JPS63169989A - Dna having genetic information on pyruvic oxidase and use thereof - Google Patents

Abstract

PURPOSE:To obtain the titled DNA useful for efficiently producing pyruvic oxidase capable of catalyazing reaction for producing acetyl phosphate, carbon dioxide and hydrogen peroxide from pyruvic acid, phosphoric acid and oxygen by applying a genetic engineering technique. CONSTITUTION:A microorganism, e.g. Lactobacillus delbrucki or Streptococcus sp., having the ability to produce pyruvic oxidase is cultivated in a liquid culture medium by a spinner culture method with aeration and the resultant culture is centrifuged to collect the microorganism. The resultant microorganism is bacteriolyzed to prepare a bacteriolytic material containing pyruvic oxidase. DNA is then separated, purified from the above-mentioned bacteriolytic material and cleaved with a restriction enzyme, e.g. EcoRI or HindIII, to provide a DNA fragment, which is linked to a vector fragment by a method for using a well-known DNA ligase to afford a recombinant DNA. The obtained recombinant DNA is then introduced into a host microorganism e.g. belonging to Escherichia coli, to provide a transformant, which is then cultivated to afford the aimed pyruvic oxidase.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a polydeoxyribonucleic acid having genetic information for a novel pyruvate oxidase.

The present invention also relates to a transformant which retains the polydeoxyribonucleic acid, pyruvate oxidase obtained by expressing the genetic information of the polydeoxyribonucleic acid in the transformant, and a method for producing the same.

(Prior art) Pyruvate oxidase catalyzes the reaction that produces acetyl phosphate, carbon dioxide, and hydrogen peroxide from pyruvate, phosphoric acid, and oxygen, and
Lactobacillus del
brucki) (Williams+s, F, R,
& I(ager.

L, P. (1966) Arch, Bioches+,
Biophys, 116 168-176] Pediococcus, Streptococcus, Aerococcus viridans
idans) (Special Publication No. 5B-40465)
It has been reported that it exists in bacteria belonging to the .

In addition, pyruvate oxidase is an oxidizing enzyme that uses pyruvate as a substrate, so it is not only used to quantify pyruvate present in body fluids such as serum, but also glutamate-oxaloacetate transaminase, glutamate-
By measuring the amount of pyruvate produced in the pyruvate production system such as pyruvate transaminase, lactate dehydrogenase, and neuraminidase-N acetylneuraminic acid aldolase, it is possible to quantify various substances that are substrates of enzymes involved in the pyruvate production system. It is extremely useful as a research reagent and clinical diagnostic reagent, such as for measuring the activities of various enzymes involved in the pyruvate production system.

[Problem that the invention seeks to solve]

The pyruvate oxidase-producing bacteria that have been reported so far have low productivity of pyruvate oxidase (not only do they have the disadvantage of being expensive in terms of production costs), but they also have the disadvantage that it is extremely difficult to remove coexisting enzymes from other species. However, in order to obtain high-quality pyruvate oxidase with high purity, the purification cost was extremely high, and it was not always satisfactory for use as a research reagent or clinical diagnostic reagent.

Furthermore, the detailed chemical structure of pyruvate oxidase, which catalyzes the reaction that produces acetyl phosphate, carbon dioxide, and hydrogen peroxide from pyruvic acid, phosphoric acid, and oxygen, which has been reported so far, has not been reported.

[Means for solving problems]

The present inventors conducted intensive studies to improve the productivity of the pyruvate oxidase, and succeeded in collecting the pyruvate oxidase gene and analyzing its next-order structure, and by applying genetic engineering techniques, As described below, we have established a highly productive manufacturing method.

