JP2001037480A - Mutated glucose-6-phosphate dehydrogenase and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new glucose-6-phosphate dehydrogenase which was mutated by modifying a part of the amino acid sequence which constitutes a protein having a glucose-6-phosphate dehydrogenase activity, has improved stability in a liquid compared with the original protein, and is useful as a reagent for clinical diagnosis and the like. SOLUTION: This is a new glucose-6-phosphate dehydrogenase which was mutated by modifying by adding, deleting, inserting, or substituting one or more amino acid(s) of the amino acid sequence which is shown by the formula and has a glucose-6-phosphate dehydrogenase activity, has improved stability in a liquid compared with the original protein, has excellent stability during storage in a solution, and is useful as a reagent for clinical diagnosis and the like. This mutated enzyme is obtained by contacting a gene coding for a wild- type enzyme with a reagent which serves as a mutagen, incorporating the obtained gene into a vector to transform a host cell with the obtained vector, followed by culturing the obtained transformant.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to wild-type glucose-
The present invention relates to a mutant glucose-6-phosphate dehydrogenase having improved liquid stability as compared with 6-phosphate dehydrogenase and a method for producing the mutant glucose-6-phosphate dehydrogenase.

[0002]

2. Description of the Related Art Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) widely exists from animals and plants to microorganisms, and is used as an enzyme for clinical test drugs for measuring creatinine kinase, glucose, ATP and the like. .
For these uses, bacteria of the genus Leuconostoc (Japanese Patent Publication No. 5-87234, Japanese Patent Publication No. 5-503)
No. 221) and those collected from microorganisms such as yeast are mainly used. However, conventional glucose-6-phosphate dehydrogenase is insufficient in stability, and particularly, nicotinamide adenine diene which is a coenzyme. Nucleotide (NA
It is known that the liquid storage stability in the presence of D) or nicotinamide adenine dinucleotide phosphate (NADP) is poor, and improvement thereof is desired.

As a method for this purpose, for example, Japanese Patent Publication No. Sho 6
In JP-A-3-43079, JP-B-3-9995, JP-A-9-313174, etc., methods using thermostable glucose-6-phosphate dehydrogenase are reported. As a method for stabilizing glucose-6-phosphate dehydrogenase itself, JP-A-7-595 describes
No. 66 and the like also report a method in which hydroxylamines and aldehydes coexist, and Japanese Patent Application Laid-Open No. 9-65877 discloses a method of chemically modifying the compound with a divalent crosslinking reagent. However, at present, these methods do not provide sufficient stability.

[0004]

SUMMARY OF THE INVENTION A glucose-6-phosphate dehydrogenase used as a reagent for clinical examination is desired to have excellent stability when stored in a solution state. The present invention provides a mutant glucose-6-phosphate dehydrogenase having improved stability by introducing a mutation into amino acids constituting wild-type glucose-6-phosphate dehydrogenase, and production of the mutant glucose-6-phosphate dehydrogenase About the law.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies to obtain glucose-6-phosphate dehydrogenase having excellent stability in a liquid state suitable for use in clinical examinations. Pseudomesenteroides (Leuconostoc pseudomesenteroides) ATCC1
A mutant glucose-protein having improved stability in a liquid form by introducing a mutation into the amino acid sequence constituting wild-type glucose-6-phosphate dehydrogenase based on the gene encoding glucose-6-phosphate dehydrogenase of H.2291 We succeeded in constructing 6-phosphate dehydrogenase, and completed the present invention.

[0006] That is, the present invention relates to a method for adding, deleting, or adding at least one amino acid of an amino acid sequence constituting a protein having glucose-6-phosphate dehydrogenase activity.
A protein having glucose-6-phosphate dehydrogenase activity mutated by insertion or substitution, wherein the stability in liquid form is improved as compared to the protein before mutation. 6-
Phosphate dehydrogenase.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The mutant glucose-6 of the present invention
Phosphate dehydrogenase has glucose-6-phosphate dehydrogenase activity in which at least one amino acid of an amino acid sequence constituting a protein having glucose-6-phosphate dehydrogenase activity is mutated by addition, deletion, insertion or substitution. It is a protein, characterized in that the liquid stability is improved as compared to glucose-6-phosphate dehydrogenase before mutation. The wild-type glucose-6-phosphate dehydrogenase before the introduction of the mutation of the present invention is not particularly limited, and includes, for example, glucose-6-phosphate dehydrogenase derived from a bacterium belonging to the genus Leuconostoc.

[0008] The above mutation can be performed according to a known method. For example, as a method for altering the function of a protein by addition, Nature Biotechnology
Vol. 17, pages 58-61 (1999). In addition, for the method using the deletion, see Biochem. Biop.
hys.Res.Commun. Vol. 248, No. 2, 372-37
See page 7, Method of insertion, FEBS Lett. No. 442
Pp. 241-245 (1999), and the method by substitution is described in Nucleic Acids Research Vol.
No. pp. 681-683 (1998), respectively.

In the present invention, as an example, Leuconostoc pseudomecenteroides (Leuconostoc pseudom
esenteroides) Glucose-6-phosphate dehydrogenase from ATCC 12291 strain was used. The properties of wild-type glucose-6-phosphate dehydrogenase are as follows, and its amino acid sequence is shown in SEQ ID NO: 1 in the sequence listing. Action: D-glucose-6-phosphate and NAD or NAD
Acts on P to produce D-glucono-δ-lactone-6-phosphate and reduced NAD or reduced NADP Optimum temperature: about 50-55 ° C Optimum pH: 7.8 Thermal stability: about 40 ° C. or lower (pH 8.0, 30 minutes) pH stability: about 5.0 to 10.0 (30 ° C., 17 hours) Km value for glucose-6-phosphate (when NAD is used as a coenzyme): Km value for about 0.1 mM NAD: about 0.1 mM Substrate specificity: High specificity for D-glucose-6-phosphate Molecular weight: about 55,000 (SDS-PAGE)

[0010] The term liquid stability in the present invention refers to, for example, the ratio of the residual enzyme activity after the enzyme is dissolved in a neutral buffer and heat-treated. As one embodiment of the present invention, 2 m
Mutant glucose-6 whose activity retention after heat treatment at 50 ° C. for 30 minutes in a 100 mM imidazole acetate buffer (pH 6.7) containing M EDTA is improved as compared with wild-type glucose-6-phosphate dehydrogenase. -A phosphate dehydrogenase. In another embodiment, 2 mM E
Mutant glucose whose activity retention after storage in a 100 mM imidazole acetate buffer (pH 6.7) containing DTA and 2 mM NADP at 40 ° C. for one week is improved as compared with wild-type glucose-6-phosphate dehydrogenase. -6
-A phosphate dehydrogenase. In still another embodiment, a mutant type having an improved activity retention rate after heat treatment at 50 ° C. for 30 minutes in a 100 mM Tris-HCl buffer (pH 8.0) as compared with wild-type glucose-6-phosphate dehydrogenase. Glucose-6-phosphate dehydrogenase.

