coli
When i) is used as the host microorganism, Lambda-gt10, Lambd
a-gt11 etc. can be used. As a plasmid,
For example, when Escherichia coli is used as a host microorganism, pBR322, pUC19,
pBluescript, pLED-M1 (Journal of Fermentat
ion and Bioenzineering, Vol. 76, 265-269 (1993)).

The host microorganism may be any microorganism as long as the recombinant vector is stable, capable of autonomous propagation and capable of expressing a foreign gene, and is generally Escherichia coli W3.
110, Escherichia coli C600, Escherichia coli HB101, Escherichia coli JM109, etc. can be used.

As a method for transferring the recombinant vector to the host microorganism, for example, when the host microorganism is Escherichia coli, a competent cell method or an electroporation method by calcium treatment can be used. By culturing the thus obtained transformant microorganism in a nutrient medium, it has been found that a large amount of hexokinase can be stably produced. Selection of the presence or absence of transfer of the target recombinant vector to the host microorganism may be performed by searching for a drug resistance marker of the vector holding the target DNA and a microorganism that simultaneously expresses hexokinase activity. For example, selection based on the drug resistance marker A microorganism that grows in a medium and produces hexokinase may be selected.

[0038] The nucleotide sequence of the hexokinase gene obtained by the above-described method is described in Science, Vol.
1205-1210 (1981)), and the amino acid sequence of hexokinase is deduced from the determined nucleotide sequence. The recombinant vector having the hexokinase gene once selected in this manner can be easily removed from a transformed microorganism and transferred to another microorganism. In addition, restriction enzymes and PCs can be used from recombinant vectors carrying the hexokinase gene.
R is used to recover DNA that is a hexokinase gene,
It can be easily ligated with another vector fragment and transferred to a host microorganism.

The culture form of the host microorganism, which is a transformant, may be selected by taking into consideration the nutritional and physiological properties of the host. Usually, liquid culture is used in many cases. Advantageously, a stirred culture is performed. As the nutrient source of the medium, those usually used for culturing microorganisms can be widely used. The carbon source may be any assimilable carbon compound, and for example, glucose, sucrose, lactose, maltose, fructose, molasses, pyruvic acid and the like are used. Any nitrogen compound can be used as the nitrogen source, and examples thereof include peptone, meat extract, yeast extract, casein hydrolyzate, and soybean meal alkaline extract. 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 culture temperature can be appropriately changed within a range in which the bacteria grow and produce hexokinase. In the case of Escherichia coli, the culture temperature is preferably about 20 to 42 ° C. The cultivation time varies somewhat depending on the conditions, but the cultivation may be terminated at an appropriate time in consideration of the time when the hexokinase reaches the maximum yield, and is usually about 6 to 48 hours. The pH of the medium can be appropriately changed within a range in which the bacteria grow and produce hexokinase, and particularly preferably, the pH is 6.0 to 6.0.
It is about 9.0.

The culture solution containing the cells producing hexokinase in the culture can be collected and used as it is, but generally, if hexokinase is present in the culture solution, filtration, centrifugation, etc. are carried out according to a conventional method. Is used after separating the hexokinase-containing solution from the microbial cells. When hexokinase is present in the cells, the cells are collected from the obtained culture by means such as filtration or centrifugation, and then the cells are destroyed by a mechanical method or an enzymatic method such as lysozyme, ED if necessary
A chelating agent such as TA and / or a surfactant is solubilized with hexokinase and separated and collected as an aqueous solution.

The hexokinase-containing solution thus obtained is precipitated by, for example, concentration under reduced pressure, membrane concentration, salting-out treatment with ammonium sulfate, sodium sulfate or the like, or fractional precipitation with an organic solvent such as methanol, ethanol, acetone or the like. It should be done. Heat treatment and isoelectric point treatment are also effective purification means. After that, purified creatinine amidohydrolase can be obtained by performing gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography, and affinity chromatography.
For example, gel filtration using Sephadex G-25 (Pharmacia Biotech), DEAE Sepharose CL-6B (Pharmacia Biotech) is added, and the mixture is separated and purified by phenyl sepharose CL6-B (Pharmacia Biotech) column chromatography. Thus, a purified enzyme preparation can be obtained. This purified enzyme preparation has been purified to the extent that it shows a substantially single band on electrophoresis (SDS-PAGE).