That is, the present invention provides a polypeptide consisting of pyruvate oxidase or S-pyruvate oxidase which is exogenous to the host and catalyzes the reaction of producing acetyl phosphate, carbon dioxide and hydrogen peroxide from pyruvate, phosphoric acid and oxygen. A polydeoxyribonucleic acid characterized by being a base sequence encoding the amino acid sequence of a polypeptide consisting of N From the terminal side, pyruvic acid, phosphoric acid and It is a polypeptide consisting of pyruvate oxidase or S-pyruvate oxidase, which catalyzes the reaction that produces acetyl phosphate, carbon dioxide, and hydrogen peroxide from oxygen, and it also produces acetyl phosphate from pyruvate, phosphoric acid, and oxygen. A recombinant DNA in which polydeoxyribonucleic acid, which is a pyruvate oxidase gene that catalyzes a reaction that produces acid, carbon dioxide, and hydrogen peroxide, or a polydeoxyribonucleic acid comprising this gene as a component, is incorporated into an expression vector is transferred into a host microorganism. Obtain a transformant, culture the transformant to express the genetic information of the polydeoxyribonucleic acid, and then use pyruvate to catalyze the reaction that produces acetyl phosphate, carbon dioxide, and hydrogen peroxide from pyruvate, phosphoric acid, and oxygen. The present invention relates to a method for producing pyruvate oxidase by collecting a polypeptide that is acid oxidase or a polypeptide having 8-pyruvate oxidase as a constituent component.

Regarding polypeptide (I), the amino acid residue represented by A consists of - or a plurality of amino acid residues,
Preferably, A includes a hydrogen atom, Met, or a signal peptide. B may be an acid amide, or may be - or more amino acid residues.

The polydeoxyribonucleic acid, which is a pyruvate oxidase gene that catalyzes the reaction that produces acetyl phosphate, carbon dioxide, and hydrogen peroxide from pyruvate, e# acid, and oxygen, or the polydeoxyribonucleic acid of which this gene is a constituent, includes at least pyruvate. Any polydeoxyribonucleic acid containing the pyruvate oxidase gene itself that catalyzes the reaction of producing acetyl phosphate, carbon dioxide, and hydrogen peroxide from acid, phosphoric acid, and oxygen may be used; for example, the pyruvate oxidase gene itself may have an N-terminus. Formula % Formula % from the side Asp Thr Asn Lys Asn Leu P
he Gly Val Asp11eLys,
Polydeoxyribonucleic acid is a base sequence encoding the amino acid sequence of a polypeptide consisting of pyruvate oxidase, which catalyzes the reaction of (If) that produces acetyl phosphate, carbon dioxide, and hydrogen peroxide from pyruvate, phosphoric acid, and oxygen. can be mentioned. Furthermore, in the amino acid sequence of the polypeptide that is pyruvate oxidase represented by formula (11), it is sufficient that the polydeoxyribonucleic acid consists of any one codon from a series of codons corresponding to each amino acid, and The polydeoxyribonucleic acid may have one or more codons other than nonsense codons at the 5' end of the polydeoxyribonucleic acid and/or one or more codons at the 3' end.
For example, as a representative example, from the 5゛ end side, the formula % formula % GCT GAA GCT GTT GCA
GCT AAT AAA GCA GGTCA
CACT GTCGTT ATCGACTGT
AAG ATT ACTCAA GAT CG
T CCA ATCCCT GTA GAA
ACA TTGAAA TTA GAT T
CA AAA CTT TACAGCGAA
GACGAA ATCAAA GCT TACA
AA GAA CGCTACGAAGCT GC
T AACTTA GTA CCA TTCA
GA GAG TACTTA GAA GCT
GAA GGCTTA G^8 TCT A
AATACATCAAA-Y (III)
[In the formula, X represents a codon or a hydrogen atom other than TAA, TAG and TG^, and Y represents a codon or a hydrogen atom] Polydeoxyribonucleic acids having a base sequence represented by the following can be mentioned.

Regarding the base sequence represented by formula (III), the codon represented by However, preferably ATC
Or polydeoxyribonucleic acid corresponding to a signal peptide can be mentioned.

The codon represented by Y may be any translation termination codon or a codon that encodes an amino acid, and may also have one or more codons that encode an amino acid at its 3'' end, but in that case It is preferable to have a translation stop codon at the 3° end of the plurality of codons.

Polydeoxyribonucleic acid comprising a pyruvate oxidase gene as a constituent, polydeoxyribonucleic acid which is a gene consisting of a base sequence encoding the amino acid sequence represented by formula (n), or polydeoxyribonucleic acid represented by formula (III>) For example, the nucleic acid is a linear expression vector obtained by separating and purifying the DNA of a microorganism that is a donor for the pyruvate oxidase gene that produces pyruvate oxidase, and then cleaving the DNA using ultrasound, restriction enzymes, etc. The blunt or cohesive ends of both DNAs are linked and closed using DNA ligase, etc., and the thus obtained recombinant DNA vector is introduced into a replicable host microorganism, and then the vector marker and pyruvate oxidase activity are used as indicators. Cultivating a microorganism carrying the recombinant DNA vector obtained by screening,
separating and purifying the recombinant DNA vector from the cultured hedron;
Next, polydeoxyribonucleic acid, which is the pyruvate oxidase gene, may be collected from the recombinant DNA vector.

