JPH10248574A - Novel lactate oxidase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel enzyme having lactate oxidase activity which can be used for quantification of lactic acid, a gene encoding the enzyme, a method for producing the enzyme by genetic engineering technology, and Provision of its use. SOLUTION: Thermal stability of about 50 ° C. or less (pH 7.5, 1
0 min), pH stability about 4.0-9.0 (25 ° C,
16 hours), optimum temperature about 35 ° C, optimum pH about 6.5
Lactic acid oxidase derived from Lactococcus bacterium and a gene encoding the enzyme, a recombinant vector containing the gene, a transformant transformed with the vector, and culturing the transformant And a method for producing lactic acid oxidase and collecting the enzyme, and a reagent for measuring lactic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel enzyme having lactate oxidase activity which can be used for quantifying lactic acid, a gene encoding the enzyme, a method for producing the enzyme by genetic engineering techniques, and a method for producing the enzyme. For use.

[0002]

2. Description of the Related Art Conventionally, lactate oxidase, which catalyzes a reaction of producing pyruvic acid and hydrogen peroxide by acting on lactic acid in the presence of oxygen, has been known as an enzyme for measuring lactic acid.
For example, it has been used with peroxidase or catalase. The lactic acid level in the body fluid is an index for various pathological conditions such as circulatory failure and liver damage. Also, the lactic acid value in the food serves as an index for quality control and the like. The enzyme of the present invention alone is used in an enzyme sensor and used for measurement of lactic acid.

[0003] Such a lactate oxidase is obtained from Aerococcus viridans (Biochemical anid).
d Biophysical Research Communications, Vol.164, 91
9-926 (1989)), Streptococcus faecalis (Str.
eptococcus faecalis) (U.S. Pat. No. 4,116,763) or a bacterium of the genus Pediococcus (Analytical Chemi).
stry, Vol. 55, 35-38 (1983)). Among them, the properties of lactate oxidase produced by Aerococcus viridans IFO12219 have been investigated (JP-B-59-101).
No. 90), Aerococcus viridans (Aerococcus)
viridans) The gene encoding lactate oxidase produced by IFO12219 has already been isolated and its amino acid sequence has been published (JP-A-2-177886).

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to isolate a novel lactate oxidase from a new bacterium which has not been produced, clone a gene encoding the enzyme, and carry out genetic engineering. Another object of the present invention is to provide a method for producing the enzyme in large quantities. Another object of the present invention is to
An object of the present invention is to derive a method for producing a novel lactate oxidase having improved physicochemical properties by modifying the enzyme.

[0005]

Means for Solving the Problems As a result of various studies to achieve the above object, the present inventors have found that Lactococcus lactis (Lactococcus lactis) is a lactic acid oxidase-producing bacterium.
tis (subsp. cremoris)) IFO3427 was selected, a novel lactate oxidase was isolated from the cells, and the gene encoding lactate oxidase was successfully isolated from chromosomal DNA extracted from the cells. A novel lactate oxidase expressed from the gene was isolated. Furthermore, the entire nucleotide sequence of the gene was determined, and the enzyme was successfully produced by genetic engineering techniques without the need to add an expensive inducer to the medium. Made it possible.

That is, the present invention is a novel lactic acid oxidase derived from the genus Lactococcus, having the following physicochemical properties. Action: Acts on lactic acid in the presence of oxygen to produce pyruvate and hydrogen peroxide. Thermal stability: about 50 ° C or less (pH 7.5, treated for 10 minutes) pH stability: about 4.0 to 9.0 (treated at 25 ° C, 16 hours)

[0007] One embodiment of the present invention is a novel lactate oxidase having the following physicochemical properties. Action: Acts on lactic acid in the presence of oxygen to produce pyruvate and hydrogen peroxide. Thermal stability: about 50 ° C. or less (pH 7.5, 10 minutes treatment) pH stability: about 4.0 to 9.0 (25 ° C., 16 hours treatment) Optimum temperature: about 35 ° C. Optimum pH: about 6 Molecular weight: about 40,000 (SDS-PAGE) About 40,000 (calculated from amino acid composition) About 160,000 (gel filtration) Isoelectric point: about 4.3 ± 0.1

Further, the present invention provides the following (a) or (b)
Lactate oxidase, which is a protein of (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) comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added, and Protein with oxidase activity

