JPH0739380A - Mutarotase gene, recombinant dna incorporated with the same and production of mutarotase - Google Patents

Abstract

PURPOSE:To provide a DNA bearing the genetic information of mutarotase, provide a recombinant DNA obtained by incorporating said DNA into a vector, and provide a method for producing mutarotase using a transformant obtained by transformation with this recombinant DNA. CONSTITUTION:The objective DNA coding amino acids derived from swine kidney. Besides, it has been confirmed that this gene is capable of producing MUT through incorporation into an appropriate manifestation system; thereby, mutarotase can be produced in great quantities at a low cost.

Description

Detailed Description of the Invention

[0001]

The present invention relates to mutarotase (hereinafter referred to as
DNA that carries the genetic information of MUT) and the DNA
DNA and recombinant DN in which vector is incorporated
The present invention relates to a method for producing MUT using a transformant transformed with A.

More specifically, the present invention provides a DNA carrying the genetic information of MUT derived from pig kidney and a method for producing the MUT by culturing transformed Escherichia coli by incorporating the DNA into a vector. Will enable the production of MUT.

The MUT produced in this way is used for application to the determination of glucose in blood. Recently, it has been found that it can be applied to the production of glucose (JP-A-3-139289).

[0004]

MUT has long been known as an enzyme that catalyzes the interconversion reaction between α-type and β-type of aldoses [Biochem. J. 4
Volume 5, p. 584 (1948)]. After that, microorganisms, plants, birds,
It is known to be widely distributed in mammals, and in particular, studies have been conducted on the properties of MUT contained in the kidneys, liver, and intestines of mammals such as cows, pigs, sheep, rabbits, and rats. .

In particular, the purification method and various properties of MUT derived from pig kidney have been clarified [J. Bioche (J. Bioche
m.) 91, 1889-1906 (1982)]. That is, the molecular weight is about 37,00
With the monomer of 0, the optimum pH is 6.5 to 7.5, the optimum temperature is 30 to 37 ° C,
The Km value for α-D-glucose is 19 mM, there are no modified amino acids such as sugar chains, and it is needle-shaped but crystallized.

[0006] However, since the MUT has been conventionally extracted from pig kidney and purified to be manufactured, it has a big problem in terms of supply and price.

The MUT gene derived from Acinetobacter calcoaceticus has been cloned and its primary structure has been reported [Nucleic Acids Research (Nucleic
Acids Res.) 14: 4309-4323 (1986)].

However, analysis of the primary structure of the pig kidney-derived MUT gene has not been reported.

[0009]

DISCLOSURE OF THE INVENTION As a result of intensive research to solve the above problems, the present inventors succeeded in purifying and isolating a DNA encoding a MUT derived from pig kidney and determining the base sequence thereof. did. Furthermore, it was confirmed that the gene can be incorporated into an appropriate expression system to produce MUT, and the present invention was completed.

[0010] Based on these results, we will open the way to efficient mass production of MUTs, and further develop MUs by protein engineering.
It also opens the way for alterations in T specificity.

[0011]

The present invention relates to a DNA encoding a MUT gene, and MUT can be produced by culturing various transformants having the gene incorporated therein.

That is, the present invention provides a DNA containing a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1. Such DNA may include various nucleotide sequences depending on the gene codon usage corresponding to each amino acid.

Those base sequences can be easily selected by those skilled in the art according to various elements of the gene expression system, for example, preferential codons depending on the type of host cell.

These genes can be isolated from each MUT-containing mammalian tissue by the method described in the following examples. Any mammalian tissue may be used as long as it contains MUT. For example, pig kidney can be used.

The present invention will be described in detail below with reference to examples, taking the case of MUT derived from pig kidney as an example.

In the case of MUTs derived from other tissues, the steps similar to those of the present invention can be carried out easily,
A variety of hosts, expression vectors, etc. used for transformation can be selected. Therefore, the present invention is not limited to the examples described below.