The hexokinase of the present invention is used as a creatine kinase measuring reagent. The creatine kinase measurement reagent contains hexokinase, buffer, magnesium ion, glucose, ADP, NAD or NADP, and a glucose-6-phosphate dehydrogenase solution. The hexokinase of the present invention is at least 60% (at 40 ° C.,
Storage for one week), the measurement reagent does not require excessive addition of hexokinase, and the measurement of creatine kinase at low cost and with high accuracy becomes possible.

[0043]

The present invention will be described below in more detail with reference to examples. In the examples, hexokinase activity was measured under the following reagents and measurement conditions. <Reagent> Reagent A 50 mM Tis-HCl buffer, pH 8.0 (including 13 mM MgCl ₂ ) Reagent B 0.67 M glucose solution (dissolved in reagent A) Reagent C 16.5 mM ATP solution (dissolved in reagent A) Reagent D 6.8 mM NAD solution (dissolved in reagent A) Reagent E Glucose-6-phosphate dehydrogenase solution (dissolved in 300 U / ml with reagent A) <Measurement conditions> First, reagent A, reagent B, reagent C, reagent D and reagent E 28: 0.5: 0.1: 0.1: 0.0
Mix at a ratio of 1 to make a reagent mixture. After preliminarily heating 3 ml of this reagent mixture at 30 ° C. for about 5 minutes, 0.1 ml of the enzyme solution was added, the reaction was started at 30 ° C., the reaction was performed for 4 minutes, and the change in absorbance per minute at 340 mm was measured. Measure with a photometer. In the blind test, distilled water is added to the reagent mixture instead of the enzyme solution, and the absorbance change is measured in the same manner. The amount of the enzyme that produces 1 micromol of NADH per minute under the above conditions is defined as 1 unit (U).

Example 1 Collection of Hexokinase from Saccharomyces pastorianus ATCC 2366 Saccharomyces pastorianus (Saccharomyces pastorianus)
rianus) ATCC2366 in 100 ml of YPD medium (1
% Polypetone, 1% yeast extract, 1% D-glucose (pH 5.3)) at 30 ° C. overnight, and then collect by centrifugation (8,000 rpm, 10 minutes) to obtain 25 mM phosphate buffer, 5 mM Glucose, 0.1 mM PMSF, pH
6.3. The cell suspension was crushed by a French press and centrifuged to obtain a supernatant. The resulting crude enzyme solution was subjected to a nucleic acid removal treatment with polyethyleneimine and an ammonium sulfate fractionation treatment, followed by Sephadex G-2.
The solution was desalted by gel filtration using 5 (Pharmacia Biotech), separated and purified by DEAE Sepharose CL-6B (Pharmacia Biotech) column chromatography, and phenyl sepharose (Pharmacia Biotech) column chromatography to obtain a purified enzyme preparation.

The sample obtained by this method was subjected to electrophoresis (S
DS-PAGE) showed almost a single band, and the specific activity at this time was about 800 U / mg protein. The enzyme activity was measured under the above conditions and operation. The results of measuring the physicochemical properties of the purified enzyme are shown below. Action: acting on D-hexose in the presence of ADP,
Produces hexose-6-phosphate and ADP. Optimum temperature: 45-50 ° C (see Fig. 1) Optimum pH: 8.0-9.0 (see Fig. 2) Thermal stability: about 40 ° C or less (store at pH 7.0 for 30 minutes, see Fig. 3) ) PH stability: about 5.5 to 7.5 (stored at 25 ° C. for 25 hours, see FIG. 4) Km value for glucose: about 0.21 mM Molecular weight: 55,000 (SDS-PAGE)

[0046]

[Table 1]

Example 2 Isolation of Chromosomal DNA Saccharomyces pasto
rianus) The chromosomal DNA of ATCC2366 was obtained by shaking the strain in 100 ml of YPD medium (1% polypetone, 1% yeast extract, 1% D-glucose (pH 5.3)) at 30 ° C. overnight, followed by centrifugation (8,000 The cells were collected by the following method (Cell 18: 1261).
Chromosomal DNA was prepared according to -1271).

