JP2602435B2 - Cloned DNA of L-Glonolactone Oxidase, Genetically Modified Vector Incorporating the Cloned DNA, and Host Cell Transformed by the Vector - Google Patents

Description

The present invention relates to a cloned DN of L-gulolactone oxidase.
A, relating to a recombinant vector incorporating the cloned DNA and a host cell transformed with the vector,
It is used for mass production of L-gulolactone oxidase, and eventually ascorbic acid, that is, vitamin C.

(Prior Art) L-Gulonolactone oxidase is a flavin enzyme having a molecular weight of 51,000, which is localized in the microsomal fraction of liver and kidney of higher animals, and is an L-ascorbic acid (vitamin C) synthesis pathway in vivo. Is a substance that acts as a catalyst in the final stage of That is, this enzyme performs an oxidation reaction using L-gulonolactone as a substrate to synthesize L-xylo-hexulolactone, and this L-xylo-hexulolactone is subsequently isomerized to L-ascorbic acid. . This L-gulonolactone oxidase is a protein bound to a membrane, its purification is not easy, and its protein structure, gene and properties have not been clarified yet.

On the other hand, ascorbic acid is a vitamin having no side effects, and it has been found that inoculation thereof is important for maintaining health. Therefore, there is a great demand for ascorbic acid as an additive in foods and beverages. Further, ascorbic acid has a strong reducing power, and therefore is in high demand as an antioxidant.

Conventionally, in order to produce this ascorbic acid, glucose is converted into d-sorbitol by high-pressure hydrogenation, fermented into d-sorbose, protected with acetone and concentrated sulfuric acid to protect hydroxy groups, and then potassium permanganate. And then treated with alcoholic hydrochloric acid to simultaneously remove acetone and esterify, followed by treatment with sodium alcoholate.
In recent years, a method for producing ascorbic acid using so-called "biotechnology" has also been proposed (Japanese Patent Laid-Open No. 60-70).
073 publication), according to this method, from Corynebacterium
The gene of 2,5-diketo-D-gluconate reductase is taken out, this gene is integrated into a plasmid, this transgenic plasmid is taken up into Eruinia herbicola , and D-glucose is directly transferred to the resulting transformant. 2,5-
The intermediate is converted to diketogluconic acid, and the intermediate is cyclized in the presence of an acid or base catalyst to form ascorbic acid.

(Problems to be Solved by the Invention and Object of the Invention) Ascorbic acid is not expected to decrease in the future even if its usefulness is considered. It is very important to develop an advantageous process.

A general-purpose method using a saccharide such as glucose as a raw material uses a combination of a fermentation method and a synthesis method, and the number of synthesis steps is relatively large. On the other hand, JP
In order to produce ascorbic acid in accordance with the method disclosed in JP-A-70073, it is necessary to appropriately control the bacterium during production of 2,5-diketogluconic acid by the transformed bacterium. To obtain it, the produced 2,5-diketogluconic acid must be separated from the cells and then subjected to a cyclization reaction.

Therefore, the problem behind the present invention is disclosed in
This method utilizes biotechnology in the same manner as the method disclosed in Japanese Patent Publication No. -70073, but is to approach the method for producing ascorbic acid from a different viewpoint from the method described in the publication.

Therefore, the present inventors have focused on L-gulolactone oxidase. This is because if the cap is used and L-gulolactone oxidase is used, ascorbic acid is produced only by mixing with L-gulonolactone as a substrate under appropriate conditions. However, what should be noted here is that L-gulolactone oxidase is localized only in the liver and kidney of higher animals, as described above, is difficult to obtain in large quantities, and is a membrane protein. It is difficult to purify.

Accordingly, an essential object of the present invention is to open the way to produce high-purity L-gulonolactone oxidase in large quantities and relatively easily, and to enable mass production of ascorbic acid. Is to provide a cloned DNA of L-gulonolactone oxidase.

