JPH02454A - Dna having genetic information of l-alpha-glycerophosphate oxidase and use thereof - Google Patents

Abstract

PURPOSE:To provide a DNA containing a base sequence coding the amino acid of polypeptide constituting L-alpha-glycerophosphate oxidase(GPO) and capable of production GPO in high efficiency. CONSTITUTION:The above DNA can be produced e.g., by the following steps utilizing a gene recombination technique. A GPO-producing microorganism is used as a donor of GPO gene and the DNA is separated from said microorganism and purified. The DNA is cut by ultrasonic wave, restriction enzyme, etc., and the cut DNA is linked and cyclized with a linearized expression vector at the blunt end or cohesive end of both DNA using a DNA ligase, etc. The obtained recombinant DNA vector is introduced into a replicative host microorgnism. The microorganisms are screened by using marker of the vector and the activity of GPO as indices and the obtained microorganism holding said recombinant DNA vector is cultured. Said recombinant vector is separated and purified from the cultured microbial cell and the objective DNA having the GPO genetic information is collected from said recombinant vector.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a transformant containing DNAX having genetic information for L-α-glycerophosphate oxidase, and a method for converting nuclear DNA by the transformant. Obtained by expressing genetic information. 35 Relating to lipedetide and its manufacturing method. .

[Conventional technology]

L-α-glycerophosphate oxidase is produced by the following reaction formula: L-α-glycerophosphate + 02 → zohydroxyacetone phosphate) + HzO2, that is, from L-α-glycerophosphate and one molecule of oxygen. It is an enzyme that catalyzes the reaction between dihydroxyacetone phosphate and one molecule of hydrogen peroxide. As described above, since L-α-glycerophosphate oxidase (hereinafter abbreviated as GPO) is an oxidase that uses ha, x, -a-glycerophosphate as a substrate,
Used for the determination of L-α-glycerophosphate. In addition, this enzyme can be used in combination with other enzymes such as IJ/♀-ase, glycerol kinase, and ATP reagents to measure components involved in the reaction, such as 1-Q-ase activity measurement, triglyceride, glycerin, ATP, etc. Because it can be easily and specifically quantified, it is a key enzyme in biochemical quantitative methods that replace conventional non-specific chemical quantitative methods, and is extremely useful in the clinical diagnostic and research fields. .

On the other hand, GPOs have long been known to exist in nature; for example, GPOs of the genus Streptococcus (5trept
ococcus), Lactobacillus sp.
acillus), Leuconostoc (Leuco
nostoc), Pediococcus end (Pedioc
occus ), Aerococc
It has been reported that it exists in bacteria belonging to the Archipelago of Biochemistry and
Biophysics, vol. 88, p. 250 (1960);
JP-A No. 53-72892; JP-A No. 55-15746].

[Problem to be solved by the invention] However, GPO produced by these conventional GPO-producing bacteria
There were several problems in its production.

In other words, these GPO-producing bacteria have low GPO productivity; when using GPO-producing bacteria of the genus Streptococcus and the family Lactobacillus, glycerol, ascorbine [, Q- There were problems such as the need to add a substance that induces GPO production, such as a keto acid; and the difficulty in removing coexisting enzymes of other species. Therefore, in order to obtain high-quality GPO with high purity, the cost becomes extremely high, which has been an obstacle to its general use as a research reagent or a clinical diagnostic reagent.

Furthermore, the detailed primary structure of the gene of GPO and the primary structure of the amino acid sequences of the constituent protein and protein have not yet been reported.

[Means for Solving the Call] Therefore, the present inventors aimed to improve the productivity of GPO, reduce the content of coexisting enzymes of other species (contaminant enzymes), and reduce the manufacturing cost (after intensive study, we found that By successfully collecting the gene and analyzing the primary structure, and applying genetic engineering techniques, we have developed a GPO that has good productivity and does not require the addition of inducers to the culture medium during production.
We established a manufacturing method and completed the present invention.

That is, the present invention provides a DNA containing a base sequence encoding the amino acid sequence of a polypeptide constituting L-α-glycerophosphate oxidase (GPO), a transformant carrying the DNA, and culturing the transformant. G obtained
Polypeptide with PO activity, G with good physical and chemical properties
The present invention provides PO and a method for producing the same.

The DNA of the present invention is produced in the following manner using, for example, genetic recombination technology. That is, from a microorganism that is a donor of a GPO gene capable of producing GPO, the DNA of the microorganism is
After separating and purifying the NA, the DNA cut using ultrasonic waves, restriction enzymes, etc. and the expression vector cut into a linear shape are combined at the blunt or cohesive ends of both DNAs.
After the recombinant DNA vector thus obtained was transferred into a replicable host microorganism, the recombinant DNA vector was screened using the vector marker and GPO activity as indicators. A microorganism carrying a DNA vector is cultured, and the recombinant D
It is produced by separating and purifying the NA vector, and then collecting the DNA of the present invention having GPO genetic information from the recombinant vector.