In the following examples, MUT activity was measured by the following method. MUT activity measurement method Enzyme activity is 147mM EDTA-2Na-NaOH buffer (pH7.4) 1.6m
l, color former (25 mg 4-aminoantipyrine, 150 mg phenol dissolved in water to a constant volume of 100 ml) 0.5 ml, glucose oxidase solution [2000 units / ml (100 mM EDTA · 2
Na-NaOH buffer (pH 7.4)] 0.5 ml, peroxidase solution [1000 units / ml (100 mM EDTA.2Na-NaOH buffer (pH 7.
4)] 0.5 ml, mixed with 3.15 ml of enzyme solution 0.05 ml 1
After holding at 0 ° C for 5 minutes, 0.2 ml of 0.5% α-D-glucose solution (prepared by adding 4 ml of water to 20 mg of α-D-glucose immediately before the reaction) was added to start the reaction.

The produced β-D-glucose is oxidized with glucose oxidase in the reaction mixture, and the hydrogen peroxide generated at that time is colored with peroxidase and a color-developing agent to increase the absorbance at 505 nm with time. taking measurement. From this change over time, the increase in absorbance per unit time was determined.
As a blind test, water is used instead of the enzyme solution.
Under the above reaction conditions, the amount of enzyme required to convert 1 μmol of α-glucose into β-glucose per minute is 1 unit.

[0019]

【Example】

Example 1 Sequence of the amino-terminal portion of MUT protein Mutarotase (derived from pig kidney: Amano Pharmaceutical Co., Ltd.) was purified by reverse phase HPLC and directly subjected to a gas phase sequencer (2 nmol was used), but no protein-derived sequence was found. could not.
Similar results were obtained for the pyridylethylated product (about 2 nmol), so it was presumed that the N-terminal amino acid of this enzyme was blocked.

Cleavage of MUT protein with protease
Determination of amino acid sequence of peptide Pyridylethylated mutarotase containing 4M urea 0.0
Dissolve in 1M Tris buffer (pH 9.0)
l) Lysyl endopeptidase was added and digested at 30 ° C. for 21 hours, and then the produced peptide was separated using a Asahipak C8P-50 column (manufactured by Asahi Kasei Kogyo) in a solvent system of 0.1% TFA-CH ₃ CN. The peptides are numbered LY-1 to LY-15 in the order of HPLC elution.

Sequence analysis was performed for each peptide. As a result, LY-8 was presumed to be a peptide containing an N-terminal sequence because it could not be analyzed by Edman reaction.

As a result of amino acid analysis, it was confirmed that LY-8 was composed of about 20 amino acids containing one arginine residue. Therefore, the peptide that does not contain arginine by the secondary decomposition with trypsin, that is, the peptide at the C-terminal side of LY-8 was determined according to the Edman method, and the peptide at the N-terminal side was analyzed by mass spectrometry to determine the amino acid sequence. (SEQ ID NO: 2)

The total primary structure of LY-9, LY-10, and LY-15 was determined as follows. (1) Secondary degradation of LY-9 with trypsin, preparation of peptide and sequencing LY-9 peptide fraction was concentrated to dryness and dissolved in 0.1M phosphate buffer (pH8.0) containing 2M urea, About 1/40 (mol / mol) trypsin was added and digested at 30 ° C for 1 hour, and then the resulting peptide was dissolved in 0.1 TFA-CH ₃ CN using a Finepak SIL3000 C ₁₃ -t7 column (manufactured by JASCO). They were separated in the system and their amino acid sequences were determined by a protein sequencer.
(SEQ ID NO: 3)

(2) Secondary Decomposition of LY-10 Peptide by V8 Protease and Preparation and Sequencing of Peptide The LY-10 peptide fraction was concentrated to dryness, and then 0.1 M ammonium hydrogen carbonate solution (pH 7.8 containing 2 M urea) was added. ), About 1/40 (m
ol / mol) V8 protease (derived from Staphylococcus aureus: manufactured by Beringer-Mannheim) was added and digested at 30 ° C. for 2 hours, and then the resulting peptide was separated in the same manner as in (1), and the protein sequencer was isolated. Its amino acid sequence was determined by. (SEQ ID NO: 4)

(3) Sequencing of LY-15 peptide Since it was thought that it consisted of about 80 amino acids from the result of amino acid analysis, it was digested with various proteases (chymotrypsin, endopeptidase, V8 protease) to give (1 ) And (2), the amino acid sequence was determined. (SEQ ID NO: 5)

Example 2 Preparation of total RNA From porcine kidney (extracted immediately after death, rapidly cut into about 2 cm square pieces, frozen in liquid nitrogen and stored at -80 ° C.), guanidinium / cesium chloride method [[Biochemistry, 13, 2633 (197
4)], [Science, 196, 1313 (197
7)], [Molecular Cloning
(Ning) (1982)]] Therefore, total RNA was extracted.