The present invention relates to a gene encoding lactate oxidase, 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) comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added, and Protein with oxidase activity

[0010] The present invention also relates to a gene encoding a lactate oxidase comprising the following DNA (c), (d) or (e). (C) DNA consisting of the nucleotide sequence of SEQ ID NO: 2 in the sequence listing (d) One or more bases are added, deleted or substituted in the above-mentioned nucleotide sequence (c), and lactic acid oxidation D encoding an amino acid sequence having enzymatic activity
NA (e) Bacterial D which hybridizes with the DNA comprising the nucleotide sequence of (c) under stringent conditions and encodes a protein having lactate oxidase activity
NA

The present invention is a recombinant vector containing a gene encoding the above lactate oxidase.

The present invention also relates to a transformant transformed by the above-mentioned recombinant vector.

Further, the present invention is a method for producing lactate oxidase, which comprises culturing the above transformant in a medium, producing lactate oxidase, and collecting the lactate oxidase.

The present invention also relates to Lactococcus lactis (subsp. Cremoris) IFO3
427. A method for producing lactate oxidase, comprising culturing 427, producing lactate oxidase, and collecting the lactate oxidase.

The present invention is a reagent for measuring lactic acid, comprising the above-mentioned lactate oxidase, peroxidase or catalase, and a reagent for detecting hydrogen peroxide.

[0016]

DETAILED DESCRIPTION OF THE INVENTION The lactate oxidase of the present invention is a lactate oxidase-producing microorganism such as Lactococcus lactis (L.
actococcus lactis (subsp.cremoris)) IFO3427
Available from However, by isolating the gene encoding lactate oxidase and isolating the lactate oxidase expressed from the gene, the lactate oxidase of the present invention can be used simply,
And it can be obtained efficiently.

The lactate oxidase of the present invention has a pH of 7.5,
It has a thermal stability of about 50 ° C. or less when treated for 0 minutes and
It is characterized by being stable at H4.0 to 9.0.

In another embodiment of the present invention, (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing or (b) an amino acid sequence represented by (a)
Alternatively, there is a protein having an amino acid sequence in which a plurality of amino acids are deleted, substituted or added, and having lactate oxidase activity. Regarding the degree of amino acid deletion, substitution, addition, without changing the basic properties,
Alternatively, it includes those whose characteristics are improved. The method for producing these mutants follows a conventionally known method.

The gene encoding the lactate oxidase of the present invention is, for example, Lactococcus lactis.
actis (subsp. cremoris) may be extracted from IFO3427 or chemically synthesized. By using the polymerase chain reaction (PCR), it is also possible to obtain a DNA fragment containing a lactate oxidase gene.

The above-mentioned gene may be, for example, (a) DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, or (b) one or more amino acids in the amino acid sequence (a) are deleted. DNA comprising a lost, substituted or added amino acid sequence and encoding a protein having lactate oxidase activity
There is. Regarding the degree of DNA deletion, substitution and addition,
Includes those that do not change the basic characteristics or improve the characteristics. The method for producing these mutants follows a conventionally known method.

Further, (c) a DNA consisting of the base sequence shown in SEQ ID NO: 2 in the sequence listing, and (d) one or more bases added, deleted or substituted in the above base sequence (c). And hybridizes under stringent conditions to DNA encoding an amino acid sequence having lactate oxidase activity or (e) a DNA comprising the nucleotide sequence of (c) above, and to exhibit lactate oxidase activity. Bacterial DNA that encodes a protein having the protein. Here, the stringent condition is × 2SSC
(300 mM NaCl, 30 mM citric acid), 65
° C for 16 hours.

As a method for obtaining the gene of the present invention, for example, Lactococcus lactis (s
ubsp. cremoris)) After isolating and purifying the chromosomal DNA of IFO3427, cutting the DNA using sonication, restriction enzyme treatment and the like, and a linear expression vector, the DNA was blunt-ended or cohesive at both ends. The recombinant vector is constructed by binding and closing with ligase or the like. After the thus obtained recombinant vector is transferred to a replicable host microorganism, screening is performed using the expression of the vector marker and the enzyme activity as indices, and the microorganism carrying the recombinant vector containing the gene encoding lactate oxidase is used. Get. Next, the microorganism is cultured, the recombinant vector is separated and purified from the cultured cells, and the lactate oxidase gene is collected from the recombinant vector.