The microorganism that is the donor of the cpo gene is not particularly limited as long as it has the ability to produce GPO, but examples include JP-A-53-72892, JP-A-55-15746;
58-165789, etc.
s faecium) F24 strain, Streptococcus-7 echaris (5treptococcus faecium)
calis) 10 CI strain, Streptococcus I
Streptococcus faecalis strain ATCC 12755, Streptococcus faecalis strain ATCC 8043, Streptococcus 7 ecaris strain ATCC 19488-, Streptococcus crelis
Streptococcus GPO production cup such as NRRL B 684 strain; Pediococcus cerevisii (Pediococcus cerevis)
iae) A T T C8042 &, Pediococcus cerevisiae ATCC8081 strain, Pediococcus cerevisiae/ie IF012230 strain, Pediococcus a.
cidilactiei) IFo strain 3885, Pediococcus bentosaceus (Pediococc
us pentosaceus ) I F 0123
18 strains, Pedeiococcus noeruvialus (Pedi)
ococcus parvulus ) I Fo
12,235 strains, Pedeiococcus homali (Pedio
coccushomari) IFo 12217
strain, Pediococcus urinaeequi (Pedio
coccus urinae-equi) ATCC29
GPO-producing bacteria of the genus Pedeiococcus such as strain 722; Lactobacillus casei (Lactobacillus casei);
ei) ATCo 7469 strain, Lactobacillus
Lactobacillus del
bruckii) NRRLB445 strain, Lactobaillus f.
ermentum) N RRL9338 strain, Lactobacillus lakemanii
Lactobacillus GPO production cup such as AT CC4797 strain; Leuconostocmese
Leuconostoc iG such as nteroides)
PO production cup; Aerococcus viridans (Aerococcus viridans) r
Examples include GPO-producing bacteria belonging to the genus Aerococcus such as strain FO12219 and strain IF012317, among which GPO-producing bacteria belonging to the genus Streptococcus are preferred.

Furthermore, a transformed microorganism imparted with GPO-producing ability by genetic recombination technology may be used as a donor of the GPO gene.

Particularly preferred GPO gene donor microorganisms include:
The present inventors isolated a strain of Streptofuccus from the soil of a farm in Yamaguchi, Susono City, Shizuoka Prefecture, G.P.
O'-53 strain is mentioned. The G P O′″-53
The strain produces GPO with better physicochemical properties than conventionally known GPO.

The taxonomic properties of the above-mentioned town-regulated CPO3-53 strain are as follows.

I. Growth status in various cultures (culture temperature 28-30℃
) ■Slant culture (ordinary agar medium + yeast extract 0.2%) Grows in a linear pattern, but the growth is rather weak (cream-colored to milky white. Does not produce soluble pigments.)

■Plane culture (ordinary agar medium + 0.2% yeast extract) Forms a round, mound-shaped fruit with a cream to milky white color. No machinable pigments are produced.

■Liquid culture g! (Tryptone Ibros (manufactured by Difco)
] It grows well and is uniformly cloudy, but it settles (the upper part becomes a bottle).

■BCP milk culture Becomes acidic.

(2) Morphological characteristics Spherical or egg-shaped bacteria, singly, in pairs, in short chains, or in long chains. It is not motile and does not form spores. size is 0
．． 8-1. Ox 1. It is Opm.

■, Physiological and biochemical properties [+: positive, -2 negative, (+): weak positive] Durham staining, ten KOH reaction, acid-fast staining, Cassis cell formation OF test (Hugh Leifson)
F Anaerobic growth 10 Growth temperature 45℃ +30℃
+ 10°C Salt tolerance NaC4 concentration 0% 5.0% 6.0% Growth pH pH9.5 pH8.0 pH4.0 0.1% Methylene V Growth in milk medium Catalase production Oxidase production Urease production (SSR, also known as Chris ) Gelatin decomposition Starch decomposition Casein decomposition Aesculin decomposition Hysolate decomposition (AP I・2OSTRgP) Cellulose decomposition Arginine decomposition Indole production + + + + + + + Hydrogen sulfide production Acetoin production (NaC1) MR test Sulfate reduction denitrification reaction (Usability test ) Citrate (also Simons, Chris)
Malate (Simons) (chris) Zolopionate (Simons) Malonate (chris) Production of gas from glucose (production of acid from sugar) Adonitol L (+) Arabinose Cellobiostalcitol Meso-Erythritol Fructose D- Galactose 10+ + 10+ + 10+ D-Glucose Glycerin Inositol Inulin Lactose Maltose Mannitol Mannose Melibiose Fructose Raffinose L(+) Rhamnose D-Li L-Susalicin L-Rubose Sorbitol Gelatin Sucrose Sucrose Trehalose + (10) + 10 10+ + + Hemolytic (API 2O5TREP) Based on the above mycological properties, the mycological characteristics of the new strain of the present invention are Durham-positive cocci to ovoid bacteria, isolated, double, short chain, or long chain. , negative for catalase and oxidase production, fermentatively decomposes minut to produce acid, non-motile, non-spore forming, size O18-1. It can be said that it is a bacterium with an OX of 1.0 μm.