The microorganism that is the donor of the pyruvate oxidase gene may be any microorganism that has the ability to produce pyruvate oxidase, for example, JP-A-54-126791, JP-A-59-159777, JP-A-59. -1
Publication No. 62877, Arch, Biochem, Bio
phys, 168-176 (1966), etc.
ctobacillus delbrucki),
Pedococcus sp.
sp) + Streptococcus sp)
eptococcus sp), Aerococcus viridans
, Lactobacillus plantarum
llus Plantarus), Lactobacillus salivarius (Lactobacillus 5a)
livarius), Leuconostoc Mesenteroides (Leuconostoc Mesenteroides)
Microorganisms such as Lactobacilaceae or Streptococceaceae such as P. roides are selected as appropriate.

Furthermore, a transformed microorganism imparted with the ability to produce pyruvate oxidase by making full use of genetic recombination technology may be used as a donor for the pyruvate oxidase gene.

DNA derived from a microorganism that is a gene donor is collected as follows. That is, one of the above-mentioned bacteria, which is a donor microorganism, is cultured with aeration in a liquid medium for about 1 to 3 days, the resulting culture is collected by centrifugation, and then the bacteria are lysed. A lysate containing the acid oxidase gene can be prepared.

Bacteriolytic methods include treatment with cell wall lytic enzymes such as lysozyme and β-glucanase, and if necessary, other enzymes such as protease and surfactants such as sodium lauryl sulfate, and physical destruction of the cell wall. Freeze-thawing or French press treatment may be performed in combination with the above-mentioned lysis method.

In order to separate and purify DNA from the lysate obtained in this way, it is possible to use a conventional method, for example, by appropriately combining methods such as protein removal treatment by phenol extraction, protease treatment, ribonuclease treatment, alcohol precipitation, and centrifugation. It can be carried out.

The isolated and purified microbial DNA can be cleaved by, for example, ultrasonication, restriction enzyme treatment, etc. However, in order to facilitate the binding of the obtained DNA fragment to the vector, restriction enzymes, especially specific nucleotides, are used. acts on the sequence, e.g. EcoRI +' 1lind
■-type restriction enzymes such as Ill and Bam1l I are suitable.

Suitable vectors are those constructed for genetic recombination from phages or plasmids that can autonomously propagate in host microorganisms.

Examples of phages include Escherichia coli (E
scherichia coli) as the host microorganism, λgt, λC1, λgt, λB, etc. can be used.

Further, as a plasmid, for example, pBR322 when Escherichia coli is used as a host microorganism.

pBR325, pAcYc184. pUc12.
pUc13. pUc18゜pUc19 etc. are Bacillus subtill.
is) as the host microorganism, pUBllo, p
C194, etc. can be used, and in addition, Duram-yin bacteria such as Escherichia coli and Satucharomyces cerevisiae can be used.
It is also possible to utilize shuttle hectors that can autonomously propagate with two or more species of host microorganisms that span the positive spectrum. It is preferable to obtain a vector fragment by cleaving such a vector with the same restriction enzyme used to cleave the microbial DNA that is the pyruvate oxidase gene donor described above.

The method for joining the microbial DNA fragment and the vector fragment may be any 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 NA ligase is used. By this action, recombinant DNA between the microbial DNA fragment and the vector fragment is created. If necessary, after annealing, the DNA can be transferred into a host microorganism and recombinant DNA can be created using in-vivo DNA ligase.

The host microorganism may be one that allows the recombinant DNA to grow stably and autonomously and that can express the characteristics of the foreign DNA. For example, the host microorganism may be Escherichia spp.
For Escherichia coli, Escherichia coli DH1, Escherichia coli HB 101゜Escherichia coli W3110.
Escherichia coli C600 etc. can be used.

As a method for transferring recombinant DNA into a host microorganism, for example, when the host microorganism belongs to the genus Escherichia, the recombinant DNA is transferred in the presence of calcium ions, and when the host microorganism belongs to the genus Bacillus, the recombinant DNA is transferred In some cases, a competent cell method or a protoplast method may be employed, and a microinjection method may also be used. It has been found that pyruvate oxidase can be stably produced.