The gene donor Lactococcus lactis (subsp. Cremoris) IF
The chromosomal DNA of O3427 is specifically collected as follows. That is, the culture obtained by stirring and culturing the donor microorganism for 1 to 3 days is collected by centrifugation,
Then, by lysing the lysate, a lysate containing the lactate oxidase gene can be prepared. 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
Can be separated and purified by a conventional method, for example, by appropriately combining methods such as protein removal treatment with phenol treatment or protease treatment, ribonuclease treatment, and alcohol precipitation treatment. The method of cutting DNA isolated and purified from microorganisms can be performed by, for example, ultrasonic treatment, restriction enzyme treatment, or the like. Preferably, a type II restriction enzyme that acts on a specific nucleotide sequence is suitable.

As the vector, a vector constructed for gene recombination from a phage or a plasmid capable of autonomous growth 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 Fermentati
on and Bioenzineering, Vol. 76, 265-269 (1993)).

Such a vector can be cleaved with the restriction enzyme used for cleaving the microbial DNA which is the lactate oxidase gene donor described above to obtain a vector fragment. It is not necessary to use the same restriction enzymes as the restriction enzymes. The method for binding the microbial DNA fragment to the vector DNA fragment may be a method using a known DNA ligase. For example, after annealing the cohesive end of the microbial DNA fragment and the cohesive end of the vector fragment, an appropriate DNA ligase is used. A recombinant vector of a microorganism DNA fragment and a vector DNA fragment is prepared by use. If necessary, after annealing, it can be transferred to a host microorganism and a recombinant vector can be prepared using in vivo DNA ligase.

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

As a method for transferring a recombinant vector to a 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.

The microorganism, which is a transformant thus obtained, can stably produce a large amount of lactate oxidase by being cultured in a nutrient medium. 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 lactate oxidase activity. A microorganism that grows in a selective medium based on the lactic acid oxidase and that produces lactate oxidase may be selected.

The nucleotide sequence of the lactate oxidase gene obtained by the above-described method is described in Science (Vol. 214, 1205).
-1210 (1981)), and the amino acid sequence of lactate oxidase was estimated from the determined base sequence. The recombinant vector having the lactate oxidase gene once selected in this manner can be easily removed from a transformed microorganism and transferred to another microorganism. In addition, DNA that is a lactate oxidase gene can be recovered from a recombinant vector having the lactate oxidase gene by restriction enzyme or PCR, coupled to 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 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 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 lactate oxidase. In the case of Escherichia coli, it 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 timing when the lactate oxidase 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 lactate oxidase, and the pH is particularly preferably about 6.0 to 9.0.

A culture solution containing cells producing lactate oxidase in the culture can be directly collected and used.
In general, when lactate oxidase is present in a culture solution according to a conventional method, it is used after separating a lactate oxidase-containing solution from microbial cells by filtration, centrifugation, or the like. If lactate oxidase is present in the cells, the cells are collected from the resulting culture by filtration or centrifugation, and then destroyed by a mechanical method or an enzymatic method such as lysozyme. If necessary, a chelating agent such as EDTA and / or a surfactant are added to solubilize the lactate oxidase, and the solution is separated and collected as an aqueous solution.

The lactate oxidase-containing solution thus obtained is concentrated under reduced pressure, membrane concentration, salting-out treatment with ammonium sulfate, sodium sulfate or the like, or fractionation with a hydrophilic organic solvent such as methanol, ethanol, acetone or the like. What is necessary is just to precipitate by a precipitation method. Heat treatment and isoelectric point treatment are also effective purification means. After that, purified lactate oxidase can be obtained by performing gel filtration with an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography, and affinity chromatography.

For example, Sephadex G-25
(Pharmacia Biotech), etc., gel filtration, DE
AE Sepharose CL-6B (Pharmacia Biotech), Octyl Sepharose CL6-B (Pharmacia Biotech)
Separation and purification can be performed by column chromatography to obtain a purified enzyme preparation. This purified enzyme preparation has been purified to the extent that it shows almost a single band by electrophoresis (SDS-PAGE).