In another embodiment, the mutant glucose-6-phosphate having an improved residual activity after storage in a serum glucose measurement reagent solution at 40 ° C. for one week as compared with wild-type glucose-6-phosphate dehydrogenase. Phosphate dehydrogenase. Here, a serum glucose measurement solution is defined as a solution in which glucose in a sample is reacted with hexokinase in the presence of ATP and magnesium ions to generate ADP and glucose-6-phosphate, and then glucose-6-phosphate and NAD Alternatively, glucose-6-phosphate dehydrogenase is allowed to act on NADP, and a change in NAD or NADP to NADH or NADPH is measured by absorbance measurement. The solution is a solution for measuring glucose. At least a pH buffer, ATP, NAD or NADP, Magnesium ion, hexokinase and glucose-6-
A solution containing phosphate dehydrogenase as a component.
The solution is recommended by the Japanese Society of Clinical Chemistry (Clinical Chemistry Volume 20,
No. 4, p. 185, December 1991) defines the desirable composition. The type and pH of the pH buffer may be appropriately selected in consideration of the stability of the substrate and the reactivity of the enzyme. For example, Tris, trisethanolamine, phosphate buffer, Good buffer, etc. ~ 50
At a concentration of 0 mM, it can be used in a pH range of 5.0 to 9.0. The amount of the other components may be set in consideration of reactivity and economy. For example, ATP is 0.2 to 10 mM, NADP is 0.2 to 10 mM, magnesium ion is 1 to 20 mM, hexokinase is used. Is 0.
3-10 U / ml, glucose-6-phosphate dehydrogenase is used in the range of 0.3-10 U / ml.

As a specific embodiment of the present invention, phenylalanine at position 66, tyrosine at position 207, asparagine at position 240, aspartic acid at position 324, and aspartic acid at position 337 in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing.
A variant glucose-6-phosphate dehydrogenase in which one or more of the sites selected from the group consisting of serine 344, lysine 344, lysine 353, and lysine 410 are substituted with another amino acid It is.

As more specific examples, phenylalanine at position 66 to tyrosine, tyrosine at position 207 to phenylalanine, asparagine at position 240 to serine,
One or more of the substitutions of the fourth aspartic acid to glutamine, 337th serine to alanine, 344th lysine to glutamine, 353rd lysine to arginine, and 410th lysine to methionine. And a mutant glucose-6-phosphate dehydrogenase.

An example of the present invention is a mutant glucose-6-phosphate dehydrogenase having the following physicochemical properties. Action: D-glucose-6-phosphate and NAD or NAD
Acts on P to produce D-glucono-δ-lactone-6-phosphate and reduced NAD or reduced NADP Optimum temperature: about 50-55 ° C Optimum pH: 7.8 Thermal stability: about 45 ° C. or lower (pH 8.0, 30 minutes) pH stability: about 4.0 to 11.0 (30 ° C., 17 hours) Km value for glucose-6-phosphate (when NAD is used as a coenzyme): Km value for about 0.1 mM NAD: about 0.1 mM Substrate specificity: High specificity for D-glucose-6-phosphate Molecular weight: about 55,000 (SDS-PAGE)

Another example of the present invention is a mutant glucose-6-phosphate dehydrogenase having the following physicochemical properties. Action: D-glucose-6-phosphate and NAD or NAD
Acts on P to produce D-glucono-δ-lactone-6-phosphate and reduced NAD or reduced NADP Optimum temperature: about 50-55 ° C Optimum pH: 7.8 Thermal stability: about 50 ° C. or lower (pH 8.0, 30 minutes) pH stability: about 4.0 to 11.0 (30 ° C., 17 hours) Km value for glucose-6-phosphate (when NAD is used as a coenzyme): Km value for about 0.3 mM NAD: about 0.1 mM Substrate specificity: High specificity for D-glucose-6-phosphate Molecular weight: about 55,000 (SDS-PAGE)

In a specific embodiment of the present invention, phenylalanine at position 66, tyrosine at position 207, asparagine at position 240, aspartic acid at position 324, and aspartic acid at position 337 in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing.
A variant glucose-6-phosphate dehydrogenase in which one or more of the sites selected from the group consisting of serine 344, lysine 344, lysine 353, and lysine 410 are substituted with another amino acid Is a gene that encodes

As a more specific example, in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, phenylalanine at position 66 is to leucine, tyrosine at position 207 is to phenylalanine, asparagine at position 240 is to serine, and
One or two or more of the substitutions of the fourth aspartic acid to glutamine, the serine at 337 to alanine, the lysine at 344 to glutamine, the lysine at 353 to arginine, and the lysine at position 410 to methionine. And a gene encoding a mutant glucose-6-phosphate dehydrogenase containing

The present invention further relates to the above-mentioned mutant glucose-6.
-A recombinant vector containing a gene encoding phosphate dehydrogenase, and a transformant obtained by transforming a host cell with the recombinant vector, further culturing the transformant,
A method for producing the glucose-6-phosphate dehydrogenase, which comprises producing a mutant glucose-6-phosphate dehydrogenase and collecting the glucose-6-phosphate dehydrogenase. Vectors and host cells include those generally used in the art.

In order to prepare a mutant glucose-6-phosphate dehydrogenase, a method for introducing a mutation into a gene encoding wild-type glucose-6-phosphate dehydrogenase can be performed by a known method. That is,
There are a method of bringing the gene DNA into contact with a drug as a mutagen, a method of irradiating ultraviolet rays, and a method of using Escherichia coli in which a gene is frequently mutated due to lack of a gene repair mechanism. Examples of a method for introducing a site-specific mutation include a PCR method using a synthetic oligonucleotide and a method using a commercially available kit.

In the present invention, the gene encoding the wild-type glucose-6-phosphate dehydrogenase before the introduction of the mutation is not particularly limited, but for example, Leuconostoc pseudo as described in SEQ ID NO: 2 in the sequence listing. Mesenteroides (Leuconostoc pseudomesenteroides) AT
And a gene encoding glucose-6-phosphate dehydrogenase derived from CC12291 strain. As another example, a protein having glucose-6-phosphate dehydrogenase activity that hybridizes with the gene in stringent conditions at 65 ° C. for 16 hours in × 2 SSC (300 mM NaCl, 30 mM citric acid) is encoded. Gene.