That is, about 2c stored at -80 ° C
A cube of m-squared pig kidney was crushed with a hammer while frozen, and then ground into powder using a mortar and pestle. 10 ml of D solution (4M guanidine thiocyanate, 25 mM sodium citrate, 0.1% mercaptoethanol). ) Was added to the Falcon tube and mixed quickly. After homogenizing with a polytron, 1 ml of 2M sodium acetate (pH 4.0), 10 ml of phenol (TE saturated), 2 ml of chloroform: isoamyl alcohol mixed solution (49: 1) were sequentially added and mixed, and shaken vigorously for 10 seconds. What was ice-cooled for 15 minutes was centrifuged (2500 xg, 40 minutes, 4 ° C). Transfer the aqueous layer to another Falcon tube, add an equal volume of phenol / chloroform (TE saturated) and mix and centrifuge (25
(00 × g, 40 minutes, 4 ° C.) (this operation was performed 4 times).

The aqueous layer was transferred to another Falcon tube, an equal amount of isopropanol was added and mixed, and the mixture was allowed to stand at -20 ° C for 60 minutes and then centrifuged (2500 xg, 40 ° C, 4 ° C). The supernatant was removed, the precipitate was dissolved in 500 μl of TE, 200 μl of lithium chloride was added, stirred, and then allowed to stand at −20 ° C. for 2 hours and centrifuged (10000
Xg, 15 minutes, 4 ° C). After removing the supernatant, add 400
It was dissolved in μl of TE, precipitated with ethanol, washed with 70% ethanol, and then dissolved in 51 μl of sterilized water to obtain total RNA. The finally obtained total RNA amount was about 1.03 mg.

Isolation of poly (A) ⁺ RNA Poly (A) ⁺ RNA was isolated from the total RNA obtained above using Oligotex-dt30 (Nippon Synthetic Rubber). That is, 100 μl of Oligotex-d was added to 400 μg of total RNA obtained above.
t30 was used. The procedure followed the attached protocol. As a result, 7.42 μg of poly (A) ⁺ RNA was recovered.

CDNA was synthesized from the poly (A) ⁺ RNA obtained in the cDNA synthesis and the library preparation using the cDNA synthesis system plus (Amersham). That is, Oligo dT was used as a primer for the synthesis of the first strand, and the RNA fragment generated by the RNase H treatment was used as a primer for the synthesis of the second strand. Next, λgt10 using adapter ligation to create a cDNA library
(Phosphatase treated with Eco RI) was used.
However, Gigapack Gold (stratagem
e) and an Eco RI- Not I- Bam HI adapter (Takara Shuzo) was used as the adapter.

It was confirmed that a 3 × 10 ⁵ pfu library was prepared by plating with NM514 as the indicator bacterium.

Screening with Oligonucleotide Probe A 38 mer mixed probe (MUT-2) shown below was prepared from the amino acid sequence partially determined in Example 1 and screened.

A A C C C C C C C CA -GA -TT -CA -ATI-AA -GGI-TT -GAT-CA -AA -TT -TG (I: Inosine) G G T T T T T T T

Λgt10 recombinant phage was plated using NM514 as a host. Next, place a nylon filter on the plate with plaque, leave it for a while, and filter paper soaked with alkaline denaturing solution (0.5M NaOH, 1.5M NaCl) and neutralizing solution (0.5M Tris-HCl pH8.0, 1.5M). NaCl)
The filters are sequentially transferred onto the filter paper soaked in and left for about 5 minutes each.