Example 3 Preparation of a DNA Fragment Containing a Gene Encoding Hexokinase and a Recombinant Vector Having the DNA Fragment Saccharomyces cerevisiae whose nucleotide sequence is known using the chromosomal DNA obtained in Example 1 as a template. (Sacch
aromyces cerevisiae) based on the nucleotide sequence encoding hexokinase.
aromyces pastorianus) Two kinds of primers (described in SEQ ID Nos. 3 and 4 in the sequence listing) were prepared to amplify the gene encoding the hexokinase of ATCC2366,
Saccharomyces pasteurianus (CR)
omyces pastorianus) A gene fragment encoding ATCC2366 hexokinase was amplified. The primers were designed such that sites for cleavage by separate restriction enzymes (NdeI at the N-terminus and BamHI at the C-terminus) were created at both ends of the amplified fragment. PCR was performed under the following reaction solution composition and amplification conditions. <Reaction composition> TaqPlus DNA polymerase (manufactured by Stratagene) 5 U / 100 μl 10-fold concentration buffer for TaqPlus DNA polymerase 10 μl / 100 μl Chromosomal DNA (template DNA) 0.01 μg / 100 μl dATP, dTTP, dGTP, dCTP 0.2 mM each Primers 1 μM each (described in SEQ ID Nos. 3 and 4 in the sequence listing) <Amplification conditions> (1) 94 ° C, 2 minutes (denaturation) (2) 94 ° C, 45 seconds (denaturation) (3) 55 ° C, 30 seconds (denaturation) (Annealing) (4) 74 ° C, 2 minutes (reaction) (30 cycles of (1) to (4) above)

About 1.5 kbp of the amplified DNA fragment was
Using a Kit (manufactured by Invitrogen), pCRTMII vector and 1 unit of T4 DNA ligase (manufactured by Toyobo) at 16 ° C.
After reacting for 12 hours, the DNA was ligated. Ligated DNA
Was transformed using competent cells of Escherichia coli JM109 (manufactured by Toyobo), and LB agar medium for selection (1% polypeptone, 0.5% yeast extract, 0.5% N
aCl, 50 μg / ml ampicillin, 0.5 mM I
PTG, 0.1 mM X-gal (pH 7.2)), and cultured at 37 ° C. for 20 hours. Among the grown colonies, white colonies were cultured in LB liquid medium (1% polypeptone, 0.5% yeast extract). , 0.5% NaCl, 50 μg
/ Ml ampicillin (pH 7.2)) at 37 ° C for 16 hours. Plasmid DN from the cultured cells according to a standard method
After preparing A, restriction enzymes NdeI and BamHI (Toyobo)
And a plasmid DNA producing an about 1.5 kbp fragment was selected.

Further, a primer (SEQ ID NO: 5 in the sequence listing) that creates a cleavage sequence with the restriction enzyme NdeI at the position of the start codon of the structural gene of β-galactosidase derived from the vector using pBluescript KS (+). And a commercially available mutation introduction kit (Transformer ™; manufactured by Clontech)
As a result, a plasmid DNA was prepared in which a sequence cut by the restriction enzyme NdeI was introduced at the position of the start codon of the structural gene of β-galactosidase. Next, this plasmid DN
A was treated with NdeI and BamHI, and a DNA fragment produced by the same restriction enzyme treatment of the plasmid DNA holding the selected PCR-amplified fragment was ligated using T4 DNA ligase, and then ligated with Escherichia coli JM109. Transform using cells (manufactured by Toyobo).
B agar medium (1% polypeptone, 0.5% yeast extract,
0.5% NaCl, 50 μg / ml ampicillin, (p
H7.2)), and cultured at 37 ° C. for 20 hours. The resulting colonies were grown on TB medium (1.2% polypeptone, 2.4% yeast extract, 0.4% glycerol, 1.25% K ₂ HPO ₄ , 0.
30% with 23% KH ₂ PO ₄ , 100 μg / ml ampicillin)
After culturing at 24 ° C. for 24 hours and screening them, a strain showing remarkable hexokinase activity could be obtained. About 1.5 kb of plasmid DNA carried by this strain
The inserted DNA fragment of p was present, and this plasmid was designated as pHXK10. FIG. 5 shows a restriction map of pHXK10.