The resulting mutant glucose-6-phosphate dehydrogenase gene is transferred to a replicable host microorganism in a state linked to a vector, and then the transformant is cultured. A vector containing the 6-phosphate dehydrogenase gene can be separated and purified, and a mutant glucose-6-phosphate dehydrogenase gene can be collected from the vector.

That is, a culture obtained by stirring and culturing a donor microorganism for, for example, 1 to 3 days is collected by centrifugation, and then lysed to obtain a mutant glucose-6.
-A lysate containing the phosphate dehydrogenase gene can be prepared. As a lysis method, for example, treatment with a lytic enzyme such as lysozyme or β-glucanase is performed, and a protease or another enzyme or a surfactant such as sodium lauryl sulfate (SDS) is used in combination as needed. Or a physical crushing method such as a French press treatment. DNA can be separated and purified from the lysate obtained in this manner by appropriately combining methods such as deproteinization by phenol treatment or protease treatment, ribonuclease treatment, and alcohol precipitation, for example. Can also.

As the vector, a vector constructed for gene recombination from a phage or a plasmid capable of autonomous propagation in a host microorganism is suitable. Examples of the phage include Escherichia coli (Escherichia coli).
When li) 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
Fermentation and Bioenzineering, Vol.76,265-269
(1993)).

The host microorganism may be any one as long as the recombinant vector can be stably and autonomously propagated and can express a foreign gene. 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 is possible to stably produce a large amount of mutant glucose-6-phosphate dehydrogenase. Selection of whether or not the target recombinant vector is transferred to the host microorganism is performed by searching for a microorganism that simultaneously expresses a drug resistance marker of the vector carrying the target DNA and a microorganism that simultaneously expresses glucose-6-phosphate dehydrogenase activity. For example, a microorganism that grows on a selective medium based on a drug resistance marker and that produces glucose-6-phosphate dehydrogenase may be selected.

The nucleotide sequence of the mutant glucose-6-phosphate dehydrogenase gene obtained by the above method is decoded by the dideoxy method described in Science (Science, Vol. 214, 1205-1210 (1981)). The amino acid sequence of the mutant glucose-6-phosphate dehydrogenase was estimated from the determined nucleotide sequence. The recombinant vector having the mutant glucose-6-phosphate dehydrogenase gene once selected in this manner can be easily removed from a transformed microorganism and transferred to another microorganism. Further, a DNA which is a mutant glucose-6-phosphate dehydrogenase gene is recovered from a recombinant vector having the mutant glucose-6-phosphate dehydrogenase gene by restriction enzyme or PCR, and ligated to another vector fragment to obtain a host. Transfer to microorganisms can also be easily performed.

The culture form of the host microorganism, which is a transformant, may be determined by taking into consideration the nutritional and physiological properties of the host. Usually, liquid culture is used in many cases. It is advantageous to perform 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, pyruvic acid and the like are used. Any available nitrogen compound may 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 phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, and specific vitamins are used as needed. The culture temperature is determined by the growth of bacteria and glucose-
Although it can be appropriately changed within the range of producing 6-phosphate dehydrogenase, in the case of Escherichia coli, it is preferably 20
~ 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 glucose-6-phosphate dehydrogenase reaches the maximum yield, and is usually about 6 to 48 hours. Medium pH
Can be appropriately changed within a range in which the bacteria grow and produce glucose-6-phosphate dehydrogenase, and the pH is particularly preferably about 6.0 to 9.0.

The culture solution containing the cells producing the mutant glucose-6-phosphate dehydrogenase in the culture can be directly collected and used, but generally, the glucose-6-phosphate dehydrogenase is cultured according to a conventional method. When present in the solution, it is used after separating the glucose-6-phosphate dehydrogenase-containing solution from the microbial cells by filtration, centrifugation or the like. When glucose-6-phosphate dehydrogenase is present in the cells, the cells are collected from the resulting culture by means such as filtration or centrifugation, and then the cells are subjected to a mechanical method or an enzyme such as lysozyme. Destroyed in a mechanical manner and, if necessary, EDTA
The glucose-6-phosphate dehydrogenase is solubilized by adding a chelating agent and / or a surfactant such as described above, and separated and collected as an aqueous solution.

[0029] The thus obtained mutant glucose-
The 6-phosphate dehydrogenase-containing solution may be precipitated by, for example, concentration under reduced pressure, membrane concentration, salting-out treatment with ammonium sulfate, sodium sulfate, or the like, or fractional precipitation using, for example, methanol, ethanol, acetone, or the like. Heat treatment and isoelectric point treatment are also effective purification means. Thereafter, by performing gel filtration with an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography, and affinity chromatography, purified glucose-6-phosphate dehydrogenase can be obtained.

For example, gel filtration using Sephadex G-25 (Amersham-Pharmacia Biotech), DEAE Sepharose CL-6B (Amersham-Pharmacia Biotech) column chromatography, phenyl Sepharose CL-6B (Amersham-Pharmacia Biotech) column chromatography, etc. Separation and purification can give a purified enzyme preparation. The purified enzyme preparation has been purified to such an extent that it shows almost a single band on SDS-PAGE.

[0031]

The present invention will be described below in more detail with reference to examples. In addition, at the substitution site of the amino acid sequence in the examples,
The types of amino acids after substitution are merely examples, and the present invention is not limited thereto, and effective amino acids can be appropriately selected and used.

In the examples, the measurement of glucose-6-phosphate dehydrogenase activity was carried out using the following reagents and measuring conditions. <Reagent> Reagent A: 55 mM Tris-HCl buffer (pH 7.
8; contains 3.3 mM MgCl ₂ ) Reagent B: 60 mM NAD ⁺ aqueous solution Reagent C: 100 mM glucose-6-phosphate aqueous solution Enzyme diluent: 55 mM Tris-HCl buffer (pH
7.8; including 0.1% BSA) <Measurement conditions> First, 2.7 ml of reagent A, 0.1 m of reagent B
1) Reagent C is mixed with 0.1 ml to prepare a reaction mixture.
After preheating at 30 ° C. for about 5 minutes, 0.1 ml of the enzyme solution was added to the reaction mixture, the reaction was started at 30 ° C., and after 5 minutes of reaction, the change in absorbance per minute at 340 nm was measured with a spectrophotometer. Measure. In the blind test, an enzyme diluent is added to the reagent mixture instead of the enzyme solution, and the absorbance change is measured in the same manner. 1 micromolar NADH per minute under the above conditions
Is defined as 1 unit (U).