The filter was replaced with 2 × SSC (0.3M NaCl, 30
Immerse it in mM citric acid) to wash, remove excess water on a paper towel and remove with a transilluminator to about 3
Irradiate UV light for a minute to fix the DNA to the filter. Put the filter on which DNA is fixed in a hybridization pack (Cosmo Bio), seal the three sides, and add 5 ml of Rapid Hybridization solution (5 x Denhard's, 5 x SSPE, 0.1% SDS, 100 µg / ml denatured DNA) per 100 cm ² of nylon filter. Seal the entrance.

Incubate the sealed at 65 ° C. for 15 minutes or longer. To this is added a synthetic oligonucleotide solution (50 μCi, 10 μl) whose ends are labeled with γ ³² P, and the mixture is mixed so that the air and bubbles are eliminated and the solution inside is uniformly distributed.
This was incubated overnight at 52 ° C with shaking. Then incubate this incubated filter with 2 x SSC-0.1% SD
Washing was performed by shaking with S at 52 ° C. for 30 minutes (three times).

After the filter was dried, it was put on an X-ray film at -80.
It was exposed to light overnight at ℃ and developed to detect the signal. This was carried out on 7 square plates (about 1 × 10 ⁴ plaques were made per plate), and 5 positive plaques (M1, M2, M3, M4, M5) were obtained.

Then, the positive plaque portion was cut out together with the agar piece including the surrounding area, and SM (1 m) containing 1 drop of chloroform was cut out.
It was suspended in l) and stored at 4 ° C. When 1 μl of a 1000-fold dilution of this was diluted with SM and secondary screening was performed (approximately 1000 plaques were made per plate), all 5 types of plates showed positive results. Results were obtained. The detection with plaque lift and synthetic oligonucleotide probe was performed according to the primary screening.

[0039] One positive single plaque from the M3 plate and two from each of the M1, M2, M4 and M5 plates (M1-A, M1-B, M2-A, M2-B, M4- A, M4-B, M5-
A total of 9 pieces of A, M5-B) were picked up with a bamboo skewer, suspended in SM (0.5 ml) containing 20 μl of chloroform, and stored at 4 ° C. Separately, pick up each single plaque with a bamboo skewer, 0.5m
It was suspended in 1 liter of sterilized water and stored at -20 ° C to be used as a template for PCR.

Confirmation of Insert Fragment Regarding 9 positive clones, primers specific to both ends of the λgt10 vector arm (λgt10 Praimer (forward,
reverse): Takara Shuzo) was used to confirm the length of the insert fragment by PCR, and the insert fragments of M1-A, M1-B, M4-A and M4-B were determined to be 600 bp, but for the others, DNA amplification was performed. It was not seen and could not be confirmed. For PCR, 1 ng of template DNA was provided,
The following reaction cycle was repeated 25 times.

94 ° C 1 minute 55 ° C 2 minutes 72 ° C 3 minutes

For the plaques that could not be confirmed, DNA was prepared using the plate assay method. The inserted DNA was excised with Eco RI and Not I, but neither was digested. Then λgt
PCR was carried out with 10 primers (forward, reverse) and subjected to agarose electrophoresis.

As a result, in M2-A and M2-B, 1.5 kb, M3 and M
For 5-A, a band was obtained near 2.5 kb. From the above, M1-A and M1-B, M2-A and M2-B, M4-A and M4-B were considered to be the same DNA, respectively, and therefore they are not distinguished.

Subcloning of M2 into Bluescript II (SK +, KS +) The cleavage fragment obtained using Hae III was electrophoresed on low melting point agarose, cut out and extracted, and then treated with Sma I. Connected to Bluescript II SK +.
E. coli JM109 competent cells were transformed, plated on LB agar medium containing IPTG and X-gal, and cultured overnight at 37 ° C. Collect the resulting white translucent colonies,
2 ml of 2xYT was inoculated.

After culturing for 5 hours, replication type (RF) DNA was prepared by the alkaline lysis method and cleaved with an appropriate restriction enzyme to confirm the fragment length of the inserted DNA. For some clones, ssDNA was prepared from phage particles produced by infection with helper phage M13KO7. The Sanger method [Proc. Natl. Acad. Sci. USA, 74, 74, using a fluorescence-labeled universal primer (-21M13, manufactured by ABI)
5483-5467 (1977)] and DNA sequencer 370A (ABI
(Manufactured by the company) was used to analyze the base sequence.