To select whether or not to transfer the target recombinant DNA into a host microorganism, it is sufficient to search for a microorganism that can simultaneously express the drug resistance marker and pyruvate oxidase of the vector holding the target recombinant DNA. A microorganism that grows on a selective medium based on the method and produces pyruvate oxidase can be selected. Once selected in this way, the recombinant DNA carrying the pyruvate oxidase gene is extracted from the transformed microorganism and used to generate pyruvate oxidase. It is also possible to easily transfer the pyruvate oxidase gene into a host microorganism.In addition, the recombinant DNA containing the pyruvate oxidase gene is cut with a restriction enzyme, etc. to cut out the pyruvate oxidase gene DNA, and the pyruvate oxidase gene DNA is cut out using a similar method. It is also possible to easily ligate the resulting vector fragments and transfer them into a host microorganism.

Furthermore, the DNA of the pyruvate oxidase mutin, which essentially has pyruvate oxidase activity, is an artificial mutant gene produced by genetic engineering from the pyruvate oxidase gene of the present invention, and this artificial mutant gene is is amplified using the method and ultimately,
This mutant gene is inserted into a vector to create recombinant DNA.
Pyruvate oxidase mutin can be produced by creating and transferring this into a host microorganism.

Furthermore, the base sequence of the pyruvate oxidase gene obtained by the above method is 5science 214120
5-1210 (1981), and the amino acid sequence of pyruvate oxidase was determined from the base sequence. On the other hand, the partial amino acid sequence constituting the N-terminal portion of the peptide, which is pyruvate oxidase, was determined as follows. That is, a pyruvate oxidase gene-donor microorganism capable of producing pyruvate oxidase is cultured in a nutrient medium to produce and accumulate pyruvate oxidase within the bacterial cells, and after completion of the culture, the obtained culture is subjected to filtration or centrifugation. The bacterial cells are collected by a method, and then the bacterial cells are destroyed by a mechanical method or an enzymatic method such as lysozyme, and if necessary, ED.
Pyruvate oxidase is solubilized by adding TA and/or a suitable surfactant, and separated and collected as an aqueous solution. The aqueous solution of pyruvate oxidase thus obtained is concentrated or treated without concentration by ammonium sulfate fractionation, gel filtration, adsorption chromatography, or ion exchange chromatography to obtain high purity pyruvate oxidase. Using high-purity pyruvate oxidase, a partial amino acid sequence constituting the N-terminal portion of a peptide that is pyruvate oxidase was determined using a liquid phase protein sequencer (manufactured by Beckman: BECKMAN System 890ME), and at least
It was confirmed that the partial amino acid sequence matched the N-terminal partial amino acid sequence of pyruvate oxidase obtained by genetic manipulation.

The culture mode of the host microorganism, which is the transformant, can be selected by considering the nutritional and physiological properties of the host.Although liquid culture is usually used in most cases, deep aeration agitation culture is used industrially. It is advantageous to do so. As the nutrient source for the medium, a wide variety of nutrients commonly used for culturing microorganisms can be used. The carbon source may be any carbon compound that can be assimilated, such as evacucrose, sucrose, lactose, maltose, fructose, IJ! Honey, pyruvic acid, etc. are used. The nitrogen source may be any available nitrogen compound, such as peptone or meat extract.

Yeast extract, casein hydrolyzate, etc. are used. Other substances include phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, and manganese.

Salts such as zinc, certain amino acids, and certain vitamins are used as needed.

The culture temperature can be changed as appropriate within a range that allows the bacteria to grow and produce pyruvate oxidase, but in the case of Escherichia coli, it is preferably about 20 to 42°C. The culture time is
Although it varies somewhat depending on the conditions, the culture should be terminated at an appropriate time when the maximum yield of pyruvate oxidase is reached (usually around 12 to 48 hours). Although it can be changed as appropriate within the range of producing oxidase, particularly preferably pH 6,
It is about 0 to 8.0.

Pyruvate oxidase in the culture can be collected and used as it is, including the culture solution containing the bacterial cells. It is used after separating the pyruvate oxidase-containing solution from the microbial cells by centrifugation or the like.