The main properties of this strain were determined by Bergey's Manual Opdeterminant.
al of Determinative Bacte
As a result of referring to the 8th edition, this strain was determined to belong to the genus Streptococcus. Streptococcus (5treptococcus)
:Hereinafter, it may be simply referred to as S.゛) The genus is 10℃ and 4
They are broadly classified based on their properties such as ability to grow at 5°C, ability to grow in 0.1% methylene night milk medium, ability to decompose gelatin, etc., and salt tolerance. A comparison of the main bacterial species of the genus Streptococcus and this bacterial strain is shown below.

In the table, Fungi (1) to (9) are as follows.

(1) Streptococcus pyogenes (S-pyog)
(2) Streptococcus eximilis (S, equi
isimilis) (3) Streptococcus equi (S, equi)
(4) Streftococcus pneumoniae (S, pne
umoniae) (5) Streftococcus su17.41J (S, 5
alivaris) (6) Streftococcus bovis (S, bovis
) (7) Streftkotsukurisufuekaris (S, f
aecalis) (8) Streftococcus-7 aecium (S, fa
(9) Streptococcus lactis (S, 1act
is) As a result of the above comparison, this strain is determined to be a strain similar to Streftococcus faecalis, Streftococcus faecium, and Streftococcus lactis. The various properties of these three bacterial strains and this strain are compared in detail and shown as a representative.

From the above comparison, the properties of this strain are S.

It matches well with faecium. However, they differ in terms of hyderein decomposition and salt tolerance (65%), which are key contributors to the classification of the genus Streptococcus.

Therefore, this bacterial strain was identified as a new strain belonging to the genus Streptococcus, and was identified as Streptococcus sp.
ococcus sp) GPOS-53, and was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as [FERM BP-212O] J.

DNA derived from a microorganism that is a gene donor is collected as follows. That is, if 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 this is lysed. Therefore, a lysate containing the GPO 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. The freeze-thaw and French press treatments may be performed in combination with the above-mentioned I'a method.

To separate and purify DNA from the lysate obtained in this way, use conventional methods such as protein removal treatment by phenol extraction, protease treatment, IJ/nuclease treatment, alcohol precipitation, centrifugation, etc., as appropriate. This is done by

In order to cleave the isolated and purified microbial DNA, for example,
This can be carried out by sonication, restriction enzyme treatment, etc., but in order to facilitate the binding of the obtained DNA fragment to the vector, restriction enzymes, especially those that act on specific nucleotide sequences, such as EcoRI, Hind Il, etc.
A method using a type restriction enzyme such as BamHI or BamHI is suitable.

Suitable vectors are those constructed for genetic recombination from phages or cilasmids that can autonomously proliferate within host microorganisms.

As a phage, for example, Escherichia 8 coli (Es
cherichia coli) as the host microorganism, λgt, λC9λgt, λB, etc. can be used.

In addition, as a plasmid, for example, when using Escherichia coli as a host microorganism, pBR322゜pBR3
25,1) ACYC184, pUc12, pUc1
3゜pUc18, pUc19, etc., when the host microorganism is Bacillus subtilis, pUBllo, pc194
It is also possible to use shuttle vectors that can autonomously proliferate in two or more host microorganisms spanning Durham's positive and negative traits, such as E. coli, Escherichia coli, and Satucharomyces cerevisiae. 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 GPO gene donor mentioned above.

The method for joining the microbial DNA fragment and the vector fragment may be any method using a known DNA IJ gauze. For example, after annealing the cohesive ends of the microbial DNA fragment and the cohesive end of the vector fragment, an appropriate method may be used. A recombinant DNA of a microbial DNA fragment and a vector fragment is created by the action of NA ligase. If necessary, recombinant DNA can also be created by transferring it to a host microorganism after incubation and 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 coli, Escherichia coli DHI, Escherichia coli HBIOI Escherichia coli W3110. Escherichia coli 0600 etc. can be used.

As a method for transferring recombinant DNA to a host microorganism, for example, when the host microorganism belongs to the genus Escherichia, the recombinant DNA is transferred in the presence of calcium ion, and when the host microorganism belongs to the genus Bacillus, the recombinant DNA is transferred. Is there a competent cell method or a method? A method such as electrical fusion transfer of recombinant DNA into a protocylast host cell can be employed, and a microinjection method may also be used.