A second object of the present invention is to provide a recombinant vector into which a cloned DNA of L-gulolactone oxidase has been incorporated.

A third object of the present invention is to provide a host cell transformed with the above-mentioned gene recombinant vector.

(Means for Solving the Problems and Achieving the Object) According to the present invention, the first object is an amino acid sequence of rat L-gulolactone oxidase. Is achieved by the cloned DNA encoding

The second object is to use the formula (Where A, C, G and T are adenine,
Means the deoxyribonucleotide having cytosine, guanine and thymine bases, and the above formula is shown as a sequence for each codon corresponding to the amino acid) or the same as the amino acid sequence designated by the nucleotide sequence. This is achieved by a recombinant vector into which a cloned DNA having another nucleotide sequence specifying the amino acid sequence has been integrated.

The third object is achieved by a host Escherichia coli which has been transformed with the above-described genetic recombinant vector.

In addition, L-gulolactone oxidase having the above-mentioned specific amino acid sequence is basically obtained by a) purifying L-gulolactone oxidase derived from rat liver by a method known per se to obtain high purity things without and, using this a step of the purified enzyme obtained antiserum by immunizing a rabbit, b) a commercially available λgt11 phage cDNA expression library made from rat liver mRNA, Esch the phage
erichia coli Y1090 (r ^-) were infected cultured, to induce fusion protein by isopropyl-beta-D-thiogalactopyranoside, and the fusion protein using antisera described above was detected by enzyme immunoassay Screening to obtain a λ phage into which the cloned DNA encoding L-gulonolactone oxidase has been integrated, and culturing the λ phage; c) cleaving the λ phage with restriction enzymes Bcl I and Sph I; A step of adding Bgl II linkers to both ends of this DNA fragment to form an insert DNA; and d) cutting the plasmid vector with the restriction enzyme Bgl II, and joining the insert DNA to the break to perform gene recombination. Reconstructing the above plasmid as a vector; e) transforming the transgenic vector into Escherichia coli or animal cells; and f) transforming the resulting plasmid. A step of producing a cultured L- gulonolactone oxidase the recombinants, g) can be prepared by methods produced are L- gulonolactone oxidase has and a step of separating and purifying.

(Examples, etc.) Next, the present invention will be described in more detail with reference examples, production examples, and test examples.

Reference Example 1 (Determination of N-terminal amino acid sequence of L-gulonolactone oxidase) L-gulonolactone oxidase derived from rat liver was prepared by a method known per se [Nishikimi, M. et al., "Arch. Biochem. Biophyl.
175: 427-435 (1976)] to obtain high purity. The purified L-gulolactone oxidase (100 μg) was subjected to Edman degradation, and the amino acid sequence of the N-terminal portion was analyzed using a gas-phase protein sequencer (Model 470A manufactured by Applied Biosystems) as follows.

Val-His-Gly-Tyr-Lys-Gly-Val-Gln-Phe-Gln-
Asn-Trp-Ala-Lys-Thr-Tyr-Gly-Cys-Ser-Pro-
Glu-Val-Tyr-Tyr-Gln-Pro-Thr-Ser-Val-Glu-
Glu-Val-Arg Production Example 1 (Preparation of cloned DNA containing L-gulonolactone oxidase) a) Preparation of anti-L-gulonolactone oxidase antiserum High-purity L from rat obtained by the method described in Reference Example 1 -Antiserum was obtained by immunizing rabbits with glonolactone oxidase.

b) Screening with cDNA library Commercially available λgt11 prepared using rat liver mRNA
Phage cDNA Expression Library (Clontec Laboratories
L-Glonolactone oxidase clone was screened by the enzyme immunization method using the above-mentioned antiserum using the above-mentioned antiserum.