When pyruvate oxidase is present in the bacterial body,
The cells of the obtained culture are collected by means such as filtration or centrifugation, and then the cells are destroyed by a mechanical method or an enzymatic method such as lysozyme, and if necessary, a chelating agent such as EDTA is added. And/or a surfactant is added to solubilize pyruvate oxidase, which is separated and collected as an aqueous solution.

The pyruvate oxidase-containing solution thus obtained is subjected to, for example, vacuum concentration, membrane concentration, salting out treatment with ammonium sulfate, sodium sulfate, etc., or hydrophilic organic solvents such as methanol and ethanol.

The precipitate may be precipitated by a fractional precipitation method using acetone or the like.Then, this precipitate is dissolved in water and dialyzed through a semipermeable membrane to remove lower molecular weight impurities. In addition, it is purified by gel filtration using an adsorbent or gel filtration agent, adsorption chromatography, or ion exchange chromatography, and the pyruvate oxidase-containing solution obtained using these methods can be purified by processing such as vacuum concentration and freeze-drying. obtained pyruvate oxidase.

The abbreviations for amino acids, peptides, nucleic acids, nucleic acid-related substances, and others described herein are based on the abbreviations commonly used in the field, and examples thereof are listed below. Furthermore, all amino acids are assumed to be in the L form.

DNA: Deoxyribonucleic acid RNA: Ribonucleic acid A: Adenine T: Thymine G; Guanine C: Cytosine Ala: Alanine Arg: Arginine Asn: Asparagine Asp: Aspartic acid Cys: Cystine Gln: Glutamine Glu: Glutamic acid cty: Glycine His: Histidine 11e: Isoleucine Leu: Leucine 1, ys: Lysine Met: Methionine Phe: Phenylalanine Pro: Proline Ser: Serin Thr: Threonine Trp :　　　　　　　　　　　) 'l”yr: Tyrosine Val: Valin Hereinafter, the present invention will be explained in detail with reference to Examples.

〔Example〕

Examples/[Isolation of chromosomal DNA] Aeroeoccus viridans (I F
The chromosomal DNA of OO/22/ri) was isolated by the following method, and the same strain was cultured in 7501 ordinary broth medium (containing 0.5% sodium thiosulfate) at 37°C overnight with shaking, followed by centrifugation (3.
Bacteria were collected at 000 rpm (000 rpm). 10% sucrose, 3 ( ) m M ) Liss hydrochloric acid (pH 0)! ;O
Suspended in solution SR1 containing mM ED=A, /d
Add the lysozyme solution Cl01197Ill) to 37
℃ for 75 minutes, then add 1 ml of 10% SDS (
To this suspension was added an equal volume of chloroform-pheno-/14+'[(/:/)
The mixture was stirred and mixed, separated into an aqueous layer and a solvent layer by centrifugation at 10.00 rpm for 3 minutes, and the aqueous layer was counted. Two times the amount of ethanol was gently layered on this water 1, and the DNA was separated by wrapping it around a glass rod while stirring slowly in a glass cabinet.

Add this to 10 rll of 10 mM) J2 hydrochloric acid (PH, r, O
) and dissolved in a solution containing 7mM EDTA (hereinafter abbreviated as TE). After treating this with an equal amount of chloroform-phenol mixture, separate the aqueous layer by centrifugation, add twice the amount of ethanol, and separate the DNA again using the above method.
．． Dissolved with 1 rnl of TE.

Example 2 - Isolation of CpACYC/Zal plasmid DNA] Escherichia co'J carrying p〇Yc/, r4Z
pM/ta/(J, Bacterio
l/311. //IA/ (=/, when grown to O, spectinomycin (final concentration 300 μV
/ml (if the plasmid contains chloramphenicol as a resistance marker) and then incubate for one more time at 37°C.
Shaking was continued for over 76 hours. Bacteria were collected by centrifugation for 3 ooorpmio, and lysozyme-8DS method and cesium chloride-ethidium bromide method (Maniort
is et al.: Molecular Cloning pp.
g otsu~ri Q Co1d Spring Harbor
Plasmid DNA was prepared according to (/ri12)).