One example of the lactate oxidase of the present invention has the following physicochemical properties. Action: Acts on lactic acid in the presence of oxygen to produce pyruvate and hydrogen peroxide. Thermal stability: about 50 ° C or less (pH 7.5, 10 minutes treatment)
(See FIG. 1) pH stability: about 4.0 to 9.0 (treated at 25 ° C. for 16 hours) (see FIG. 2) Optimum temperature: about 35 ° C. (see FIG. 3) Optimum pH: about 6.5 ７7.5 (see FIG. 4) Molecular weight: about 40,000 (SDS-PAGE) about 40,000 (calculated from amino acid composition) about 160,000 (gel filtration) Isoelectric point: about 4.3 ± 0.1

Aerococcus viridans I whose lactate oxidase and properties of the present invention are known
Table 1 shows a comparison of properties with lactic acid oxidase produced by FO12219. The homology between the amino acid sequence of the lactate oxidase of the present invention and the only amino acid sequence of the lactate oxidase produced by the well-known Aerococcus viridans IFO12219 is 51.8%,
The comparison between the two is shown in FIG. The lactate oxidase of the present invention is a novel enzyme having different properties from known enzymes that catalyze the same reaction.

[0037]

[Table 1]

The reagent for measuring lactic acid of the present invention contains the above-mentioned lactate oxidase, peroxidase or catalase and a reagent for detecting hydrogen peroxide. Conventionally known peroxidases or catalase can be used. The hydrogen peroxide detection reagent is a reagent for detecting hydrogen peroxide generated by lactate oxidase with peroxidase or catalase, and includes, for example, 4-aminoantipyrine and a phenol derivative or an aniline derivative. The present invention is not limited to these hydrogen peroxide detection reagents.

[0039]

The present invention will be described below in more detail with reference to examples. In the examples, lactate oxidase activity was measured under the following reagents and measurement conditions. <Reagent> 50 mM DL-lactic acid, 50 mM 3-dimethylglutaric acid, 1.5 mM 4-aminoantipyrine,
Aqueous solution containing 3.1 mM N-dimethylaniline, 2.5 U / ml peroxidase, 0.2 μM flavin adenine dinucleotide (pH 6.5)

<Measurement Conditions> After preliminarily heating 1 ml of the reagent at 37 ° C. for about 5 minutes, adding 0.02 ml of the enzyme solution, starting the reaction at 37 ° C., and reacting for 10 minutes, followed by 0.25% dodecyl The reaction is stopped by adding 2 ml of an aqueous solution of sodium sulfate, and the change in absorbance at 565 nm is measured with a spectrophotometer. In the blind test, distilled water is added to the reagent instead of the enzyme solution, and the change in absorbance is measured in the same manner. 1 under the above conditions
The amount of enzyme that decomposes 1 micromol of lactic acid per minute is defined as 1 unit (U).

Example 1 Isolation of Chromosomal DNA Lactococcus lactis (sub
sp. cremoris)) The chromosomal DNA of IFO3427 was isolated by the following method. The strain was cultured in a Brain Heart Infusion medium (manufactured by Difco) with shaking at 37 ° C. overnight, and then collected by centrifugation (8,000 rpm, 10 minutes). 1
After washing the cells with a solution containing 5 mM sodium citrate and 0.15 M sodium chloride, 20% sucrose, 50 mM Tris-HCl (pH 7.6), 1 mM ED
Suspend in 5 ml of a solution containing TA, add 1 ml of lysozyme solution (100 mg / ml), and incubate at 37 ° C. for 30 minutes.
Then 11 ml of 1% lauroyl sarcosine acid, 0.1
A solution containing M EDTA (pH 9.6) was added. To this suspension was added about 100% of ethidium bromide solution and about 100% of cesium chloride, and the mixture was stirred and mixed, and the DNA was collected by ultracentrifugation at 55,000 rpm for 20 hours. The fractionated DNA is 1m
10 mM Tris-HCl containing M EDTA, pH 8.0
The solution was dialyzed against a solution (hereinafter abbreviated as TE) to obtain a purified DNA sample. This was treated with an equal volume of a chloroform / phenol solution, and the aqueous layer was separated by centrifugation, and twice the amount of ethanol was added.
In TE.