As a result, a sequence homologous to the mutarotase partial sequence containing the probe sequence was found in one of the Hae III fragments of M2. Therefore, M2 was determined to be a clone encoding the mutarotase gene.

DNA amplified by PCR of M2 was treated with Eco RI, Not
Cleavage with I and Bam HI confirms the resulting fragment and produces a fragment that appears to contain the full length
Disconnects using the Eco RI, Bluescript I was Eco RI processing
It was ligated to I SK + and transformed into JM109 in the same manner as described above to prepare replication type (RF) DNA.

This was cleaved with several restriction enzymes having a cleavage site at the multi-cloning site to prepare a restriction enzyme map. (Shown in Figure 1)

Various deletion clones cleaved with the respective restriction enzymes were prepared, and the clones were appropriately treated with Bl.
After ligation to uescript II and transformation into JM109, the nucleotide sequence was analyzed. For the analysis, the Sanger method and DNA sequencer 370A, which were fluorescently labeled as described above, were used.

By these operations, the entire nucleotide sequence of M2 (SEQ ID NO: 6) was determined, and as a result it was confirmed that it consisted of 1434 bp. The determined nucleotide sequence is considered to include the entire open reading frame of mutarotase, and the amino acid sequence was deduced from this, and Example 1 was obtained.
It showed perfect agreement with the partial amino acid sequence obtained in.

Example 3 Construction of expression plasmid MUT revealed by protein sequencer
To the oligonucleotide corresponding to the N-terminal amino acid sequence of
A primer # 1 was prepared by modifying it to serve as an Nco I recognition site. Further, a DNA fragment considered to contain the MUT gene was amplified by the above-mentioned PCR method using the primers # 1 and # 2 shown below. Primer # 1 5'-TTC CCC ATG GTT TCA GTT ACA A
GA-3 '(24mer) primer # 2 λgt10 (forward) (described in Example 2)

After amplification, the reaction solution was confirmed by agarose gel electrophoresis to confirm the amplified DNA fragment, which was extracted and collected using the MERMAID Kit. As a result, a DNA fragment of about 1.4 kb was synthesized.

Here, the primer # 1 is the restriction enzyme NocI.
The primer # 2 contains the restriction enzyme Hind III sites, so the DNA fragment obtained was
By digesting with Nco I and Hind III, Nco I site and Hind III site can be introduced at both ends of the DNA fragment, respectively.

Restriction enzymes ( Nco I and Hi
cut with nd III), further expression vector (pTrc99A: Pharmacia) similarly cut and ligated with Nco I and Hind III, thereby obtaining a plasmid PTRM08. The ligation reaction was performed using a ligation kit (Takara Shuzo) according to the instructions attached to the kit.

After ligation, E. coli JM10 was transformed.
A known method using 9 competent cells, such as [Molecular Cloning (198
9)].

The transformant thus obtained was spread on an LB agar medium and cultured overnight to pick up a single colony, and a plasmid DNA (dsDNA) prepared by a known method, for example, an alkali-lysis method was used. It was confirmed that it was cloned by cutting with a restriction enzyme.

Cultivation of transformants and extraction of MUT One platinum loop was inoculated into 100 ml of LB medium (containing 1 mM IPTG), and cultured with shaking at 37 ° C. for 14.5 hours (OD ₅₉₀ = 3.
Five). Collect the collected cells in TS buffer [20 mM Tris buffer (pH
8), 30 mM NaCl] and then suspended in TS buffer. The suspended bacterial cells were treated with lysozyme and ultrasonically disrupted by a conventional method, and the supernatant obtained by centrifugation was used as a crude extract.

The MUT activity and protein amount (Bradford method) of the enzyme fraction outside the cells and inside the cells were measured for the recombinants cultured under the conditions of non-recombinant and expression inducer (IPTG). Table 1 shows the respective results in 1 ml of the culture solution.

[0059]

[Table 1]

As a result, the E. coli wild strain (JM109) had M
No UT activity is observed. In the recombinant, M
UT was induced by 1 mM IPTG approximately 8-fold. The specific activity of MUT derived from pig kidney was 584 units / mg.