Example 3. [Creation of plasmid pOXIj containing hirupate oxidase (pop) gene] (i)
A, viridans chromosome D prepared in Example X
NA2μt (about 05μ?) and 100 times the amount of EωRI cutting buffer<! ;00mM trinuhydrochloric acid (pH -6)
, 70 mM Mgcl □ , /M
NaCl, 7QmM mercaptoethanol 1V
) / A”t, EcoRI (Takara Shuzo 70
unit/, g)/pL, aqueous layer pL were mixed and cut at 37°C/hour. Separately prepared plasmid pA
Approximately 0.3 μ2 of CYC/g'l DNA was cleaved with Eco RI using the same method, and 0 units of alkaline phosphatase (hereinafter sometimes abbreviated as BAP, manufactured by Takara Shuzo) was added, and the mixture was incubated at 5°C. /hour processed.

Mix these two kinds of DNA solutions cut with EcoRI, and then! /10 amount of 3M sodium acetate was added, and the mixture was further treated with a chloroform-phenol mixture in an amount equal to the total amount, and the aqueous layer was separated by centrifugation. Twice the volume of ethanol was added to this aqueous layer, and the DNA was precipitated by centrifugation, followed by drying under reduced pressure. After dissolving in water μt, 700 times the amount of Ligenyonpatufa (0!; M Tris-HCl (pH 7))
, O,/M MgCl2. Q, /M dithiothreitol, 10mM spermidine.

10mMATP) 10pL and T4! DNA ligase/
μL (manufactured by Takara Shuzo Co., Ltd./7junit) was added, mixed, and left overnight at ≠°C. This DNA solution was treated with chloroform-phenol, and the ethanol precipitate was collected and dried under reduced pressure.
Dissolved in μL of TE.

(iI) 10Od BHI medium (Brain Hear
Escherichia coli W3/10 strain in the logarithmic growth phase cultured with tInfusion (manufactured by Difco) C received from the National Institute of Genetics, stock number ME777g,
ATCC2732! ;'J was collected by centrifugation.
/ 0.000 rpm, 2 minutes)ttoral
of ice-cold 3QmM potassium acetate, 101% RhCl,
/ Om M CaCl2,j Q m MnCl
A solution containing 2 and 75% glycerin (pI (5 g
). After standing at 0°C for S minutes, centrifuge, discard the supernatant, and add JmMMOPS buffer (manufactured by Dotite) to <z>. ; m M CaCl2, iomMR
A mixture containing bCl and 75% glycerin (pH 6)
, 5) and left at 0°C for 1S minutes to obtain competent cells.

+=+ 10 µt of the DNA solution prepared in (i) was added to 200 µt of this E. coli suspension, and the mixture was allowed to stand at °C for 30 minutes. Add BHI medium/d and incubate at 37°C for 0 minutes,
This 100μt was added to tetracycline (75μt/1l
lj) was plated on a BHI agar plate and cultured overnight at 37°C to obtain transformants. This transformant is POP
Medium (composition: peptone 5?, meat extract 2?, yeast extract 3 y.

NaC1/t,, K2) LP01/? , Mg
50.0.3; ff, peroxidase! ;00
IU, FAD7g3~, Ginseng 0/? , thiamine pyrophosphate≠2qη, pyruvic acid/d, agar 1
The plate was replicated (5v, distilled water/l, pH 70) and further cultured overnight at 37°C.

When we examined the transformants of approximately ttsoo colonies, we obtained a strain with brown discoloration around the colony. Submission No. 207/No., F E
RM-i isol)7/''. After pure isolation, this bacterium was cultured overnight at 37°C in BHI medium, and the productivity of pyruvate oxidase was examined using the pyruvate oxidase activity measurement method described below. As a result, it had a pyruvate oxidase activity of approximately 3 u/ml. Ta.

The plasmid possessed by this strain was isolated in the same manner as in Example 2, and the plasmid containing the pyruvate oxidase gene and the pACYC/, r≠ gene was named pOXI3.

Method for measuring pyruvate oxidase activity The method for measuring the activity of pyruvate oxidase of the present invention is as follows.

0, j M potassium pyruvate 0. /ml
O,j M phosphate buffer (pH 71 (1), 2
tnt02%≠-aminoantipyrine 0. /m10
2% N,N-dimethylaniline O2go/(7mM
gC]2! ;OuL/Qm
M Thiaminopyrofonufate 20 uL Perio'hV Dase CI/-3U/ml) O,/m
lmMFAD 10! d.