Example 2 Preparation of a DNA Fragment Containing a Gene Encoding Lactate Oxidase and a Recombinant Vector Having the DNA Fragment 5 μg of the DNA obtained in Example 1 was partially digested with a restriction enzyme Sau3AI (manufactured by Toyobo) to obtain 2 kbp. After digestion into the above fragments, pBluescri digested with restriction enzyme BamHI (Toyobo)
1 μg of pt was reacted with 1 unit of T4-DNA ligase (manufactured by Toyobo) at 16 ° C. for 12 hours to ligate DNA.
The ligated DNA was transformed using competent cells (manufactured by Toyobo) of Escherichia coli JM109, and an agar medium for detecting lactate oxidase activity [1.0% polypeptone, 0.5% yeast extract, 0.5% NaCl, 0.1% lactic acid, 5U / ml peroxidase (manufactured by Toyobo), 0.01% o-dianisidine, 50 µg / ml ampicillin, 1.5% agar]. Lactate oxidase activity was detected using, as an indicator, colonies that grew in the above medium and were stained brown.

Approximately 10,000 transformant colonies were obtained per μg of the DNA used. As a result of the above screening, one colony stained brown was found.
This strain was cultured in an LB liquid medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl (pH 7.2), 50 μg / ml ampicillin), and the lactate oxidase activity was measured. The plasmid carried by this strain contains about 3 kbp
Of the inserted DNA fragment,
LCO3. FIG. 6 shows a restriction map of pLCO3.

Example 3 Determination of Nucleotide Sequence A subclone was prepared using various restriction enzymes for an inserted DNA fragment of about 3 kbp of pLCO3. Various subclones can be prepared by using a sequencing kit (Radioa
ctive Sequencing Kit) (manufactured by Toyobo) to determine the nucleotide sequence. 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 is about 40,000, and Lactococcus lactis (subsp. Cremoris) IFO3
The molecular weight was almost identical to the molecular weight of 427 lactate oxidase.

Example 4 Preparation of Transformant Competent cells of Escherichia coli JM109 (manufactured by Toyobo) were transformed with pLCO3 to obtain a transformant Escherichia coli JM109 (pLCO3).

Example 5 Escherichia coli JM109 (p
Production of lactate oxidase from LCO3) 500 ml of LB medium was dispensed into a 2 L flask, and
After autoclaving at 15 ° C for 15 minutes, the mixture was allowed to cool, and 0.5 ml of 50 mg / ml ampicillin (manufactured by Nacalai Tesque), which had been sterile-filtered separately, was added. This culture medium was inoculated with 5 ml of a culture solution of Escherichia coli JM109 (pLCO3) which had been cultured with shaking at 30 ° C. for 7 hours in an LB medium, followed by aeration and agitation culture at 37 ° C. for 18 hours. The lactic acid activity at the end of the culture was about 10 U / ml.

The above cells were collected by centrifugation, and 20 mM
Suspended in phosphate buffer, pH 7.0. The cell suspension was crushed by a French press and centrifuged to obtain a supernatant. After performing the nucleic acid removal treatment and the ammonium sulfate fractionation treatment on the resulting crude enzyme solution with polyethyleneimine, Sephadex
(Sephadex) Desalting by gel filtration with G-25 (Pharmacia Biotech), DEAE Sepharose CL-6B (Pharmacia Biotech) column chromatography, Octyl Sepharose CL6-B (Pharmacia Biotech)
Separation and purification were performed by column chromatography to obtain a purified enzyme preparation. The lactate oxidase preparation obtained by this method shows almost a single band by electrophoresis (SDS-PAGE),
The specific activity at this time was about 150 U / mg-protein.

The properties of lactate oxidase obtained by the above method are shown below. Action: Acts on lactic acid in the presence of oxygen to produce pyruvate and hydrogen peroxide. Thermal stability: about 50 ° C. or less (pH 7.5, 10 minutes treatment) pH stability: about 4.0 to 9.0 (25 ° C., 16 hours treatment) Optimum temperature: about 35 ° C. Optimum pH: about 6 Molecular weight: about 40,000 (SDS-PAGE) About 40,000 (calculated from amino acid composition) About 160,000 (gel filtration) Isoelectric point: about 4.3 ± 0.1

Example 7 Lactococcus lactis (sub)
sp. cremoris)) IFO3427 was cultured in brain heart infusion medium at 37 ° C for about 1 to 3 days,
The culture was collected by centrifugation to collect bacteria, and then lysed to produce lactate oxidase, and the lactate oxidase was collected.

[0050]

Industrial Applicability According to the present invention, a novel lactate oxidase gene is isolated from a bacterium belonging to the genus Lactococcus, and a method for producing the enzyme by genetic engineering techniques has been established. It can be used for quantification. Further, a method for producing a novel lactate oxidase having improved physicochemical properties can be derived by modifying the gene.