REFERENCE EXAMPLE Cloning and Expression of Glucose-6-Phosphate Dehydrogenase Gene Leukonostock pseudomesenteroides ATCC12291 strain was
After culturing at 26 ° C. of ctobacilli MRS Broth (manufactured by Difco), the cells were collected by centrifugation, and chromosomal DNA was prepared from the cells according to a conventional method. Next, the chromosomal DNA was partially digested with a restriction enzyme Sau3A1 (manufactured by Toyobo Co., Ltd.), and a fragment of 3 to 7 Kb was recovered by agarose gel electrophoresis.
On the other hand, pBluescript KS (+) (manufactured by Toyobo Co., Ltd.)
The fragment was cut with amH1 (manufactured by Toyobo), further treated with alkaline phosphatase (manufactured by Toyobo), and ligated with the above DNA fragment using T4 DNA ligase (manufactured by Toyobo).

The ligated DNA was used to transform Escherichia coli JM109, and LB agar medium (1
% Polypeptone, 0.5% yeast extract, 1% NaCl,
1.5% agar, 100 μg / ml ampicillin (pH
7.2)), and cultured at 37 ° C. for 18 hours. The LB liquid medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 100 μg /
ml of ampicillin (pH 7.2)) at 37 ° C. for 16 hours with shaking, and screening was performed using glucose-6-phosphate dehydrogenase activity as an index.
A transformant having a glucose-6-phosphate dehydrogenase activity of 1.54 U / ml per ml was obtained. Using the plasmid DNA of this transformant as a template, the nucleotide sequence of the known glucose-6-phosphate dehydrogenase gene of Leuconostoc mesenteroides (Table 5-5)
No. 03221), a DNA fragment was amplified by a PCR method using a gene encoding a glucose-6-phosphate dehydrogenase protein and two kinds of primers that amplify about 100 bp upstream of the gene. PCR was performed under the following reaction solution composition and conditions.

<Composition of reaction solution> KOD Dash DNA polymerase (manufactured by Toyobo)
5 U / 100 μl 10-fold concentration KOD Dash DNA polymerase buffer 10 μl / 100 μl Plasmid DNA 50 ng dATP, dTTP, dGTP, dCTP 0.2 each
mM primers 100 pM each <Amplification conditions> (1) 94 ° C., 30 seconds (2) 60 ° C., 2 seconds (3) 74 ° C., 1 minute (1 cycle from (1) to (3)
(0 cycles)

The amplified DNA fragment was digested with the restriction enzyme EcoR.
PBluescript KS (+) digested with V and ligated with T4 DNA ligase, and used to construct Escherichia coli JM10.
9 and transformed on a LB agar medium for selection (1% polypeptone, 0.5% yeast extract, 1% NaCl, 1.5% agar, 100 μg / ml ampicillin (pH 7.2)), and Incubated for 18 hours. The grown colonies were cultured with shaking in LB liquid medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 100 μg / ml ampicillin (pH 7.2)) at 37 ° C. for 16 hours.
As a result of screening a strain expressing glucose-6-phosphate dehydrogenase activity, 1 strain per 1 ml of the culture solution was determined.
A transformant showing 50 U / ml of glucose-6-phosphate dehydrogenase activity was obtained. The plasmid DNA extracted from this transformant was named pG6D66. p
The nucleotide sequence of the glucose-6-phosphate dehydrogenase gene of G6D66 was determined using a DNA sequencer (ABI PRISM ™).
310 Genetic Analyzer; manufactured by PERKIN-ELMER)
Was determined. The nucleotide sequence is represented by SEQ ID NO: 1
And the amino acid sequence of glucose-6-phosphate dehydrogenase deduced from the nucleotide sequence is shown in SEQ ID NO: 2 in the sequence listing.

Example 1 Preparation of mutant glucose-6-phosphate dehydrogenase A recombinant plasmid pG6D66 containing a wild-type glucose-6-phosphate dehydrogenase gene, a synthetic oligonucleotide represented by SEQ ID NO: 3 in the Sequence Listing, and a complementary oligonucleotide QuickChangeTM Site-Dir based on synthetic oligonucleotides
Using the expected Mutagenesis Kit (manufactured by STRATAGENE), mutagenesis was performed according to the protocol, and the nucleotide sequence was determined in the same manner as in the Reference Example.
A recombinant plasmid (pG6D66M1) encoding a mutant glucose-6-phosphate dehydrogenase in which phenylalanine at position 66 in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing was substituted with leucine was obtained.

PG6D66 and SEQ ID NO: 4 in the sequence listing
A mutant glucose-6 in which the tyrosine at position 207 in the amino acid sequence of SEQ ID NO: 2 in the sequence listing is substituted with phenylalanine in the same manner as described above, on the basis of the synthetic oligonucleotide described and the synthetic oligonucleotide complementary thereto. -A recombinant plasmid (pG6D66M2) encoding phosphate dehydrogenase was obtained.

Based on pG6D66 and the synthetic oligonucleotide represented by SEQ ID NO: 5 in the Sequence Listing and the synthetic oligonucleotide complementary thereto, asparagine at position 324 of the amino acid sequence represented by SEQ ID NO: 2 in the same manner as described above. A recombinant plasmid (pG6D66M3) encoding a mutant glucose-6-phosphate dehydrogenase in which the acid was substituted with glycine was obtained.

Based on pG6D66 and the synthetic oligonucleotide represented by SEQ ID NO: 6 in the Sequence Listing and the synthetic oligonucleotide complementary thereto, the serine at position 337 of the amino acid sequence represented by SEQ ID NO: 2 was produced in the same manner as described above. A recombinant glucose (pG6D) encoding a mutant glucose-6-phosphate dehydrogenase in which
66M4).

Further, based on pG6D66M2 and the synthetic oligonucleotide represented by SEQ ID NO: 7 in the sequence listing and the synthetic oligonucleotide complementary thereto, the 207th amino acid sequence represented by SEQ ID NO: 2 in the sequence listing was produced in the same manner as described above. In which the tyrosine is substituted with phenylalanine and the asparagine at position 240 is substituted with serine.
A recombinant plasmid (pG6D66M5) encoding phosphate dehydrogenase was obtained.

Based on pG6D66M2 and the synthetic oligonucleotide described in SEQ ID NO: 8 in the Sequence Listing and the synthetic oligonucleotide complementary thereto, the tyrosine at position 207 of the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing was obtained in the same manner as described above. Is a recombinant plasmid encoding a mutant glucose-6-phosphate dehydrogenase in which lysine at position 353 is replaced by arginine (pG6
D66M6).