Distilled water 0.22 ml Collect the reaction solution/Q ml of the above composition into a test tube, and heat at 37°C.
After prewarming for 3 minutes, add 20 μt of enzyme solution and heat to 37°C.
, the reaction was carried out for 10 minutes, and after the reaction, 0. /MED
Add TA (pH'75) to stop the expansion, and then add 7 ml of distilled water to it.
Colorimetric determination is performed at a wavelength of 5 nm. / μmol in 7 minutes
The activity of e to produce hydrogen peroxide was expressed as /unit (U).

Example≠, [Mapping of pOXI3 and determination of base sequence of pOp gene] Escherichia coli W3/10 pOX 3 strains KaupA
CY C/ g 'I,! : POX engineering 3 planumid DNA was prepared in the same manner.

Restriction enzyme C1a I for pOX engineering 3 DNA.

EcoRV, H4nd I [I, Scal,
Pst, l, Pvu 11.

A cleavage map was created using Xbar (all manufactured by Takara Shuzo) I (Pa (manufactured by Toyobo). The results are shown in Figure 1.
The base sequence of A was determined by the dideoxy method using M/3 phage (
Science 2/'I/20! ; /210
-@■ Shown.

Fruit melt, (Production of pyruvate oxidase) Escherichia coli W3/10pOXI3 strain 201B
The cells were cultured in HI medium (manufactured by Difco) at 37°C/g using a Jafer meter.

Bacteria were collected by centrifugation for rpmlQ minutes. After washing with physiological saline, it was suspended in 2 tons of iomMv acid buffer (pH 70). Lysozyme //Ilf/ml, EDTA-
, 2Na -2mM, ) Lito 7X-
100 was added to give a concentration of 0.7%, the mixture was incubated at 37°C for 30 minutes, and the supernatant was separated by centrifugation at 3.00 rpm for 10 minutes.

This supernatant liquid / tp is stuck and salting out (IaO% ~ Otsu 6
%) total 6%, the precipitate was collected by centrifugation (5000 rpm 30 minutes). This precipitate is 2001111/Qm
It was dissolved in M phosphate buffer (pH 70, containing 10 IJM FAD) and desalted with Sephadex G-25. After this, ion exchange chromatography was performed using DEAE-Sepharose CL-Otsu B, and the active fraction was separated and desalted.
A powder sample was obtained by freeze-drying. This enzyme preparation was measured by the pyruvate oxidase activity measurement method described above and was found to have a specific activity of 5/U/■.

Example B, [Determination of the amino acid sequence of the amino terminal part of pyruvate oxidase isolated and purified from a recombinant plant] The pyruvate oxidase isolated and purified in Example S was analyzed using a Beckman amino acid sequencer (Beckman).
The sequence of 10 amino acids from the amino terminus was examined using a System (gOME).

As a result, N-terminal Met exists in about gθ%, and from the 7th position of the amino acid sequence
It was confirmed that the pyruvate oxidase gene and the amino acid sequence of pyruvate oxidase were clarified by the present invention. An efficient method for producing pyruvate oxidase has been provided.In addition, a more efficient method for producing pyruvate oxidase can be provided by using the pyruvate oxidase gene of the present invention and various genetic engineering techniques. It is.

[Brief explanation of the drawing]

Figure 1 is a schematic diagram showing the structure of the p-0XI3 vector, and Figure 2 (shown across Figures 2-1 and 2-2) is
The coding strand (5') of the pyruvate oxidase gene DNA
-3') and the respective sequences of the resulting translation products are shown.

Claims (13)