Example 3 Determination of Nucleotide Sequence The inserted DNA fragment of about 1.5 kbp of pHXK10 was sequenced using a Radioactive Sequencing Kit (manufactured by Toyobo) in accordance with a conventional method. The determined base sequence and amino acid sequence are shown in the sequence listing. The molecular weight of the protein determined from the amino acid sequence was 53,998.
Moreover, the pHXK10 inserted DNA fragment does not contain a portion other than the hexokinase gene. That is, NdeI and BamHI restriction sites are present at both ends of the inserted DNA fragment, respectively, and NdeI is the initiation codon (AT
Overlapping with G), BamHI is located immediately after the stop codon (TGA) of the hexokinase gene.

Example 4 Preparation of Transformant Competent cells of Escherichia coli JM109 (manufactured by Toyobo) were transformed with pHXK10 to obtain a transformant Escherichia coli JM109 (pHXK10).

Example 5 Escherichia coli JM10
9 (pHKX10)) 500 ml of TB medium was dispensed into a 2 L flask, and
The mixture was autoclaved at 15 ° C for 15 minutes, allowed to cool, and then 0.5 ml of 50 mg / ml ampicillin (manufactured by Nacalai Tesque), which had been separately sterilized and filtered, was added. The culture medium was inoculated with 5 ml of a culture solution of Escherichia coli JM109 (pHXK10) previously shake-cultured at 30 ° C. for 16 hours in an LB medium containing 1% glucose, and cultured at 30 for 24 hours with aeration and stirring. Hexokinase activity at the end of the culture is about 30 U / ml
Met. The above cells were collected by centrifugation, and 25 mM phosphate buffer, 5 mM glucose, 0.1 mM PMS
F, pH 6.3. The cell suspension was crushed by a French press and centrifuged to obtain a supernatant. After subjecting the obtained crude enzyme solution to a nucleic acid removal treatment with polyethyleneimine and an ammonium sulfate fractionation treatment, Sephadex (Sephade
x) Desalting was performed by gel filtration using G-25 (Pharmacia Biotech), and DEAE Sepharose CL-6B was used.
(Pharmacia Biotech) column chromatography and phenyl sepharose (Pharmacia Biotech) column chromatography were separated and purified to obtain purified enzyme preparations. The sample obtained by this method showed a substantially single band by electrophoresis (SDS-PAGE), and the specific activity at this time was about 800 U / mg protein.

Using the hexokinase obtained by the above method, the following creatine kinase measurement solution was prepared, and its storage stability at 40 ° C. was evaluated using a commercially available hexokinase (Saccharkinase).
omyces genus, Toyobo Co., Ltd., commercially available product A) and glucokinase (Bacillu
FIG. 6 shows the results of comparison with s stearothermophilus-derived, Unitika, commercial product B). The creatine kinase measurement reagent was prepared in accordance with the method recommended by the Japanese Society for Clinical Chemistry (Clinical Chemistry, Vol. 19, No. 2, June 1990). From FIG.
It was confirmed that the hexokinase of the present invention exhibited excellent stability in a creatine kinase measurement solution.

[0055] pH (30 ℃) 6.60 imidazole acetate buffer 115 mM EDTA 2.3 mM magnesium acetate 11.3 mM N-acetyl -L- cysteine 23 mM ADP 2.3mM AMP 5.8mM P ' , 5 5 - Jiadenomin 5'Pentalyn acid 11.5 μM D-glucose 23 mM NADP 2.3 mM hexokinase 3450 U / l, 30 ° C. Glucose-6-phosphate dehydrogenase 1725 U / l, 30 ° C.

[0056]

According to the present invention, a novel hexokise having excellent stability in a creatine kinase measurement solution is isolated,
A method for producing the enzyme by a genetic engineering technique has been established, and it has become possible to supply the enzyme in a large amount and to quantify creatinine, which is stable in a creatine kinase measurement solution with high purity.