Furthermore, the amount of MUT expressed by the recombinant was about 22% of the total protein mass of the intracellular enzyme fraction.

Confirmation of MUT from transformant When the whole protein of E. coli, which is a transformant, was developed by SDS-PAGE, the same molecular weight as that of the purified enzyme derived from pig kidney (about
It was confirmed that the protein showing 37000) was expressed in large amounts. When this was transferred to a PVDF membrane and the amino acid sequence was analyzed by a protein sequencer, the same sequence as the MUT derived from pig kidney was found. However, about 30% of the proteins had a methionine corresponding to the start codon at the N-terminus.

[0063]

INDUSTRIAL APPLICABILITY According to the present invention, a method for inexpensively producing a large amount of mutarotase has been established.

[0064]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 341 Sequence type: Amino acid sequence Val Ser Val Thr Arg Ser Val Phe Gly Asp Leu Pro Ser Gly Ala 15 Gly Thr Val Glu Lys Phe Gln Leu Gln Ser Asp Gln Leu Arg Val 30 Asp Ile Ile Ser Trp Gly Cys Thr Ile Thr Ala Leu Glu Val Lys 45 Asp Arg Gln Gly Arg Ala Ser Asp Val Val Leu Gly Phe Ala Glu 60 Leu Lys Glu Tyr Leu Gln Lys His Pro Tyr Phe Gly Ala Val Val 75 Gly Arg Val Ala Asn Arg Ile Ala Lys Gly Thr Phe Thr Leu Asp 90 Gly Lys Glu Tyr Lys Leu Ala Ile Asn Asn Gly Pro Asn Ser Leu 105 His Gly Gly Val Arg Gly Phe Asp Lys Val Leu Trp Thr Pro Arg 120 Val Leu Ser Asn Gly Ile Glu Phe Ser Arg Val Ser Pro Asp Gly 135 Glu Glu Gly Tyr Pro Gly Glu Leu Lys Val Trp Val Thr Tyr Thr 150 Leu Asp Gly Gly Glu Leu Val Val Asn Tyr Arg Ala Gln Ala Ser 165 Gln Thr Thr Pro Val Asn Leu Thr Asn His Ser Tyr Phe Asn Leu 180 Ala Gly Gln Gly Ser Pro Asn Ile Tyr Asp His Glu Val Thr Ile 195 Glu Ala Asp Ala Phe Leu Pro Val Asp Glu Thr Leu Ile Pro Thr 210 Gly Glu Ile Ala Pro Val Gln Gly Thr Ala Phe Asp Leu Arg Lys 225 Pro Val Glu Leu Gly Lys His Leu Gln Glu Phe His Ile Asn Gly 240 Phe Asp His Asn Phe Cys Leu Lys Arg Ser Lys Glu Lys Gln Phe 255 Cys Ala Arg Val His His Ala Gly Ser Gly Arg Val Leu Glu Val 270 Tyr Thr Thr Gln Pro Gly Ile Gln Phe Tyr Thr Gly Asn Phe Leu 285 Asp Gly Thr Leu Lys Gly Lys Thr Gly Ala Val Tyr Pro Lys His 300 Ser Gly Phe Cys Leu Glu Thr Gln Asn Trp Pro Asn Ala Val Asn 315 Gln Pro His Phe Pro Pro Val Leu Leu Lys Pro Gly Glu Glu Tyr 330 Asn His Thr Thr Trp Phe Val Phe Ser Val Ala 341

SEQ ID NO: 2 Sequence length: 20 Sequence type: Amino acid sequence Val Ser Val Thr Arg Ser Val Phe Gly Asp Leu Pro Ser Gly Ala 15 Gly Thr Val Glu Lys 20

SEQ ID NO: 3 Sequence length: 30 Sequence type: Amino acid sequence Val Leu Trp Thr Pro Arg Val Leu Ser Asn Gly Ile Glu Phe Ser 15 Arg Val Ser Pro Asp Gly Glu Glu Gly Tyr Pro Gly Glu Leu Lys 30

SEQ ID NO: 4 Sequence length: 37 Sequence type: Amino acid sequence Gln Phe Cys Ala Arg Val His His Ala Gly Ser Gly Arg Val Leu 15 Glu Val Tyr Thr Thr Gln Pro Gly Ile Gln Phe Tyr Thr Gly Asn 30 Phe Leu Asp Gly Thr Leu Lys 37