Further, based on pG6D66M5 and each of the synthetic oligonucleotides described in SEQ ID NOs: 3, 5 and 8 in the sequence listing and the synthetic oligonucleotides complementary thereto, in the same manner as described above, the sequence described in SEQ ID NO: 2 in the sequence listing was obtained. A variant in which the phenylalanine at position 66 is substituted with leucine, the tyrosine at position 207 is substituted with phenylalanine, the asparagine at position 240 is substituted with serine, the aspartic acid at position 324 is substituted with glycine, and the lysine at position 353 is substituted with arginine. Recombinant plasmid encoding glucose-6-phosphate dehydrogenase (pG6D66M7)
I got

Further, based on pG6D66M2 and a synthetic oligonucleotide described in SEQ ID NO: 9 in the sequence listing and a synthetic oligonucleotide complementary thereto, the 207th amino acid sequence in SEQ ID NO: 2 in the sequence listing was produced in the same manner as described above. A recombinant plasmid (pG6D66M8) encoding a mutant glucose-6-phosphate dehydrogenase in which tyrosine was replaced by phenylalanine and lysine at position 344 was replaced by glutamine was obtained.

PG6D66M2 and SEQ ID NO: 10 in the sequence listing
Based on the synthetic oligonucleotide described and the synthetic oligonucleotide complementary thereto, the tyrosine at position 207 in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing is replaced with phenylalanine and the lysine at position 410 is replaced with methionine in the same manner as described above. Recombinant plasmids (pG) encoding each substituted mutant glucose-6-phosphate dehydrogenase
6D66M9).

PG6D66, pG6D66M1, pG6
D66M2, pG6D66M3, pG6D66M4, p
G6D66M5, pG6D66M6, pG6D66M
7. Escherichia coli competent cells (Escherichia coli JM109; manufactured by Toyobo Co., Ltd.) were transformed with the recombinant plasmids pG6D66M8 and pG6D66M9, and the transformants were obtained.

Example 2 Production of Mutant Glucose-6-phosphate Dehydrogenase 100 ml of LB medium was dispensed into a 500 ml Sakaguchi flask, autoclaved at 121 ° C. for 20 minutes, allowed to cool, and then 100 μl of separately sterile-filtered ampicillin was filtered. / Ml. Escherichia coli JM109 (pG6D66M1) which had been cultured for 16 hours at 30 ° C. in LB containing 100 μl / ml ampicillin in this medium.
Was inoculated with 1 ml of the medium, and cultured with aeration and stirring at 30 ° C. for 18 hours. The glucose-6-phosphate dehydrogenase activity at the end of the culture was about 1040 U / ml of the culture solution.
ml.

The above cells were collected by centrifugation, and 2 mM
20 mM phosphate buffer (pH 7.5) containing EDTA
The suspension was disrupted by sonication and centrifuged to obtain a crude enzyme solution. After the obtained crude enzyme solution was fractionated with ammonium sulfate, it was desalted with Sephadex G-25 (Amersham-Pharmacia), Q-Sepharpse (Amersham-Pharmacia) ion exchange column chromatography, and Superdex 200 (Pharmacia Biotech) gel filtration column. Separation and purification were performed by chromatography to obtain a purified enzyme preparation. The sample obtained by this method showed a substantially single band on SDS-PAGE, and the specific activity at this time was about 730 U / mg protein. This mutant was named G6D66M1.

PG6D66, pG6D66M2, pG6
D66M3, pG6D66M4, pG6D66M5, p
G6D66M6, pG6D66M7, pG6D66M
8. Each Escherichia coli J by pG6D66M9
For the M109 transformant, a purified enzyme preparation was obtained in the same manner as described above. G6D66, G6D66M2, G6D66M3, G6D
66M4, G6D66M5, G6D66M6, G6D6
Named 6M7, G6D66M8, G6D66M9.

Example 3 Comparison of thermostability between mutant glucose-6-phosphate dehydrogenase and wild-type glucose-6-phosphate dehydrogenase (1) Various mutant glucose-6-phosphate dehydrogenases obtained in Example 2 (G6D66M1, G6D66M2, G6
D66M3, G6D66M4) and wild-type glucose-
FIG. 1 shows the results of comparing the change over time in the residual activity ratio when 6-phosphate dehydrogenase (G6D66) was heat-treated at 50 ° C. in 100 mM imidazole acetate buffer (pH 6.7) containing 2 mM EDTA. . FIG.
As is clear from the above, the mutant glucose-6-
Phosphate dehydrogenase is shown to have improved stability compared to wild type.

Example 4 Comparison of thermostability between mutant glucose-6-phosphate dehydrogenase and wild-type glucose-6-phosphate dehydrogenase (2) Various mutant glucose-6-phosphate dehydrogenases obtained in Example 2 (G6D66M2, G6D66M5, G6
D66M6, G6D66M7) and wild-type glucose-
FIG. 2 shows the results of comparing the change over time in the residual activity ratio when 6-phosphate dehydrogenase (G6D66) was heat-treated at 52 ° C. in 100 mM imidazole acetate buffer (pH 6.7) containing 2 mM EDTA. . FIG.
As is clear from the above, the mutant glucose-6-
It is shown that the stability of phosphate dehydrogenase is additively improved as compared with the wild type by appropriately combining mutations.

Example 5 Comparison of storage stability between mutant glucose-6-phosphate dehydrogenase and wild-type glucose-6-phosphate dehydrogenase (1) Mutant glucose-6-phosphate dehydrogenase obtained in Example 2 G6D66M2, G6D66M8) and wild-type glucose-6-phosphate dehydrogenase (pG6D
66) is 2 mM EDTA and 2 mM NADP, respectively.
100 mM imidazole acetate buffer (pH 6.
FIG. 3 shows the results of comparing the time-dependent changes in the residual activity ratio when stored at 40 ° C. in 7). As is clear from FIG. 3, it is shown that the mutant glucose-6-phosphate dehydrogenase of the present invention has improved liquid storage stability in the presence of the coenzyme NADP as compared with the wild type.

Example 6 Comparison of storage stability between mutant glucose-6-phosphate dehydrogenase and wild-type glucose-6-phosphate dehydrogenase (2) Mutant glucose-6-phosphate dehydrogenase obtained in Example 2 G6D66M2, G6D66M9) and wild-type glucose-6-phosphate dehydrogenase (pG6D
66) in each of the following glucose measuring reagents at 37 ° C.
FIG. 4 shows the results of comparing the time-dependent changes in the residual activity ratio when the cells were stored in the above-mentioned manner. As is clear from FIG. 4, it is shown that the mutant glucose-6-phosphate dehydrogenase of the present invention has improved liquid storage stability in a glucose measurement reagent as compared with the wild type.