[0057]

[Sequence list] SEQ ID NO: 1 Sequence length: 486 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Organism name: Saccharomyc
es pastorianus) Strain name: ATCC2366 Sequence Met Val His Leu Gly Pro Lys Lys Pro Gln Ala Arg Lys Gly Ser Met 1 5 10 15 Ala Asp Val Pro Lys Glu Leu Met Gln Gln Ile Glu Asn Phe Glu Lys 20 25 30 Ile Phe Thr Val Pro Thr Glu Thr Leu Gln Ala Val Thr Arg His Phe 35 40 45 Ile Ser Glu Leu Glu Lys Gly Leu Ser Lys Lys Gly Gly Asn Ile Pro 50 55 60 Met Ile Pro Gly Trp Val Met Asp Phe Pro Thr Gly Lys Glu Ser Gly 65 70 75 80 Asp Phe Leu Ala Ile Asp Leu Gly Gly Thr Asn Leu Arg Val Val Leu 85 90 95 Val Lys Leu Gly Gly Gly Asp Arg Thr Phe Asp Thr Thr Gln Ser Lys Tyr 100 105 110 Arg Leu Pro Asp Ala Met Arg Thr Thr Gln Asn Pro Asp Glu Leu Trp 115 120 125 Glu Phe Ile Ala Asp Ser Leu Lys Ala Phe Ile Asp Glu Gln Phe Pro 130 135 140 Gln Gly Ile Ser Glu Pro Ile Pro Leu Gly Phe Thr Phe Ser Phe Pro 145 150 155 160 Ala Ser Gln Asn Lys Ile Asn Glu Gly Ile Leu Gln Arg Trp Thr Lys 165 170 175 Gly Phe Asp Ile Pro Asn Ile Glu Asn His Asp Val Val Pro Met Leu 180 185 190 Gln Lys Gln Ile Thr Lys Arg Asn Ile Pro Ile Glu Val Val A la Leu 195 200 205 Ile Asn Asp Thr Thr Gly Thr Leu Val Ala Ser Tyr Tyr Thr Asp Pro 210 215 220 Glu Thr Lys Met Gly Val Ile Phe Gly Thr Gly Val Asn Gly Ala Tyr 225 230 235 240 Tyr Asp Val Cys Ser Asp Ile Glu Lys Leu Gln Gly Lys Leu Ser Asp 245 250 255 Asp Ile Pro Pro Ser Ala Pro Met Ala Ile Asn Cys Glu Tyr Gly Ser 260 265 270 270 Phe Asp Asn Glu His Val Val Leu Pro Arg Thr Arg Tyr Asp Ile Thr 275 280 285 Ile Asp Glu Glu Ser Pro Arg Pro Gly Gln Gln Thr Phe Glu Lys Met 290 295 300 300 Ser Ser Gly Tyr Tyr Leu Gly Glu Ile Leu Arg Leu Ala Leu Met Asp 305 310 315 320 Met Tyr Lys Gln Gly Phe Ile Phe Lys Asn Gln Asp Leu Ser Lys Phe 325 330 335 Asp Lys Pro Phe Val Met Asp Thr Ser Tyr Pro Ala Arg Ile Glu Glu 340 345 350 Asp Pro Phe Glu Asn Leu Glu Asp Thr Asp Asp Leu Phe Gln Asn Glu 355 360 365 Phe Gly Ile Asn Thr Thr Val Gln Glu Arg Lys Leu Ile Arg Arg Leu 370 375 380 Ser Glu Leu Ile Gly Ala Arg Ala Ala Arg Leu Ser Val Cys Gly Ile 385 390 395 400 Ala Ala Val Cys Gln Lys Arg Gly Tyr Lys Thr Gly His Ile A la Ala 405 410 415 Asp Gly Ser Val Tyr Asn Arg Tyr Pro Gly Phe Lys Glu Lys Ala Ala 420 425 430 Asn Ala Leu Lys Asp Ile Tyr Gly Trp Thr Gln Thr Ser Leu Asp Asp 435 440 445 445 Tyr Pro Ile Lys Ile Val Pro Ala Glu Asp Gly Ser Gly Ala Gly Ala 450 455 460 Ala Val Ile Ala Ala Leu Ala Gln Lys Arg Ile Ala Glu Gly Lys Ser 465 470 475 480 Leu Gly Ile Ile Gly Ala 485