[0051]

[Sequence list]

SEQ ID NO: 1 Sequence length: 366 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Organism name: Lactococcus lactis
tis (subsp. cremoris)) Strain name: IFO 3427 sequence Glu Lys Thr Tyr Gln Ala Gly Thr Asn Glu Gly Ile Val Asp Phe Ile 1 5 10 15 Asn Met Glu Asp Leu Glu Ile Ala Ala Ser Gln Val Ile Pro Ala Gly 20 25 30 Gly Tyr Gly Tyr Ile Ser Ser Gly Ala Gly Asp Leu Phe Thr Tyr Gln 35 40 45 Glu Asn Glu Arg Ala Phe Asn His Gln Leu Ile Ile Pro His Val Leu 50 55 60 Arg Asp Val Glu Leu Pro Asp Thr Thr Thr His Phe Asp Glu Glu Thr 65 70 75 80 Leu Thr Ala Pro Ile Ile Met Ala Pro Val Ala Ala His Gly Leu Ala 85 90 95 His Val Lys Ala Glu Lys Ala Ser Ala Lys Gly Val Ala Asp Phe Gly 100 105 110 Thr Ile Tyr Thr Ala Ser Ser Tyr Ala Ser Cys Thr Leu Glu Glu Ile 115 120 125 Arg Glu Ala Gly Gly Glu Lys Ala Pro Gln Trp Phe Gln Phe Tyr Met 130 135 140 Ser Lys Asp Asn Glu Ile Asn Leu Asp Ile Leu Glu Val Ala Lys Arg 145 150 155 160 Asn Gly Ala Lys Ala Ile Val Leu Thr Ala Asp Ala Thr Val Gly Gly 165 170 175 Asn Arg Glu Thr Asp Arg Arg Asn Gly Phe Thr Phe Pro Leu Pro Met 180 185 190 Pro Ile Val Gln Ala Tyr Gln Ser Gly Val Gly Gln Thr Met Asp Ala 195 200 205 Val Tyr Lys Ser Ser Lys Gln Lys Leu Ser Pro Lys Asp Val Glu Phe 210 215 220 Ile Ala Ala His Ser Asp Leu Pro Val Tyr Val Lys Gly Val Gln Ser 225 230 235 240 Glu Glu Asp Val Tyr Arg Ser Leu Glu Ser Gly Ala Gly Gly Ile Trp 245 250 255 Val Ser Asn His Gly Gly Arg Gln Leu Asp Gly Gly Pro Ala Ala Phe 260 265 270 Asp Ser Leu Gln Tyr Val Ala Asp Ala Val Asp Lys Arg Val Pro Ile 275 280 285 Val Phe Asp Ser Gly Val Arg Arg Gly Gln His Val Phe Lys Ala Ile 290 295 300 Ala Ser Gly Ala Asp Leu Val Ala Ile Gly Arg Pro Val Ile Tyr Gly 305 310 315 320 Leu Ser Leu Gly Gly Ser Thr Gly Val Arg Gln Val Phe Asp Phe Phe 325 330 335 Lys Thr Glu Leu Glu Met Val Met Gln Leu Ala Gly Thr Gln Thr Val 340 345 350 Glu Asp Ile Lys Lys Ile Lys Leu Arg Glu Asn Arg Phe Ile 355 360 365 366