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 GPO of the recombinant DNA vector that holds the target DNA fragment. Microorganisms that grow on a selective medium and produce GPO can be selected based on resistance markers.

The recombinant DNA carrying the GPO gene selected in this way can be removed from the transformed microorganism and transferred to other host microorganisms. In addition, the GPO gene DNA is cut out from the Mi1% DNA carrying the GPO gene using a restriction enzyme, and the DNA is then ligated to the end of another open vector vector obtained by cutting in a similar manner. Recombinant DNA having these characteristics can also be produced and introduced into other 7a-based microorganisms.

Furthermore, the DNA encoding a GPO mutin with essential KGPO activity means an artificial mutant gene produced from the GPO gene of the present invention by genetic engineering techniques, and this artificial mutant gene can be produced by site-specific base conversion method and target gene. Regarding the GPO mutin DNA, which is obtained using various genetic engineering methods such as replacing a specific DNA fragment with an artificially displaced DNA fragment, and which has particularly excellent properties among the artificially mutated genes thus obtained, Finally, this mutin DNA is inserted into a vector to create a recombinant DNA.
GPO mutin can be produced by creating A and transferring it to a host microorganism.

The base sequence of the DNA of the present invention thus obtained is Sc+e
nce 214, 12O5-1210 (1981) K can be deciphered and determined by the dideoxy method shown. For example, the base sequence of the GPO gene in a plasmid obtained using Streptococcus sp. PO3-53 strain as the GPO gene donor and Escherichia coli as the host microorganism is shown in Figure 2 (1) to (5).
It is as follows.

In Figures 2 (1) to (5), the doublet indicated by
, may further have one or more codons encoding amino acids at its 5' end, but preferably corresponds to ATG or a signal peptide. Mention may be made of nucleic acids. The codon represented by Y may be any translation stop 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' terminal side, In that case, it is preferable to add a translation stop codon to the 3' end of the plurality of codons.

Furthermore, the amino acid sequence of a polypeptide produced by expressing the DNA of the present invention can be predicted and determined from the base sequence of the DNA. The partial amino acid sequence constituting the N-terminal portion of the peptide can be determined by the following procedure.

That is, a GPO gene-donating microorganism with apo-producing ability is cultured in a nutrient medium to produce and accumulate ic GPO within the microbial cell, and culture! ! After a long period of time, the cells of the obtained culture are collected by means such as Pd2 or centrifugation, and then the cells are disrupted mechanically or by a chemical method such as lysozyme, and if necessary, treated with EDTA. And/or by adding an appropriate surfactant etc., GPO becomes a town drama,
Separated and collected as an aqueous solution. G obtained by this lateral pressure
High purity GPO can be obtained by concentrating or without concentrating the water bath solution of PO by ammonium sulfate fractionation, dull filtration, adsorption chromatography, ion exchange chromatography, and high purity GPO can be used to convert liquid phase Sequencer (Ben2fufu company!l!!: BECKMAN
The partial amino acid sequence constituting the N-terminal portion of the peptide, which is System890MFJ) K Yotsu GPO, is determined. In addition, it was confirmed that the partial amino acid sequence was identical to the N-terminal partial amino acid sequence of GPO obtained by genetic manipulation.
The amino acid sequences determined as described above from the base sequence shown in (5) are as shown in FIG. 1 (1) to (6). Among the amino acid sequences shown in FIG. 1 (1) to (6), the amino acid residue indicated by A consists of one or more amino acid residues, and A is preferably L7 or a hydrogen atom or a hydrogen atom. Met or signal-eftide.

What is represented by B may be an acid amide (
It may also be one or more amino acid residues.

The transformant thus obtained stably produces a larger amount of peptide having GPO activity than when cultured in a nutrient medium.

The culture conditions for the host microorganism, which is a form-transformer, should be selected by taking into account the nutritional and physiological properties of the host. It is advantageous to culture.As a nutrient source for the medium,
A wide variety of materials commonly used for culturing microorganisms can be used. As a carbon source, any carbon compound that can be assimilated is sufficient (
For example, sucrose, sucrose, lactose, maltose, lactose, lactose, etc. are used.

The nitrogen source may be any available nitrogen compound, such as peptone, meat extract, yeast extract, casein hydrolyzate, and the like. Others include phosphates, carbonates, sulfur rvl salts, magnesium, calcium, potassium, iron,
Salts such as manganese and zinc, specific amino acids, and specific vitamins are used as needed, but conventional CPO
The addition of GPO inducers such as glycerol, ascorbic acid or a-keto acids, which are required in the a production process, is not required in the process of the present invention.

The culture temperature can be changed as appropriate within a range that allows the bacteria to grow and produce GPO, but in the case of Escherichia coli, it is preferably 2.
The temperature is about 0 to 42°C. The culture time varies depending on the conditions, but the culture should be finished at an appropriate time, usually at 12 to 48 hours.
Time is hidden.