SEQ ID NO: 2 Sequence length: 1464 Sequence type: nucleic acid Topology: linear Sequence type: genomic DNA / synthetic DNA Origin Organism name: Saccharomyc
es pastorianus) Strain name: ATCC2366 Sequence ATG GTT CAT TTA GGT CCA AAG AAA CCA CAG GCC AGA AAG GGT TCC ATG 48 Met Val His Leu Gly Pro Lys Lys Pro Gln Ala Arg Lys Gly Ser Met 1 5 10 15 GCC GAT GTG CCA AAG GAA TTG ATG CAA CAA ATT GAG AAT TTT GAA AAA 96 Ala Asp Val Pro Lys Glu Leu Met Gln Gln Ile Glu Asn Phe Glu Lys 20 25 30 ATT TTC ACT GTT CCA ACT GAA ACT TTA CAA GCC GTT ACC AGG CAT TTC 144 Ile Phe Thr Val Pro Thr Glu Thr Leu Gln Ala Val Thr Arg His Phe 35 40 45 ATT TCC GAA TTG GAA AAG GGT TTG TCC AAG AAG GGT GGT AAC ATT CCA 192 Ile Ser Glu Leu Glu Lys Gly Leu Ser Lys Lys Gly Gly Asn Ile Pro 50 55 60 ATG ATT CCA GGT TGG GTT ATG GAT TTC CCA ACT GGT AAG GAA TCC GGT 240 Met Ile Pro Gly Trp Val Met Asp Phe Pro Thr Gly Lys Glu Ser Gly 65 70 75 80 GAT TTC TTG GCC ATT GAT TTG GGT GGT ACC AAC TTG AGA GTT GTC TTA 288 Asp Phe Leu Ala Ile Asp Leu Gly Gly Thr Asn Leu Arg Val Val Leu 85 90 95 GTC AAG TTG GGC GGT GAC CGT ACC TTT GAC ACC ACT CAA TCT AAG TAC 336 Val Lys Leu Gly Gly Asp Arg Thr Phe Asp Thr Thr Gln Ser Lys Tyr 100 105 110 AGA TTA CCA GAT GCT ATG AGA ACT ACT CAA AAT CCA GAC GAA TTG TGG 384 Arg Leu Pro Asp Ala Met Arg Thr Thr Gln Asn Pro Asp Glu Leu Trp 115 120 125 GAA TTT ATT GCC GAC TCT TTG AAA GCC TTT ATT GAT GAG CAA TTC CCA 432 Glu Phe Ile Ala Asp Ser Leu Lys Ala Phe Ile Asp Glu Gln Phe Pro 130 135 140 CAA GGT ATC TCT GAG CCA ATT CCA TTG GGT TTC ACC TTT TCT TTC CCA 480 Gln Gly Ile Ser Glu Pro Ile Pro Leu Gly Phe Thr Phe Ser Phe Pro 145 150 155 160 GCT TCT CAA AAC AAA ATC AAT GAA GGT ATC TTG CAA AGA TGG ACT AAA 528 Ala Ser Gln Asn Lys Ile Asn Glu Gly Ile Leu Gln Arg Trp Thr Lys 165 170 175 GGT TTT GAT ATT CCA AAC ATT GAA AAC CAC GAT GTT GTT CCA ATG TTG 576 Gly Phe Asp Ile Pro Asn Ile Glu Asn His Asp Val Val Pro Met Leu 180 185 190 CAA AAG CAA ATC ACC AAG AGG AAT ATC CCA ATT GAA GTT GTT GCT TTG 624 Gln Lys Gln Ile Thr Lys Arg Asn Ile Pro Ile Glu Val Val Ala Leu 195 200 205 ATA AAC GAC ACT ACC GGT ACT TTG GTT GCT TCT TAC TAC ACT GAC CCA 672 Ile Asn Asp Thr Thr Gly Thr Leu Val Ala Ser Tyr Tyr Thr Asp Pro 210 215 220 GAA ACT AAG ATG GGT GTT ATC TTC GGT ACT GGT GTC AAT GGT GCT TAC 