Tris-HCl buffer 100 mM Magnesium acetate 5 mM ATP 1.2 mM NADP 1.2 mM Hexokinase 1000 U / l (30 ° C.) Glucose-6-phosphate dehydrogenase 1000
U / l (30 ° C) pH 7.5 (25 ° C)

[0055]

As described above, according to the present invention, a protein having glucose-6-phosphate dehydrogenase activity is mutated and improved to obtain a mutant glucose-6-phosphate dehydrogenase having excellent stability in liquid form. it can. Further, by using genetic engineering technology, the mutant glucose-6-phosphate dehydrogenase can be supplied in high purity and in large quantities.

[0056]

[Sequence list] SEQ ID NO: 1 Sequence length: 486 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Organism: Leuconostoc pseudomesenteroides Strain name: ATCC12291 Sequence Met Val Ser Glu Ile Lys Thr Leu Val Thr Phe Phe Gly Gly Thr Gly 1 5 10 15 Asp Leu Ala Lys Arg Lys Leu Tyr Pro Ser Val Phe Asn Leu Tyr Lys 20 25 30 Lys Gly Tyr Leu Gln Lys His Phe Ala Ile Val Gly Thr Ala Arg Gln 35 40 45 Ala Leu Asn Asp Asp Glu Phe Lys Gln Leu Val Arg Asp Ser Ile Lys 50 55 60 Asp Phe Thr Asp Asp Gln Ala Gln Ala Glu Ala Phe Ile Glu His Phe 65 70 75 80 Ser Tyr Arg Ala His Asp Val Thr Asp Ala Ala Ser Tyr Ala Val Leu 85 90 95 Lys Glu Ala Ile Glu Glu Ala Ala Asp Lys Phe Asp Ile Asp Gly Asn 100 105 110 Arg Ile Phe Tyr Met Ser Val Ala Pro Arg Phe Phe Gly Thr Ile Ala 115 120 125 Lys Tyr Leu Lys Ser Glu Gly Leu Leu Ala Asp Thr Gly Tyr Asn Arg 130 135 140 Leu Met Ile Glu Lys Pro Phe Gly Thr Ser Tyr Asp Thr Ala Ala Glu 145 150 155 160 Leu Gln Asn Asp Leu Glu Asn Ala Phe Asp Asp Asn Gln Leu Phe Arg 165 170 175 Ile Asp His Tyr Leu Gly Lys Glu Met Val Gln Asn Ile Ala Ala Leu 180 185 190 Arg Phe Gly Asn Pro Ile Phe Asp Ala Ala Trp Asn Lys Asp Tyr Ile 195 200 205 Lys Asn Val Gln Val Thr Leu Ser Glu Val Leu Gly Val Glu Glu Arg 210 215 220 Ala Gly Tyr Tyr Asp Thr Ala Gly Ala Leu Leu Asp Met Ile Gln Asn 225 230 235 240 His Thr Met Gln Ile Val Gly Trp Leu Ala Met Glu Lys Pro Glu Ser 245 250 255 Phe Thr Asp Lys Asp Ile Arg Ala Ala Lys Asn Ala Ala Phe Asn Ala 260 265 270 270 Leu Lys Ile Tyr Asp Glu Ala Glu Val Asn Lys Tyr Phe Val Arg Ala 275 280 285 Gln Tyr Gly Ala Gly Asp Ser Ala Asp Phe Lys Pro Tyr Leu Glu Glu 290 295 300 Leu Asp Val Pro Ala Asp Ser Lys Asn Asn Thr Phe Ile Ala Gly Glu 305 310 315 320 Leu Gln Phe Asp Leu Pro Arg Trp Glu Gly Val Pro Phe Tyr Val Arg 325 330 335 Ser Gly Lys Arg Leu Ala Ala Lys Gln Thr Arg Val Asp Ile Val Phe 340 345 350 Lys Ala Gly Thr Phe Asn Phe Gly Ser GluGln Glu Ala Gln Glu Ala 355 360 365 Val Leu Ser Ile Ile Ile Asp Pro Lys Gly Ala Ile Glu Leu Lys Leu 370 375 380 Asn Ala Lys Ser Val Glu Asp Ala Phe Asn Thr Arg Thr Ile Asp Leu 385 390 395 400 Gly Trp Thr Val Ser Asp Glu Asp Lys Lys Asn Thr Pro Glu Pro Tyr 405 410 415 Glu Arg Met Ile His Asp Thr Met Asn Gly Asp Gly Ser Asn Phe Ala 420 425 430 Asp Trp Asn Gly Val Ser Ile Ala Trp Lys Phe Val Asp Ala Ile Ser 435 440 445 Ala Val Tyr Thr Ala Asp Lys Ala Pro Leu Glu Thr Tyr Lys Ser Gly 450 455 460 Ser Met Gly Pro Glu Ala Ser Asp Lys Leu Leu Ala Ala Asn Gly Asp 465 470 470 475 480 Ala Trp Val Phe Lys Gly 485