The pH of the culture medium can be changed as appropriate within a range that allows the bacteria to grow and produce GPO, but is particularly preferably about pH 6 to 8.

If GPO in the culture is present in the culture solution, the culture solution containing the bacterial cells can be collected and used as is, but in general, the culture solution containing the bacterial cells can be collected by filtration, centrifugation, etc. according to conventional methods. GPO after separating GPO and microbial cells
fIJ fluid is used.

If GPO is present in the bacterial cells, collect the bacterial cells by filtering or centrifuging the resulting culture,
Next, this bacterial cell is destroyed by a mechanical method or an enzymatic method such as lysozyme, and if necessary, a medium rate agent such as EDTA and/or a surfactant is added to solubilize the GPO, and the GPO is separated and collected as an aqueous solution. .

The GPO-containing solution obtained by maintaining the pressure is subjected to, for example, vacuum concentration, membrane concentration, salting out treatment with ammonium sulfate, sodium sulfate, etc., or fractional precipitation with a hydrophilic organic solvent such as methanol, ethanol, acetone, etc. All you have to do is let it settle. This precipitate can then be dissolved in water and dialyzed through a semipermeable membrane to remove lower molecular weight impurities. In addition, it is purified by dull filtration using an adsorbent or gel filtration agent, adsorption chromatography, or ion exchange chromatography, and the GPO-containing solution obtained using these methods is purified by processing such as vacuum concentration and freeze-drying. You can get GPO.

The method for measuring the activity of the GPO thus obtained is as follows.

2O0mM PIPES-NaOH loose #[(1)H6
,5) 300mM L-Q-glycerophosphate 5 u/
rat'2 oxidase 1.5mM 4-aminoantipyrine 0.05% TritonX-10
0L OmM DAO8'' $13.5-zomethoxy-N-ethyl-N-(2-hydroxy-3-sulfofavirun-aniline sodium salt) The reaction gLO- having the above composition was taken into a test tube.

After preheating at 37°C for 5 minutes, 20μt of # raw layer was added and the reaction was carried out at 37°C for 5 minutes. After one reaction, α0 of 2.011t was added.
After the reaction is stopped by adding 5% SDS, colorimetric determination is performed at a wavelength of 600 nm. The activity that produced 1 μmote of hydrogen peroxide per minute was defined as 1 rate (U).

Calculation formula 1a8: Molecular absorption coefficient of quinone dye 1j! L (ex”/
itmoLe)X: Enzyme , nucleic acid-related substances, and others are based on the common abbreviations in those fields, and examples thereof are listed below. Furthermore, all amino acids are assumed to have 5 forms.

DNA: Deoxylime Nucleic Acid RNA: Lyse Nucleic Acid A: Adenine T: Thymine G Nigeanine C: Cytosine A 1 a: Alanine Arg: Arginine Asn: Asuno9 Ragin A! 1p: Asuno9 laginate Cys nigistein G ln: Glutamine Glu: Glutamate G1y Nigericin Hf S: Histidine I le: Inleucine Lau: Leucine Lys: Lysine M et: Methionine Phe: Phenylalanine Pro: F-L Ser: Serine Thr: Threonine Trp: Tryptophan Tyr: Tyrosine Vat: Valine [Example] The present invention will be described in detail below with reference to Examples, but the present invention is not limited by these in any way.

Example 1 [Isolation of chromosomal DNA] 5treptococcus Sp, G P OS
-53 chromosomal DNA was isolated by the following method. Transfer the same strain to 150 ml of plain heart infusion medium (
Difc.

After culturing with shaking overnight at 37° C. in BHI medium (manufactured by BHI Medium), the cells were collected by centrifugation (rotating at 3 ooo for 10 minutes). 10 chinatucarose, 50mM Tris-HCl (pH 8,0) 50
Suspend 5 dK in a solution containing mM EDTA, add 1-norizozyme solution (10 capes/ml), and incubate at 37 °C for 1 dK.
The mixture was incubated for 5 minutes and then 1 d of 10% 5DS (sodium dodecyl sulfate) solution was added. An equal volume of chloroform-phenol mixture (1:1) was added to this suspension, stirred and mixed, and centrifuged at 10,000 rpm for 3 minutes to separate into an aqueous layer and a solvent layer, and the aqueous layer was separated. Gently add 5 layers of 2 times the amount of ethanol to this aqueous layer, and slowly stir with a glass rod.
NA was separated by wrapping it with glass 41DC. This is 1
0-10mM Tris salt 1'f (pH 8,0), 1m
It was dissolved in a solution containing MEDTA (hereinafter abbreviated as Tg). After treating this with an equal volume of chloroform-phenol mixture, separate the aqueous layer by centrifugation, add twice the amount of ethanol, and separate another #' DNA using the above method.
Dissolved with TE.