720 Glu Thr Lys Met Gly Val Ile Phe Gly Thr Gly Val Asn Gly Ala Tyr 225 230 235 240 TAC GAT GTT TGT TCC GAT ATC GAA AAG CTA CAA GGA AAA CTA TCT GAT 768 Tyr Asp Val Cys Ser Asp Ile Glu Lys Leu Gln Gly Lys Leu Ser Asp 245 250 255 GAC ATT CCA CCA TCT GCT CCA ATG GCC ATC AAC TGT GAA TAC GGT TCC 816 Asp Ile Pro Pro Ser Ala Pro Met Ala Ile Asn Cys Glu Tyr Gly Ser 260 265 270 TTC GAT AAT GAA CAT GTC GTT TTG CCA AGA ACT AGA TAC GAT ATC ACC 864 Phe Asp Asn Glu His Val Val Leu Pro Arg Thr Arg Tyr Asp Ile Thr 275 280 285 ATT GAT GAA GAA TCT CCA AGA CCA GGC CAA CAA ACC TTT GAA AAA ATG 912 Ile Asp Glu Glu Ser Pro Arg Pro Gly Gln Gln Thr Phe Glu Lys Met 290 295 300 TCT TCT GGT TAC TAC TTA GGT GAA ATT TTG CGT TTG GCC TTG ATG GAC 960 Ser Ser Gly Tyr Tyr Leu Gly Glu Ile Leu Arg Leu Ala Leu Met Asp 305 310 315 320 ATG TAC AAA CAA GGT TTC ATC TTC AAG AAC CAA GAC TTG TCT AAG TTC 1008 Met Tyr Lys Gl n Gly Phe Ile Phe Lys Asn Gln Asp Leu Ser Lys Phe 325 330 335 GAC AAG CCT TTC GTC ATG GAC ACT TCT TAC CCA GCC AGA ATC GAG GAA 1056 Asp Lys Pro Phe Val Met Asp Thr Ser Tyr Pro Ala Arg Ile Glu Glu 340 345 350 GAT CCA TTC GAG AAC CTA GAA GAT ACC GAT GAC TTG TTC CAA AAT GAG 1104 Asp Pro Phe Glu Asn Leu Glu Asp Thr Asp Asp Leu Phe Gln Asn Glu 355 360 365 TTC GGT ATC AAC ACT ACT GTT CAA GAA CGT AAA TTG ATC AGA CGT TTA 1152 Phe Gly Ile Asn Thr Thr Val Gln Glu Arg Lys Leu Ile Arg Arg Leu 370 375 380 TCT GAA TTG ATT GGC GCT AGA GCT GCT AGA TTG TCC GTT TGT GGT ATT 1200 Ser Glu Leu Ile Gly Ala Arg Ala Ala Arg Leu Ser Val Cys Gly Ile 385 390 395 400 400 GCT GCT GTC TGT CAA AAG AGA GGT TAC AAG ACC GGT CAC ATC GCT GCA 1248 Ala Ala Val Cys Gln Lys Arg Gly Tyr Lys Thr Gly His Ile Ala Ala 405 410 415 GAC GGT TCC GTT TAC AAC AGA TAC CCA GGT TTC AAA GAA AAG GCT GCC 1296 Asp Gly Ser Val Tyr Asn Arg Tyr Pro Gly Phe Lys Glu Lys Ala Ala 420 425 430 AAT GCT TTG AAG GAC ATT TAC GGC TGG ACT CAA ACC TCA CTA GAC GAC 1344 Asn Ala Leu Lys Asp Ile Tyr Gly Trp Thr Gln Thr Ser Leu Asp Asp 435 440 445 TAC CCA ATC AAG ATT GTT CCT GCT GAA GAT GGT TCC GGT GCT GGT GCC 1392 Tyr Pro Ile Lys Ile Val Pro Ala Glu Asp Gly Ser Gly Ala Gly Ala 450 455 460 GCT GTT ATT GCT GCT TTG GCC CAA AAA AGA ATT GCT GAA GGT AAG TCT 1443 Ala Val Ile Ala Ala Leu Ala Gln Lys Arg Ile Ala Glu Gly Lys Ser 465 470 475 475 480