SEQ ID NO: 2 Sequence length: 1098 Sequence type: nucleic acid Topology: linear Sequence type: genomic DNA Origin Organism name: Lactococcus lac
tis (subsp. cremoris)) Strain name: IFO 3427 sequence GAA AAA ACG TAT CAA GCA GGT ACA AAT GAA GGA ATA GTG GAT TTT ATC 48 Glu Lys Thr Tyr Gln Ala Gly Thr Asn Glu Gly Ile Val Asp Phe Ile 1 5 10 15 AAT ATG GAA GAT TTA GAA ATA GCC GCT TCA CAA GTG ATT CCA GCT GGT 96 Asn Met Glu Asp Leu Glu Ile Ala Ala Ser Gln Val Ile Pro Ala Gly 20 25 30 GGT TAT GGT TAC ATC AGC AGT GGT GCT GGC GAC TTA TTT ACT TAT CAA 144 Gly Tyr Gly Tyr Ile Ser Ser Gly Ala Gly Asp Leu Phe Thr Tyr Gln 35 40 45 GAA AAT GAA CGG GCA TTC AAT CAT CAA CTG ATT ATT CCT CAT GTA TTA 192 Glu Asn Glu Arg Ala Phe Asn His Gln Leu Ile Ile Pro His Val Leu 50 55 60 AGA GAT GTA GAA TTA CCC GAT ACA ACT ACA CAT TTT GAC GAA GAA ACG 240 Arg Asp Val Glu Leu Pro Asp Thr Thr Thr His Phe Asp Glu Glu Thr 65 70 75 80 CTG ACT GCA CCA ATC ATC ATG GCA CCA GTT GCT GCT CAT GGC TTA GCT 288 Leu Thr Ala Pro Ile Ile Met Ala Pro Val Ala Ala His Gly Leu Ala 85 90 95 CAT GTG AAG GCG GAG AAA GCT TCT GCA AAA GGT GTA GCA GAT TTT GGG 336 His Val Lys Ala Glu Lys Ala Ser Ala L ys Gly Val Ala Asp Phe Gly 100 105 110 ACG ATC TAT ACA GCA AGT TCT TAT GCT TCT TGT ACT TTA GAA GAG ATA 384 Thr Ile Tyr Thr Ala Ser Ser Tyr Ala Ser Cys Thr Leu Glu Glu Ile 115 120 125 AGA GAA GCA GGA GGG GAG AAA GCA CCG CAA TGG TTT CAA TTT TAT ATG 432 Arg Glu Ala Gly Gly Glu Lys Ala Pro Gln Trp Phe Gln Phe Tyr Met 130 135 140 AGC AAA GAT AAT GAA ATC AAT TTG GAT ATT CTT GAA GTG GCC AAG CGT 480 Ser Lys Asp Asn Glu Ile Asn Leu Asp Ile Leu Glu Val Ala Lys Arg 145 150 155 160 AAT GGA GCA AAA GCA ATC GTA TTG ACA GCG GAT GCA ACA GTT GGA GGA 528 Asn Gly Ala Lys Ala Ile Val Leu Thr Ala Asp Ala Thr Val Gly Gly 165 170 175 AAC CGT GAA ACA GAC CGC AGA AAT GGG TTT ACC TTC CCA CTG CCA ATG 576 Asn Arg Glu Thr Asp Arg Arg Asn Gly Phe Thr Phe Pro Leu Pro Met 180 185 190 CCA ATC GTT CAG GCT TAT CAA TCA GGA GTA GGC CAA ACG ATG GAT GCT 624 Pro Ile Val Gln Ala Tyr Gln Ser Gly Val Gly Gln Thr Met Asp Ala 195 200 205 GTG TAC AAA TCT TCC AAA CAG AAG CTT AGT CCA AAA GAT GTA GAA TTT 672 Val Tyr Lys Ser Ser Lys G ln Lys Leu Ser Pro Lys Asp Val Glu Phe 210 215 220 ATT GCT GCT CAT TCT GAT TTG CCA GTT TAT GTG AAA GGT GTC CAG TCA 720 Ile Ala Ala His Ser Asp Leu Pro Val Tyr Val Lys Gly Val Gln Ser 225 230 235 240 GAA GAA GAT GTC TAT CGT TCA TTA GAA TCG GGT GCT GGC GGA ATC TGG 768 Glu Glu Asp Val Tyr Arg Ser Leu Glu Ser Gly Ala Gly Gly Ile Trp 245 250 255 GTA TCT AAT CAT GGC GGC CGT CAA CTA GAT GGT GGT CCT GCA GCG TTT 816 Val Ser Asn His Gly Gly Arg Gln Leu Asp Gly Gly Pro Ala Ala Phe 260 265 270 GAT TCG TTG CAA TAT GTG GCA GAT GCA GTA GAT AAA CGT GTA CCA ATC 864 Asp Ser Leu Gln Tyr Val Ala Asp Ala Val Asp Lys Arg Val Pro Ile 275 280 285 GTG TTT GAC AGC GGT GTA CGA CGC GGA CAG CAT GTT TTC AAA GCA ATC 912 Val Phe Asp Ser Gly Val Arg Arg Gly Gln His Val Phe Lys Ala Ile 290 295 300 GCA TCA GGT GCA GAT CTA GTT GCT ATT GGC AGA CCC GTT ATT TAT GGA 960 Ala Ser Gly Ala Asp Leu Val Ala Ile Gly Arg Pro Val Ile Tyr Gly 305 310 315 320 TTG TCT TTA GGA GGA AGC ACA GGA GTC CGT CAA GTG TTT GAT TTC TTT 1008 Leu Ser Leu Gly Gly Ser Thr Gly Val Arg Gln Val Phe Asp Phe Phe 325 330 335 AAA ACA GAA TTG GAA ATG GTC ATG CAA TTA GCT GGA ACA CAA ACA GTA 1056 Lys Thr Glu Leu Glu Met Val Met Gln Leu Ala Gly Thr Gln Thr Val 340 345 350 GAA GAT ATC AAA AAA ATA AAA TTG CGT GAG AAT CGT TTT ATT 1098 Glu Asp Ile Lys Lys Ile Lys Leu Arg Glu Asn Arg Phe Ile 355 360 365 366