Example 2 [Isolation of pACYC184 plasmid DNA] Escherichia coli pM harboring pAcYc184
191 (J, Bacteriol 134, 1141 (1
981) :ATCC37033) was cultured with shaking in 1z BHI medium (manufactured by Difco). Turbidity is ODsgo
When d increases to = 1.0, spectenomyce/(
(final concentration 300μr/*) was added, and further heated to 37°C for 16
Shaking was continued for more than an hour. Bacteria were collected by centrifugation at 3000 rpm for 10 minutes, and the cells were collected using the lysozyme-8DS method and the cesium chloride-ethyzomepromide method (Maniortis
et al.: Molecular Cloning pp 8
6-94 Co1d Spring Harbor (
Plasmid DNAy5! according to (1982) ). : Prepared.

Example 3 (Preparation of cilasmid pGPO81 having L-α-glycerophosphate oxidase gene) (1) 5treptococcus prepared in Example 1
sp. chromosomal DNA (approximately 0.5 /d ) and 100 times the volume of M buffer (maniortis et al.
Molecular CIoning+ I) pl 04
) 1 pts Hind I[I (Zen Shuzo 1.
Mix 1 μt of Qunit/μt) and 6 μt of water, 37
C. for 2 hours. Plasmid pACYC made by another KA
l 84 DNA approximately 0.3μ? Hi using a similar method
Cut with ndlll, and then add alkaline phosphatase (hereinafter abbreviated as BAP. Zenshuzo 1!!)
0.6 unit was added and treated at 65°C for 1 hour. Mix these two types of DNA solutions cut with Hind m, add the same amount of 3M sodium acetate, treat with a chloroform-7 enol mixture equal to the total amount, and separate the aqueous layer by centrifugation. did. 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 89 μt of water, 10 times the concentration of lidar stain/
Buffer (0.5M Trix hydrochloric acid (pH 7.6),
0.1M MgCl2, 0.1M dithiothreitol, 10mM spermidine, 10mM ATP)
10pl and 1μt of T4 DNA ligase (Zen Shuzo 350
unit) was added, mixed, and left overnight at 4°C. This DN
The AI solution was treated with chloroform-phenol, and the ethanol precipitate was collected and dried under reduced pressure, and then dissolved in 1 μt of TE.

(g) 100 m of BHI medium (Brain Hear
Escherichia coli DH1 strain in the logarithmic growth phase was cultured in 1" Yarnal Opmolecular Pyrozoan (J, Mol.

BioL, 557 (1983): Zoenetech Stock Center, provided by Yale University School of Medicine] was collected by centrifugation at 10,000 rpm, 2
40 d of ice-cold 30 mM potassium acetate, 1
00mM RbC2110mM CaCt! , 50
It was suspended in a solution containing mM MnCl2 and 15% glycerin (pH 5,8). After standing at 0°C for 5 minutes, centrifuge, discard the supernatant, and add 4Ilt 10mM M0Ps buffer (manufactured by Dotite), 75mM CaC4, 10mM
A solution containing RbC4 and 15% glycerin (pH
6, 5) and left at 0°C for 15 minutes to form competent cells.

(fil) To 200 μt of this E. coli suspension, 10 μt of the DNA solution prepared in (1) was added and left at θ° C. for 30 minutes. After adding BHI medium 1- and incubating at 37°C for 90 minutes, 100 μt of this was added to chloram 72 dicol (25 μf
/rat) on BHI agar plates and incubated at 37°C.
The cells were cultured overnight to obtain transformants. GP
O8 medium (composition is peptone 59, meat extract 2F, yeast extract 5F, NaC2I PXK2HPO41P,
MgSO40,5F,)♀-oxidase 500 IU
, cyanidisin 0.1F, potassium glycerophosphate 15
2. Agar 152, distilled water 11 SpH 7,0) was added to Zolate RG-V Delica and further cultured overnight at 37°C.

When about 5,000 colonies of transformants were examined, we obtained 4 strains with brown color around the colony, and one of these strains was named Escherichia coli DH1pGPO81 strain [Feikoken Joyori No. 2133, FERM BP-21
33). After pure isolation, this strain was cultured overnight at 37°C in BHI medium, and GPO productivity was examined by a GPO activity measurement method, and it was found to have a GPO activity of about 0.5 u/ml.

The plasmid DNA possessed by this strain was isolated as in Example 2, and pACYC18 containing the GPO gene was isolated.
The cilasmid containing the 4 genes was named pGPO81.

Example 4 Mapping of CpGPO81 and GPO
Determination of gene base sequence] pACY from Escherichia-Furi DH1pGPO81 strain
pGPOS 1 cilasmid DN in a similar manner to C184
A was prepared. Limit amount f for pGPO8I DNA
:BarnH1% BgLU, Cta I, Eco
Rr.