That is, the λgt11 phage was transformed into Escherichia coli Y1090 (r
^- ), Mixed with a soft agar medium, spread on an LB agar medium (containing 100 μg of ampicillin) and solidified. This was cultured at 42 ° C. for 3 hours to form a plaque, and a nitrocellulose filter impregnated with 10 mM isopropyl-β-D-thiogalactopyranoside (IPTG) solution was placed on the plaque. After culturing at 37 ° C for 2 hours, the fusion protein induced by IPTG was adsorbed to the filter. This filter was washed with TBST (50 mM Tris-HCl, pH7.
Wash gently with 9,150mM NaCl, 0.05% Tween-20), 20%
TBST containing fetal calf serum was allowed to act for 30 minutes.

This was allowed to act for 16 hours with TBST prepared by diluting the anti-L-gulolactone oxidase antiserum described in the above section a) 1000 times. Next, after the filter was sufficiently washed with TBST, TBST diluted 3000-fold with goat anti-rabbit IgG (manufactured by Bio-Rad) labeled with peroxidase derived from horseradish was allowed to act for 2 hours. TBST to Tween-2
After thoroughly washing the filter with the solution from which 0 has been removed,
Positive clones were detected by developing color using hydrogen peroxide and 4-chloro-1-naphthol as substrates.
The same screening was repeated for positive clones to purify them into single clones. The phage were subjected to liquid culture in an LB medium and then precipitated by polyethylene glycol [Maniatis, T. et al., "Molecular Cloning" A Laborator.
y Manual, Cold Spring Harbor Laboratory, New York (1
982)].

c) Preparation of cloned DNA The λ phage DNA obtained in b) above was subjected to restriction enzyme EcoR.
Cleavage with I yields the desired cloned DNA containing the base sequence encoding the amino acid sequence of L-gulolactone oxidase.

When the cloned DNA is cleaved with an appropriate restriction enzyme known per se, cloned DNA fragments having various chain lengths can be obtained, and these fragments can be used for various studies.

Production Example 2 (Preparation of Genetically Modified Vector) λgt1 into which cloned DNA encoding L-gulonolactone oxidase obtained in the above Production Example 1 section c) was incorporated.
After the phage is cleaved by the action of Bcl I and Sph I, which are restriction enzymes that do not cut the protein-coding region,
This DNA fragment was recovered by agarose gel electrophoresis. 67 mM potassium phosphate (pH 7.4), 6.7 mM MgCl ₂ , 1 mM 2 containing E. coli DNA polymerase (Klenow fragment) and 50 μM each of dNTPs (dATP, dCTP, dGTP, dTTP)
-The end of the fragment was made blunt by applying a mercaptoethanol solution to the above DNA fragment.

Next, a Bgl II linker was added to both ends of the DNA fragment using a DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.), and the Bgl II linker-added DNA was recovered by agarose gel electrophoresis.

On the other hand, plasmid pKSV-10 (manufactured by Pharmacia Japan Co., Ltd.) was digested with Bgl II.
II linker-added DNA was bound.

Escherichia coli was transformed with the obtained vector to transform Escherichia coli, ampicillin-resistant bacteria were selected from the transformants, and a plasmid was removed from the bacteria to obtain a desired genetically modified vector.

In this recombinant vector, SV40 of pKSV-10 was used.
DNA encoding L-gulolactone oxidase is arranged in the correct direction downstream of the promoter, and therefore, this vector is a vector capable of expressing L-gulonolactone oxidase.

Production Example 3 (Preparation of Transformed Host Cells) After mouse LKT ^- cells were cultured in Dulbecco's modified Eagle medium, 1.5 × 10 ⁶ cells were placed per 60 mm diameter petri dish.

On the other hand, 50 mM HEPES (pH 7.1), 280 mM NaCl and 15 mM NaCl
₂ 5 μg of plasmid pKSVGO in 1.25 ml of a solution consisting of HPO ₄
(Genetically recombinant vector finally obtained in Production Example 2), 2.5 μg of the thymidine kinase gene, 5 μg of a salmon sperm DNA solution (1.1 ml) and a 2M CaCl ₂ solution (0.15 ml) were mixed, and the mixture was stirred at room temperature for 30 minutes. The treatment liquid was prepared by leaving it to stand.