SEQ ID NO: 2 Sequence length: 1461 Sequence type: Nucleic acid Topology: Linear Sequence type: Genomic DNA / Synthetic DNA Origin Organism: Leuconostoc pseudomesenteroites (Leuconostoc pseudomesenteroides) : ATCC12291 origin sequence ATG GTT TCA GAA ATC AAG ACG TTA GTA ACT TTC TTT GGT GGC ACT GGT 48 Met Val Ser Glu Ile Lys Thr Leu Val Thr Phe Phe Gly Gly Thr Gly 1 5 10 15 GAC TTG GCC AAG CGT AAG CTT TAC CCA TCA GTT TTC AAT CTT TAT AAA 96 Asp Leu Ala Lys Arg Lys Leu Tyr Pro Ser Val Phe Asn Leu Tyr Lys 20 25 30 AAA GGC TAC TTG CAA AAG CAT TTT GCC ATT GTT GGA ACG GCC CGT CAA 144 Lys Gly Tyr Leu Gln Lys His Phe Ala Ile Val Gly Thr Ala Arg Gln 35 40 45 GCC CTC AAT GAT GAC GAA TTC AAA CAA TTG GTT CGT GAT TCA ATT AAA 192 Ala Leu Asn Asp Asp Glu Phe Lys Gln Leu Val Arg Asp Ser Ile Lys 50 55 60 GAT TTC ACT GAC GAT CAA GCA CAA GCT GAG GCG TTC ATC GAA CAT TTC 240 Asp Phe Thr Asp Asp Gln Ala Gln Ala Glu Ala Phe Ile Glu His Phe 65 70 75 80 TCA TAC CGT GCA CAC GAC GTA ACA GAT GCT GCT TCA TAC GCT GTT TTA 288 Ser Tyr Arg Ala His Asp Val Thr Asp Ala Ala Ser Tyr Ala Val Leu 85 90 95 AAA GAG GCG ATT GAA GAA GCT GCC GAC AAA TTT GAT ATC GAT GGC AAC 336 Lys Glu Ala Ile Glu Glu Ala Ala Asp Lys Phe Asp Ile Asp Gly Asn 100 105 110 CGC ATT TTC TAT ATG TCA GTT GCG CCA CGT TTC TTT GGT ACA ATT GCC 384 Arg Ile Phe Tyr Met Ser Val Ala Pro Arg Phe Phe Gly Thr Ile Ala 115 120 125 AAA TAT CTT AAG TCA GAA GGC CTA CTA GCT GAC ACT GGT TAC AAC CGT 432 Lys Tyr Leu Lys Ser Glu Gly Leu Leu Ala Asp Thr Gly Tyr Asn Arg 130 135 140 TTG ATG ATT GAA AAG CCT TTC GGT ACA TCA TAT GAC ACA GCT GCC GAA 480 Leu Met Ile Glu Lys Pro Phe Gly Thr Ser Tyr Asp Thr Ala Ala Glu 145 150 155 160 CTC CAA AAT GAC TTG GAA AAC GCA TTT GAT GAT AAC CAA CTA TTC CGT 528 Leu Gln Asn Asp Leu Glu Asn Ala Phe Asp Asp Asn Gln Leu Phe Arg 165 170 175 ATT GAC CAC TAC CTT GGT AAG GAA ATG GTT CAA AAC ATT GCT GCC CTT 576 Ile Asp His Tyr Leu Gly Lys Glu Met Val Gln Asn Ile Ala Ala Leu 180 185 190 CGC TTT GGT AAC CCA ATT TTC GAT GCT GCT TGG AAC AAG GAT TAC ATC 624 Arg Phe Gly Asn Pro Ile Phe Asp Ala Ala Tla Asp Lys Asp Tyr Ile 195 200 205 AAG AAC GTT CAA GTA ACA TTG TCA GAA GTC TTG GGT GTC GAA GAA CGT 672 Lys Asn Val Gln Val Thr Leu Ser Glu Val Leu Gly Val Glu Glu Arg 210 215 220 GCC GGC TAC TAT GAC ACA GCC GGT GCA TTG CTT GAC ATG ATT CAA AAC 720 Ala Gly Tyr Tyr Asp Thr Ala Gly Ala Leu Leu Asp Met Ile Gln Asn 225 230 235 240 CAC ACC ATG CAA ATT GTT GGT TGG TTA GCC ATG GAA AAA CCA GAA TCA 768 His Thr Met Gln Ile Val Gly Trp Leu Ala Met Glu Lys Pro Glu Ser 245 250 255 TTC ACT GAC AAA GAC ATT CGT GCC GCT AAA AAC GCA GCC TTT AAT GCT 816 Phe Thr Asp Lys Asp Ile Arg Ala Ala Lys Asn Ala Ala Phe Asn Ala 260 265 270 270 TTG AAG ATC TAT GAT GAA GCA GAA GTT AAC AAA TAC TTT GTT CGT GCA 864 Leu Lys Ile Tyr Asp Glu Ala Glu Val Asn Lys Tyr Phe Val Arg Ala 275 280 285 CAA TAT GGT GCC GGT GAT TCA GCT GAC TTC AAG CCA TAC CTT GAA GAA 912 Gln Tyr Gly Ala Gly Asp Ser Ala Asp Phe Lys Pro Tyr Leu Glu Glu 290 295 300 TTA GAC GTA CCT GCT GAT TCT AAA AAC AAT ACC TTC ATC GCC GGC GAA 960 Leu Asp Val Pro Ala Asp Ser Lys Asn Asn Thr Phe Ile Ala Gly Glu 305 310 315 320 TTG CAA TTT GAT TTG CCA CGT TGG GAG GGT GTC CCA TTC TAT GTC CGT 1008 Leu Gln Phe Asp Leu Pro Arg Trp Glu Gly Val Pro Phe Tyr Val Arg 325 330 335 TCA GGT AAG CGC TTA GCT GCT AAA CAG ACA CGG GTT GAT ATC GTC TTT 1056 Ser Gly Lys Arg Leu Ala Ala Lys Gln Thr Arg Val Asp Ile Val Phe 340 345 350 AAG GCT GGC ACG TTT AAC TTT GGT TCA GAA CAA GAA GCA CAA GAA GCT 1104 Lys Ala Gly Thr Phe Asn Phe Gly Ser Glu Gln Glu Ala Gln Glu Ala 355 360 365 GTC TTG TCA ATT ATC ATT GAT CCA AAG GGT GCT ATC GAA TTG AAG TTG 1152 Val Leu Ser Ile Ile Ile Asp Pro Lys Gly Ala Ile Glu Leu Lys Leu 370 375 380 380 AAC GCC AAG TCA GTT GAA GAT GCT TTC AAC ACA CGT ACA ATT GAC TTA 1200 Asn Ala Lys Ser Val Glu Asp Ala Phe Asn Thr Arg Thr Ile Asp Leu 385 390 395 400 400 GGT TGG ACT GTA TCT GAC GAA GAT AAG AAG AAC ACG CCA GAA CCA TAC 1248 Gly Trp Thr Val S er Asp Glu Asp Lys Lys Asn Thr Pro Glu Pro Tyr 405 410 415 GAA CGT ATG ATT CAC GAC ACT ATG AAT GGT GAT GGC TCT AAC TTC GCT 1296 Glu Arg Met Ile His Asp Thr Met Asn Gly Asp Gly Ser Asn Phe Ala 420 425 430 GAC TGG AAT GGC GTT TCA ATC GCT TGG AAG TTC GTT GAT GCT ATT TCA 1344 Asp Trp Asn Gly Val Ser Ile Ala Trp Lys Phe Val Asp Ala Ile Ser 435 440 445 445 GCC GTT TAT ACC GCA GAT AAA GCA CCA CTT GAA ACT TAC AAG TCG GGC 1392 Ala Val Tyr Thr Ala Asp Lys Ala Pro Leu Glu Thr Tyr Lys Ser Gly 450 455 460 TCA ATG GGT CCT GAA GCA TCC GAT AAA TTA TTG GCT GCC AAT GGT GAT 1440 Ser Met Gly Pro Glu Ala Ser Asp Lys Leu Leu Ala Ala Asn Gly Asp 465 470 475 480 GCT TGG GTG TTT AAA GGT TAA 1461 Ala Trp Val Phe Lys Gly 485

SEQ ID NO: 3 Sequence length: 37 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence CGTGATTCAA TTAAAGATTT GACTGACGAT CAAGCAC 37