SEQ ID NO: 3 Sequence length: 38 Sequence type: nucleic acid Number of strands: single-stranded Sequence type: synthetic DNA Topology: linear sequence GGGGGCATAT GGTTCATTTA GGTCCAAAGA AACCACAG 38

SEQ ID NO: 4 Sequence length: 39 Sequence type: number of nucleic acid chains: single-stranded Sequence type: synthetic DNA Topology: linear sequence GGGAGGGATC CTCATTAAGC GCCAATGATA CCAAGAGAC 39

SEQ ID NO: 5 Sequence length: 38 Sequence type: nucleic acid Number of strands: single-stranded Sequence type: synthetic DNA Topology: linear sequence CAATTTCACA CAGGAAACCA TATGACCATG ATTACGCC 38

[Brief description of the drawings]

FIG. 1 is a diagram showing the optimal temperature of the hexokinase of the present invention.

FIG. 2 is a graph showing the optimal pH of the hexokinase of the present invention.

FIG. 3 shows the thermostability of the hexokinase of the present invention.

FIG. 4 shows the pH stability of the hexokinase of the present invention.

FIG. 5 is a diagram showing a restriction map of a plasmid, pHXK10.

FIG. 6 is a graph showing the residual activity of the hexokinase of the present invention and a commercially available hexokinase (glycokinase) in a creatinine kinase measurement reagent at pH 6.6, 40 ° C., for 0 to 14 days.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // (C12N 15/09 ZNA C12R 1:85) (C12N 1/21 C12R 1:19) (C12N 9/12 C12R 1:85) ) (C12N 9/12 C12R 1:19)

Claims (14)

[Claims] 1. A novel hexokinase having the following physicochemical properties. Action: Acts on D-hexose in the presence of ATP and magnesium to produce D-hexose-6-phosphate and ADP. Stability in creatine kinase assay solution: at least 6
0% (Store at 40 ° C for 1 week) 2. A novel hexokinase having the following physicochemical properties. Action: Acts on D-hexose in the presence of ATP and magnesium to produce D-hexose-6-phosphate and ADP. Stability in creatine kinase assay solution: at least 6
0% (stored at 40 ° C for 1 week) Optimum temperature: about 45 to 50 ° C Optimum pH: about 8.0 to 9.0 Molecular weight: 53,998 (value obtained from amino acid composition) 55,000 ( SDS-PAGE) 3. The novel hexokinase according to claim 2, which further has the following physicochemical properties. Thermal stability: about 40 ° C. or less (pH 7.0, 30 minutes) pH stability: about 5.5 to 7.5 (25 ° C., 20 hours) Km value for glucose: about 0.21 mM Km value for ATP: about 0.074 mM substrate specificity: D-glucose, D-fructose, D-
Acts on mannose, D-galactose, 2-deoxy-D-glucose. 4. Saccharomyces pasteurianus (Sacch)
aromyces pastorianus) The novel hexokinase according to claim 2, which is an enzyme produced by ATCC2366. 5. A novel hexokinase which is a protein of the following (a) or (b): (A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing; (b) an amino acid sequence (a) consisting of an amino acid sequence in which one or more amino acids have been added, deleted or substituted, and a hexokinase Active protein 6. A novel hexokinase having the amino acid sequence set forth in SEQ ID NO: 1. 7. A gene encoding hexokinase, which is a protein of the following (a) or (b): (A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing; (b) an amino acid sequence (a) consisting of an amino acid sequence in which one or more amino acids have been added, deleted or substituted, and a hexokinase Active protein 8. A gene encoding hexokinase having the amino acid sequence of SEQ ID NO: 1. 9. The following (c), (d) or (e) DN:
A gene encoding hexokinase consisting of A. (C) DNA consisting of the nucleotide sequence of SEQ ID NO: 2 in the Sequence Listing (d) In the nucleotide sequence of (c), one or more bases are added, deleted or substituted, and the hexokinase activity is Encoding an amino acid sequence having
A: a bacterial DN which hybridizes with a DNA consisting of the nucleotide sequence of (e) and (c) under stringent conditions and encodes a protein having hexokinase activity
A 10. A recombinant vector containing a gene encoding the hexokinase according to claim 7, 8, or 9. 11. A transformant obtained by transforming a host cell with the recombinant vector according to claim 10. 12. A method for producing hexokinase, comprising culturing the transformant according to claim 11, producing hexokinase, and collecting the hexokinase. 13. Saccharomyces pasteurianus (Sac
(charomyces pastorianus) ATCC2366,
A method for producing hexokinase, which comprises producing hexokinase and collecting the hexokinase. 14. A hexokinase, buffer, glucose, magnesium, ADP, NAD or NADP or NADP according to any one of claims 1 to 6 and glucose-.
A reagent for measuring creatine kinase comprising a 6-phosphate dehydrogenase solution.