A cleavage map was created using Pst lX5al I and Xho I (all produced by Zenshuzo IB). The result is the third
Shown in the figure. The base sequence of the DNA containing the GPO gene was determined using the dideoxy method (Science
.

214.12O5-1210 (1981)]. The nucleotide sequence of the obtained GPO gene is shown in Figure 2 (1
) to (5), X=ATG and Y=TAA. Moreover, the amino acid sequence determined from the base sequence is A=Met in FIG. 1 (1) to (6).

B=OH.

Example 5 [Escherichia coli D H1pGPO81
Production of L-Q-glycerophosphate oxidase using Escherichia coli DH1pGPO81 strain (2°1f)
The cells were cultured in BHI medium (manufactured by Difco) at 37° C. for 18 hours using a shear 77 meter and collected by centrifugation at sOOOrpm for 10 minutes. After washing with 2 tons of physiological saline, 2 tons (7) 1
It was suspended in 0mM phosphate buffer (pH 7.0). Lysozyme 'tl'? /lnt, 2mM EDTA-2Na, and 0.1% Lydon x-ioo were added, incubated at 37°C for 30 minutes, and separated by centrifugation at 000 rpm for 10 minutes.

Ammonium sulfate salting out (40 to 66 t) was performed on 1.9 ml of this supernatant, and the precipitate was collected by centrifugation (5000 rpm, 30 minutes). This precipitate was dissolved in 2O0-10mM phosphate buffer (DH7.0, containing 1opM F'AD) and desalted with Sephadex G-25. After this,
Ion-exchange chromatography was performed using DEAE-Sepharose CL-6B, the active fraction was separated, desalted, and lyophilized as a powder. 27%).

The physicochemical properties of the 10-glycerophos-7ate oxidase thus obtained were as follows.

(1) Action: Catalyzes the following reactions.

L-α-glycerophosphate 10O2 → dihydroxyacetone phosphate + HzOz (2) Substrate 0 isomerism:
It has group 'X specificity for L-α-glycerophosphate.

(3) Optimum pH; this enzyme 11 (5, Ou/m ),
100mM glycine-hydrochloric acid buffer (pH 2, o~
S, O), acetate buffer (pH 4,0-6.0)3.
3-dimethylglutamic acid (DMG)-NaOH buffer (pH 5.0-7.0), phosphate buffer (p
H6,0~a,o), Tris-HCl buffer (p
After adjusting the tA with each buffer (H7.0 to 9.0) and reacting the substrate with the enzyme, the absorbance at 500 nm was measured according to the enzyme activity side method described above. 5
．． It was 5-7.5.

(4) pH stability: This enzyme solution (5u/ml)
100mM acetate buffer (pH 4,0-6.=O)
, 3゜3-dimethylglutamineg-NaOH buffer (pH 5.0-7.0), phosphate buffer (
pH6.0-8.0),? Lithium hydrochloric acid buffer 7-(+)
H7, O ~ 9.0) were adjusted with each buffer and treated at 50°C for 5 minutes, respectively, and was determined from the measured value of the original residual activity. As a result, the pH stability was 6.0 to 8.0.

<5>Km value; 5.5±0.5mM (for measurement of L-α-glycerol) (6) Isoelectric point; pH 4-0±0.5 (focal electrophoresis using Gearia amphorite) (7)
Thermostability: 0.1M of this enzyme solution (5u/me)
Adjusted with phosphate buffer (pH 6,5) and at each temperature ((
From the measured value of residual activity when treated for 5 minutes, 40
It showed 100% residual activity up to C.

(8) Enzyme stability over time; this enzyme solution (5u/g) was added to 0
．． 1 M MES-NaOH buffer (pH 6,5
) and treated at 37° C. for various elapsed time periods.The measured residual activity showed a residual activity of 90 or more after 24 hours had elapsed.

This characteristic showed the enzyme stability over time in a solution, which is required in actual clinical diagnosis, and the present enzyme was extremely useful.

〔Effect of the invention〕

By using the DNA and transformant of the present invention, L-α-chryserophosphate oxidase can be produced more efficiently without adding glycerol, ascorbic acid, α-keto acid, etc. Ta.

Furthermore, the present invention has revealed the GPO gene.