SEQ ID NO: 4 Sequence length: 41 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GCTGCTTGGA ACAAGGATTT CATCAAGAAC GTTCAAGTAA C41

SEQ ID NO: 5 Sequence length: 38 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GCCGGCGAAT TGCAATTTGG TTTGCCACGT TGGGAGGG 38

SEQ ID NO: 6 Sequence length: 38 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GTCCCATTCT ATGTCCGTGC AGGTAAGCGC TTAGCTGC 38

SEQ ID NO: 7 Sequence length: 39 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GCTTGACATG ATTCAAAACA CCACCATGCA AATTGTTGG 39

SEQ ID NO: 8 Sequence length: 36 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence CGGGTTGATA TCGTCTTTAG GGCTGGCACG TTTAAC 36

SEQ ID NO: 9 Sequence length: 39 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GGTAAGCGCT TAGCTGCTCA ACAGACACGG GTTGATATC 39

SEQ ID NO: 10 Sequence length: 39 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GTATCTGACG AAGATAAGAT GAACACGCCA GAACCATAC 39

[Brief description of the drawings]

FIG. 1 shows the thermostability of the mutant glucose-6-phosphate dehydrogenase of the present invention (one mutation).

FIG. 2 shows the thermostability of the mutant glucose-6-phosphate dehydrogenase of the present invention (combination of multiple mutations).

FIG. 3 is a graph showing the storage stability of the mutant glucose-6-phosphate dehydrogenase of the present invention in the presence of NADP.

FIG. 4 is a diagram showing the storage stability of a mutant glucose-6-phosphate dehydrogenase of the present invention in a glucose measurement reagent.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 9/04 C12N 5/00 A // (C12N 15/09 ZNA C12R 1:01) (C12N 1/21 (C12R 1:19) (72) Inventor Shizuo Hattori 10-24 Toyo-cho, Tsuruga-shi, Fukui Toyo Spinning Co., Ltd. Inside Tsuruga Bio-Laboratory (72) Inventor Yoshihisa Kawamura 10-24 Toyo-cho, Tsuruga-shi, Fukui Toyo Bosei F-term in Tsuruga Bio-Laboratory, Inc. (reference) 4B024 AA03 AA11 BA08 CA01 CA03 CA20 DA06 EA04 GA11 GA19 GA25 HA03 4B050 CC04 DD02 EE01 FF03E FF04E FF11E FF12E FF13E LL03 LL05 4B065 AA01Y AA26B ABC BC01 BC01 BD01 BD14 BD15 CA28 CA46

Claims (18)

[Claims] 1. A glucose-6-phosphate dehydrogenase activity mutated by adding, deleting, inserting or substituting at least one amino acid of an amino acid sequence constituting a protein having a glucose-6-phosphate dehydrogenase activity. A mutant glucose-6-phosphate dehydrogenase, which has improved stability in liquid form as compared to the protein before mutation. 2. The mutant glucose-6-phosphate dehydrogenase according to claim 1, wherein at least one amino acid of the amino acid sequence constituting the protein having glucose-6-phosphate dehydrogenase activity is substituted with another amino acid. . 3. The mutant glucose-6-phosphate dehydrogenase according to claim 2, wherein at least one amino acid of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing is substituted with another amino acid. 4. An amino acid sequence of a protein which is encoded by a DNA which hybridizes with a DNA consisting of the sequence shown in SEQ ID NO: 2 under stringent conditions and has glucose-6-phosphate dehydrogenase activity. 3. The mutant glucose-6 according to claim 2, wherein one amino acid is substituted with another amino acid.
-Phosphate dehydrogenase. 5. The amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, phenylalanine at position 66, tyrosine at position 207, asparagine at position 240, aspartic acid at position 324, serine at position 337, lysine at position 344. The mutated glucose-6-phosphate according to claim 3 or 4, wherein one or a combination of two or more selected from the group consisting of lysine at position 353 and lysine at position 410 is substituted with another amino acid. Acid dehydrogenase. 6. The mutant glucose-6 according to claim 3, wherein the phenylalanine at position 66 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is a substitution site.
-Phosphate dehydrogenase. 7. The mutant glucose-6-phosphate dehydrogenase according to any one of claims 3 to 5, wherein the tyrosine at position 207 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing is a substitution site. 8. The mutated glucose-6- according to any one of claims 3 to 5, wherein asparagine at position 240 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is a substitution site.
Phosphate dehydrogenase. 9. The mutant glucose-6 according to any one of claims 3 to 5, wherein the aspartic acid at position 324 of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is a substitution site.
-Phosphate dehydrogenase. 10. The mutant glucose-6-phosphate dehydrogenase according to any one of claims 3 to 5, wherein serine at position 337 of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing is a substitution site. 11. The mutant glucose-6-phosphate dehydrogenase according to any one of claims 3 to 5, wherein the lysine at position 344 of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing is a substitution site. 12. The mutant glucose-6-phosphate dehydrogenase according to any one of claims 3 to 5, wherein lysine at position 353 of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is a substitution site. 13. The mutant glucose-6-phosphate dehydrogenase according to any one of claims 3 to 5, wherein the lysine at position 410 in the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing is a substitution site. 14. The method according to claim 1, which has the following physicochemical properties.
14. The mutant glucose-6-phosphate dehydrogenase according to any one of items 13 to 13. Action: D-glucose-6-phosphate and NAD or NAD
Acts on P to produce D-glucono-δ-lactone-6-phosphate and reduced NAD or reduced NADP Optimum temperature: about 50-55 ° C Optimum pH: 7.5-8.0 heat Stability: about 45 to 50 ° C or less (pH 8.0, 30 minutes) pH stability: about 4.0 to 11.0 (30 ° C, 17 hours) Km value for glucose-6-phosphate (glucose-
Km value for 6-phosphate (when NAD is used as a coenzyme): about 0.1 to 0.3 mM Km value for NAD: about 0.1 to 0.3 mM Substrate specificity: D-glucose-6-phosphate High specificity for acid Molecular weight: about 55,000 (SDS-PAGE) A glucose-6-phosphate dehydrogenase gene encoding the mutant glucose-6-phosphate dehydrogenase according to any one of claims 1 to 14. 16. The mutant glucose- according to claim 15,
A recombinant vector comprising a gene encoding 6-phosphate dehydrogenase. A transformant obtained by transforming a host cell with the recombinant vector according to claim 16. 18. A mutant, comprising culturing the transformant according to claim 17, producing mutant glucose-6-phosphate dehydrogenase, and collecting the mutant glucose-6-phosphate dehydrogenase. Glucose-
A method for producing 6-phosphate dehydrogenase.