0.5 ml of the treated solution was added to the above-mentioned cell-containing petri dish, left at room temperature for 30 minutes, 5 ml of Dulbecco's modified Eagle's medium was added, and the cells were cultured for 5 hours under conditions of 5% CO ₂ and 37 ° C. After replacing Dulbecco's modified Eagle medium,
After culturing for 24 hours, the medium is replaced with a HAT medium containing hypoxanthine (15 μg / ml), aminopterin (1 μg / ml) and thymidine (5 μg / ml), and the culture is continued. 2
After 4 weeks, the cell colonies formed were separated to obtain the desired transformed host cells.

Reference Example 2 (Production of L-gulolactone oxidase) The transformed host cells obtained in Production Example 3 were grown by culturing in Dulbecco's modified Eagle medium, and the culture supernatant and the cultured cells were collected. The culture supernatant is directly subjected to column chromatography, and the cultured cells are suspended in 1% Triton X-100 solution and then subjected to column chromatography to separate and purify L-gulonolactone oxidase. did.

In this reference example, as described in reference example 1, L-gulolactone oxidase derived from rat liver was prepared as a raw material, and λ phage DNA described in section c) of production example 1 was treated with restriction enzyme EcoRI. Cloned gene DN of L-gulonolactone oxidase prepared by cleavage
Since A is integrated into the plasmid of the host cell, this host cell produces L-gulolactone oxidase, but the amino acid sequence is the same as that of rat liver, but the nucleotide sequence is different. If the cloned DNA of the synthesized L-gulolactone oxidase is integrated into the plasmid of the host cell, the host cell will produce a substance similar to this enzyme instead of L-gulonolactone oxidase derived from rat liver. .

Test Example 1 (Preparation of Restriction Enzyme Map) The cDNA insert (insert) fragment of λ phage (λgt11) cut with the restriction enzyme EcoRI described in section c) of Production Example 1 was fractionated by agarose gel electrophoresis. The corresponding portion of the gel was cut out and a DNA fragment was extracted therefrom. This D
The NA fragment and the plasmid pUC19 cut with EcoRI were ligated using a DNA ligation kit (Takara Shuzo). Using the obtained recombinant plasmid, in the same manner as in Production Example 3, except that Escherichia
and the cells of a coli JM109, to obtain a transformed host cell. This host cell is cultured, and the plasmid D is lysed by the alkali-SDS lysate method (the method of Maniatis, T., supra).
NA was prepared. Obtained plasmid DNA (insert DNA
Was cleaved with various restriction enzymes, and the length of the DNA fragment was examined by agarose gel electrophoresis to create a restriction enzyme map. It turned out to have.

Test Example 2 (Determination of Base Sequence of Plasmid DNA and Determination of Amino Acid Sequence of L-Gulonolactone Oxidase Part) Plasmid DNA mentioned in Test Example 1 was subjected to restriction enzyme EcoRI.
The fragment obtained by cleavage and subcloned into phage M13 mp18 by, Esch by the resulting recombinant vector
E. coli JM109 was transformed. The plasmid of the obtained transformant is digested with the restriction enzyme EcoRI, and among those having an insert DNA of about 1.3 kb, the RF DNA of the above phage is prepared, and a deletion kit for kilosequence (Takara Shuzo Co., Ltd.) Using a exonuclease III and a mung bean nuclease [Henikoff, S. "Gene" 28: 351-359 (1984)
Yanisch-Perron, C. et al., "Gene" Vol. 33, No. 103-11.
9 (1985)].

On the other hand, using various restriction enzymes, the EcoRI cut fragment of DNA is further cut into fragments having various region lengths,
Each of these fragments was subcloned into phage M13 mp18.