[Brief description of the drawings]

FIG. 1 is a diagram showing the thermal stability of a lactate oxidase of the present invention.

FIG. 2 is a diagram showing the pH stability of the lactate oxidase of the present invention.

FIG. 3 is a diagram showing an optimum temperature of a lactate oxidase of the present invention.

FIG. 4 is a graph showing the optimum pH of the lactate oxidase of the present invention.

FIG. 5: Amino acid sequence of lactate oxidase of the present invention and Aerococcus viridans IFO1
It is a figure which shows the comparison with the amino acid sequence of lactate oxidase which 2219 produces.

FIG. 6 shows a restriction map of pLCO3.

Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // (C12N 15/09 ZNA C12R 1:01) (C12N 1/21 C12R 1:19) (C12N 9/04 C12R 1:01) (C12N 9/04 C12R 1:19)

Claims (13)

[Claims] 1. A novel lactate oxidase derived from the genus Lactococcus, having the following physicochemical properties. Action: Acts on lactic acid in the presence of oxygen to produce pyruvate and hydrogen peroxide. Thermal stability: about 50 ° C or less (pH 7.5, treated for 10 minutes) pH stability: about 4.0 to 9.0 (treated at 25 ° C, 16 hours) 2. A novel lactate oxidase having the following physicochemical properties. Action: Acts on lactic acid in the presence of oxygen to produce pyruvate and hydrogen peroxide. Thermal stability: about 50 ° C. or less (pH 7.5, 10 minutes treatment) pH stability: about 4.0 to 9.0 (25 ° C., 16 hours treatment) Optimum temperature: about 35 ° C. Optimum pH: about 6 Molecular weight: about 40,000 (SDS-PAGE) About 40,000 (calculated from amino acid composition) About 160,000 (gel filtration) Isoelectric point: about 4.3 ± 0.1 3. A lactate oxidase 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) comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added, and Protein with oxidase activity 4. The lactate oxidase according to claim 1, which has an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. 5. A gene encoding lactate oxidase which is the following protein (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) comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added, and Protein with oxidase activity 6. A gene encoding lactate oxidase, which is a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. 7. The following (c), (d) or (e) DN:
A gene encoding lactate oxidase consisting of A. (C) DNA consisting of the nucleotide sequence of SEQ ID NO: 2 in the sequence listing (d) One or more bases are added, deleted or substituted in the above-mentioned nucleotide sequence (c), and lactic acid oxidation D encoding an amino acid sequence having enzymatic activity
NA (e) Bacterial D which hybridizes with the DNA comprising the nucleotide sequence of (c) under stringent conditions and encodes a protein having lactate oxidase activity
NA 8. A gene encoding lactate oxidase containing a DNA consisting of the nucleotide sequence of SEQ ID NO: 2 in the sequence listing. 9. A recombinant vector containing a gene encoding the lactate oxidase according to claim 5, 6, 7, or 8. 10. A transformant obtained by transforming a host cell with the recombinant vector according to claim 9. 11. A method for producing lactate oxidase, comprising culturing the transformant according to claim 10, producing lactate oxidase, and collecting the lactate oxidase. 12. Lactoco lactis (Lactoco)
ccus lactis (subsp. cremoris) A method for producing lactate oxidase, which comprises culturing IFO3427, producing lactate oxidase, and collecting the lactate oxidase. 13. A reagent for measuring lactic acid, comprising the lactate oxidase according to claim 1 and peroxidase or catalase and a reagent for detecting hydrogen peroxide.