[Brief explanation of the drawing]

FIG. 1 (1) to (6) are diagrams showing the amino acid sequence of GPO expressed by the GPO gene of the present invention as a whole, and FIG.
It is a drawing showing the base sequence of the PO gene. FIG. 3 is a schematic diagram showing the structure of plasmid pGPOS1. In Figure 1 (1) to (6) or Figure 2 (1) to (5), A
represents an amino acid residue or a hydrogen atom, B represents an amino acid residue or a hydroxy group, Xf″1TAA, TA
Indicates a codon or 5' end group other than G and TGA;
Y represents a codon or a 3' terminal group. that's all

Claims (15)

[Claims] (1) DNA containing a base sequence encoding the amino acid sequence of a polypeptide constituting L-α-glycerophosphate oxidase. (2) D according to claim 1, wherein the L-α-glycerophosphate oxidase has the following physicochemical properties:
N.A. (a) Action: Catalyzes the following reactions. L-α-glycerophosphate + O_2 → dihydroxyacetone phosphate + H_2O_2 (b) Substrate specificity; has substrate specificity for L-α-glycerophosphate. (c) Optimum pH; 5.5-7.5 (d) pH stability; 6.0-8.0 (3) The amino acid sequence of the polypeptide constituting L-α-glycerophosphate oxidase is shown in Figures 1 (1) to (6) from the N-terminal side [In the figure, A indicates an amino acid residue or a hydrogen atom, B represents an amino acid residue or -OH]
The DNA according to claim 1, which is represented by: (4) The base sequence is shown in Figure 2 (1) to (5) from the 5' end.
) [In the figure, X represents a codon or 5' end group other than TAA, TAG and TGA, and Y represents a codon or 3' end group].
A. (5) A transformant, which is foreign to the host and retains DNA containing a base sequence encoding the amino acid sequence of a polypeptide constituting L-α-glycerophosphate oxidase. (6) The transformant according to claim 5, wherein the L-α-glycerophosphate oxidase has the following physicochemical properties. (a) Action: Catalyzes the following reactions. L-α-glycerophosphate + O_2 → dihydroxyacetone phosphate + H_2O_2 (b) Substrate specificity; Has substrate specificity for L-α-glycerophosphate. (c) Optimum pH; 5.5-7.5 (d) pH stability; 6.0-8.0 (7) The amino acid sequence of the polypeptide constituting L-α-glycerophosphate oxidase is shown in Figures 1 (1) to (6) from the N-terminal side [In the figure, A indicates an amino acid residue or a hydrogen atom, B represents an amino acid residue or -OH]
The transformant according to claim 5, which is represented by: (8) The base sequence is shown in Figure 2 (1) to (5) from the 5' end.
) [In the figure, X represents a codon or 5' end group other than TAA, TAG and TGA, and Y represents a codon or 3' end group]. (9) The transformant according to claim 5, wherein the transformant is a microorganism belonging to Escherichia coli. (10) The transformant is E. coli DH1.p
The transformant according to claim 5, which is GPOS1 strain [FERMBP-2133]. (11) Figure 1 (1) to (6) from the N-terminal side [A in the figure]
represents an amino acid residue or a hydrogen atom, B represents an amino acid residue or -OH] A polypeptide constituting L-α-glycerophosphate oxidase. (12) L-α-glycerophosphate oxidase having the following physicochemical properties. (a) Action: Catalyzes the following reactions. L-α-glycerophosphate + O_2 → dihydroxyacetone phosphate + H_2O_2 (b) Substrate specificity; has substrate specificity for L-α-glycerophosphate. (c) Optimum pH; 5.5-7.5 (d) pH stability; 6.0-8.0 (e) Km value; 5.5±0.5mM (using L-α-glycerophosphate as a substrate) (f) Isoelectric point; pH 4.0 ± 0.5 (g) Molecular weight; 195,000 ± 10,000 (by gel filtration method) (h) Thermal stability; ) Enzyme stability over time; more than 90% residual activity after 24 hours at 37°C in MES-NaOH buffer (pH 6.5) (13) Genetic information of the DNA is obtained by culturing a transformant containing DNA containing a base sequence encoding the amino acid sequence of a polypeptide constituting L-α-glycerophosphate oxidase that is foreign to the host. 1. A method for producing L-α-glycerophosphate oxidase, which comprises expressing L-α-glycerophosphate oxidase and collecting a polypeptide constituting L-α-glycerophosphate oxidase from the culture. (14) The production method according to claim 13, wherein the L-α-glycerophosphate oxidase has the following physical and chemical properties. (a) Action: Catalyzes the following reactions. L-α-glycerophosphate + O_2 → dihydroxyacetone phosphate + H_2O_2 (b) Substrate specificity; has substrate specificity for L-α-glycerophosphate. (c) Optimum pH; 5.5-7.5 (d) pH stability; 6.0-8.0 (15) The DNA containing the base sequence encoding the amino acid sequence of the polypeptide constituting L-α-glycerophosphate oxidase is L-α belonging to the genus Streptococcus.
-The method according to claim 13, wherein the DNA is derived from an α-glycerophosphate oxidase-producing bacterium. (16) Claim 1 wherein the host is a microorganism belonging to Escherichia coli.
3. The manufacturing method described in 3.