[Claims] (1) Polydeoxyribonucleic acid foreign to the host is converted from pyruvate, phosphoric acid and oxygen to acetyl phosphate,
A polydeoxyribonucleic acid characterized by being a base sequence encoding the amino acid sequence of a polypeptide consisting of pyruvate oxidase that catalyzes a reaction that produces carbon dioxide and hydrogen peroxide, or a polypeptide having the pyruvate oxidase as a constituent component. (2) Polydeoxyribonucleic acid, which is foreign to the host, has the following formula from the N-terminal side: The polydeoxyribonucleic acid according to claim 1, which comprises a base sequence encoding the amino acid sequence of a polypeptide which is pyruvate oxidase represented by the following formula or a polypeptide having the pyruvate oxidase as a constituent component. (3) Polydeoxyribonucleic acid, which is foreign to the host, is expressed from the 5' end side (there is a gene sequence) [wherein, X represents a codon or hydrogen atom other than TAA, TAG, and TGA, and Y represents a codon or 2. The polydeoxyribonucleic acid according to claim 1, which is a base sequence encoding the amino acid sequence of a polypeptide consisting of pyruvate oxidase represented by the following formula (indicating a hydrogen atom) or a polypeptide consisting of the pyruvate oxidase as a constituent component. (4) A polypeptide or pyruvate oxidase in which the host microorganism is foreign to the host and catalyzes a reaction that produces acetyl phosphate, carbon dioxide, and hydrogen peroxide from pyruvate, phosphoric acid, and oxygen; A transformant characterized by retaining polydeoxyribonucleic acid, which is a base sequence encoding the amino acid sequence of a polypeptide comprising as a constituent component. (5) From the N-terminal side, the host microorganism has the following formula: [There is a gene sequence] [In the formula, A represents an amino acid residue or a hydrogen atom, [There is a gene sequence] B represents an amino acid residue or -OH] The transformant according to claim 4, which retains a polydeoxyribonucleic acid consisting of a base sequence encoding the amino acid sequence of a polypeptide that is pyruvate oxidase represented by or a polypeptide of which the pyruvate oxidase is a constituent component. . (6) The host microorganism, starting from the 5' end, is expressed by the formula [there is a gene sequence] [wherein, X represents a codon or hydrogen atom other than TAA, TAG, and TGA, and Y represents a codon or a hydrogen atom]. 5. The transformant according to claim 4, which retains a polydeoxyribonucleic acid which is a base sequence encoding the amino acid sequence of a polypeptide consisting of pyruvate oxidase or a polypeptide having the pyruvate oxidase as a constituent component. (7) The transformant according to claim 4, wherein the host microorganism belongs to the genus Escherichia. (8) The transformant according to claim 7, wherein the host microorganism belonging to the genus Escherichia is a microorganism belonging to the genus Escherichia coli. (9) From the N-terminal side, amino acids represented by the formula [Gene sequence is available] [In the formula, A represents an amino acid residue, a hydrogen atom, or an acetyl group, and B represents an amino acid residue, -OH or -NH_2] A polypeptide comprising pyruvate oxidase or a polypeptide comprising pyruvate oxidase as a constituent component, which catalyzes a reaction that produces acetyl phosphate, carbon dioxide, and hydrogen peroxide from the sequence pyruvate, phosphoric acid, and oxygen. (10) Polydeoxyribonucleic acid, which is a pyruvate oxidase gene that catalyzes the reaction that produces acetyl phosphate, carbon dioxide, and hydrogen peroxide from pyruvate, phosphoric acid, and oxygen, or a polydeoxyribonucleic acid containing this gene as a component, into an expression vector. The incorporated recombinant DNA is transferred into a host microorganism to obtain a transformant, the transformant is cultured to express the genetic information of the polydeoxyribonucleic acid, and then pyruvic acid, phosphoric acid, and oxygen are converted to acetyl phosphate and dioxide. A method for producing pyruvate oxidase, which comprises collecting a polypeptide that is pyruvate oxidase that catalyzes a reaction that produces carbon and hydrogen peroxide, or a polypeptide that is a constituent of the pyruvate oxidase. (11) Polydeoxyribonucleic acid, which is a pyruvate oxidase gene that catalyzes the reaction that produces acetyl phosphate, carbon dioxide, and hydrogen peroxide from pyruvate, phosphoric acid, and oxygen, or a polydeoxyribonucleic acid of which this gene is a component, is present at the N-terminus. A polypeptide that is pyruvate oxidase or the pyruvate oxidase represented by the formula [gene sequence is available] [in the formula, A represents an amino acid residue or a hydrogen atom, and B represents an amino acid residue or -OH] Claim 10 is characterized in that it is a polydeoxyribonucleic acid consisting of a base sequence encoding the amino acid sequence of a polypeptide consisting of
Manufacturing method described in section. (12) The production method according to claim 10, wherein the polydeoxyribonucleic acid is a pyruvate oxidase gene derived from a microorganism belonging to the Lactobacillaceae or Streptococceae family, or contains the gene as a constituent component. (13) The production method according to claim 12, wherein the pyruvate oxidase gene derived from a microorganism belonging to the Streptococcus family is a polydeoxyribonucleic acid obtained from the pyruvate oxidase-producing bacterium of the genus Aerococcus. .