The above-mentioned deletion mutant and subclone
For the DNA fragments, the dideoxy-chain terminator method [Sanger, F. "Science" Vol. 214, No. 1205-1210]
(1981)] [Mizusawa, S. et al. “Nucleic Aci
ds Res., Vol. 14, pp. 1319-1324 (1986)]. The results are as shown in FIG. 1, in which the dotted arrows indicate The determination direction and the region of the base sequence for the DNA fragment of the translation mutant are shown, and the solid arrow indicates the determination direction and the region of the base sequence for the DNA fragment of the subclone.

The nucleotide sequence finally determined by joining the nucleotide sequences determined for each fragment was as shown in FIG. 2 and had a region length of 2120 bp. In this base sequence, the region from base number 4 to base 1320 is a region encoding L-gulonolactone oxidase, which starts from the start codon ATG and ends with the stop codon TAA within the open reading frame. It was present and had 439 amino acids. The amino acid sequence designated by the base sequence at the N-terminus of the cloned DNA (the underlined amino acid sequence in FIG. 2) is the N-terminal amino acid sequence of rat L-gulolactone oxidase (as analyzed in Reference Example 1). This cloned DNA nucleotide sequence was
-Glonolactone oxidase.

(Effects of the Invention) The present invention provides a cloned DNA of rat-derived L-gulonolactone oxidase, an expression vector into which the cloned DNA has been incorporated, and a host cell transformed with the vector.

Therefore, by culturing the transformed cells, L-gulolactone oxidase can be produced easily and in large quantities. Since the amino acid sequence was determined, rat L-
Various DNAs having the same amino acid sequence as glonolactone oxidase and different nucleotide sequences can be prepared.
Can be used to produce various substances having the same action as rat L-gulolactone oxidase.

Furthermore, ascorbic acid (vitamin C) is produced very easily when the produced L-gulolactone oxidase is allowed to act on L-gulonolactone. Therefore, the present invention greatly facilitates the production of ascorbic acid. To

[Brief description of the drawings]

FIG. 1 shows the DNA region cloned according to the present invention, including the nucleotide sequence encoding the amino acid sequence of rat L-glonolactone oxidase, and the restriction enzymes used for sequencing the region; FIG. 2 is a drawing showing a strategy for determining a nucleotide sequence, and FIG. 2 is a drawing showing a nucleotide sequence and an amino acid sequence constituting an open reading frame in the cloned DNA region shown in FIG.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical display location C12R 1:19) (C12N 5/10 C12R 1:91) (C12N 9/04 C12R 1:19) (C12N 9/04 C12R 1:91)

Claims (5)

(57) [Claims] 1. The amino acid sequence of rat L-gulolactone oxidase A cloned DNA, which encodes (2) (Where A, C, G and T are adenine,
Means the deoxyribonucleotide having cytosine, guanine and thymine bases, and the above formula is shown as a sequence for each codon corresponding to the amino acid) or the same as the amino acid sequence designated by the nucleotide sequence. 2. The cloned DNA according to claim 1, wherein the cloned DNA has another nucleotide sequence specifying an amino acid sequence. 3. The expression (Where A, C, G and T are adenine,
Means the deoxyribonucleotide having cytosine, guanine and thymine bases, and the above formula is shown as a sequence for each codon corresponding to the amino acid) or the same as the amino acid sequence designated by the nucleotide sequence. A genetically modified vector comprising a cloned DNA having another nucleotide sequence specifying an amino acid sequence. 4. The genetic recombination according to claim 3, wherein the vector is an expression vector that expresses a protein encoded by the DNA incorporated in the vector in a host cell. vector. (5) (Where A, C, G and T are adenine,
Means the deoxyribonucleotide having cytosine, guanine and thymine bases, and the above formula is shown as a sequence for each codon corresponding to the amino acid) or the same as the amino acid sequence designated by the nucleotide sequence. It is characterized by being Escherichia coli or an animal cell transformed by a recombinant vector into which a cloned DNA having another nucleotide sequence specifying the amino acid sequence has been integrated. Host cells.