SEQ ID NO: 5 Sequence length: 86 Sequence type: Amino acid sequence Val Trp Val Thr Tyr Thr Leu Asp Gly Gly Glu Leu Val Val Asn 15 Tyr Arg Ala Gln Ala Ser Gln Thr Thr Pro Val Asn Leu Thr Asn 30 His Ser Tyr Phe Asn Leu Ala Gly Gln Gly Ser Pro Asn Ile Tyr 45 Asp His Glu Val Thr Ile Glu Ala Asp Ala Phe Leu Pro Val Asp 60 Glu Thr Leu Ile Pro Thr Gly Glu Ile Ala Pro Val Gln Gly Thr 75 Ala Phe Asp Leu Arg Lys Val Glu Leu Gly Lys 86

SEQ ID NO: 6 Sequence length: 1434 Sequence type: Nucleic acid Sequence GGCGGGAGTT CGAGGGACCT GGAGCAACTA CACACCCCAA GCTTTCCTAA TG 52 GTT TCA GTT ACA AGA TCC GTG TTT GGA GAC CTC CCC TCG GGG GCA 97 Val Ser Val Thr Arg Ser Val Phe Gly Asp Leu Pro Ser Gly Ala 15 GGG ACG GTG GAA AAG TTC CAG CTG CAA TCA GAC CAG CTG AGA GTG 142 Gly Thr Val Glu Lys Phe Gln Leu Gln Ser Asp Gln Leu Arg Val 30 GAC ATC ATC TCC TGG GGC TGC ACC ATC ACG GCC CTG GAG GTC AAA 187 Asp Ile Ile Ser Trp Gly Cys Thr Ile Thr Ala Leu Glu Val Lys 45 GAC AGG CAG GGC AGA GCC TCA GAC GTG GTG CTT GGC TTT GCT GAA 232 Asp Arg Gln Gly Arg Ala Ser Asp Val Val Leu Gly Phe Ala Glu 60 TTG AAA GAG TAC CTC CAA AAA CAT CCC TAC TTT GGA GCA GTG GTT 277 Leu Lys Glu Tyr Leu Gln Lys His Pro Tyr Phe Gly Ala Val Val 75 GGC AGA GTG GCA AAC CGA ATT GCC AAA GGA ACA TTC ACA TTG GAT 322 Gly Arg Val Ala Asn Arg Ile Ala Lys Gly Thr Phe Thr Leu Asp 90 GGG AAG GAG TAT AAG CTG GCC ATC AAC AAC GGG CCC AAC AGC CTT 367 Gly Lys Glu Tyr L ys Leu Ala Ile Asn Asn Gly Pro Asn Ser Leu 105 CAT GGA GGA GTC AGA GGA TTT GAT AAG GTG CTC TGG ACC CCT CGG 412 His Gly Gly Val Arg Gly Phe Asp Lys Val Leu Trp Thr Pro Arg 120 GTC CTG TCA AAT GGC ATC GAG TTC TCG AGG GTC AGT CCA GAT GGT 457 Val Leu Ser Asn Gly Ile Glu Phe Ser Arg Val Ser Pro Asp Gly 135 GAG GAA GGC TAC CCT GGA GAG TTA AAA GTC TGG GTG ACA TAC ACG 502 Glu Glu Gly Tyr Pro Gly Glu Leu Lys Val Trp Val Thr Tyr Thr 150 CTG GAT GGT GGG GAG CTC GTG GTC AAC TAT CGA GCA CAG GCC AGT 547 Leu Asp Gly Gly Glu Leu Val Val Asn Tyr Arg Ala Gln Ala Ser 165 CAG ACC ACC CCA GTC AAT CTG ACC AAC CAC TCT TAT TTC AAC CTG 592 Gln Thr Thr Pro Val Asn Leu Thr Asn His Ser Tyr Phe Asn Leu 180 GCG GGC CAG GGT TCC CCA AAT ATA TAT GAC CAT GAA GTC ACT ATA 637 Ala Gly Gln Gly Ser Pro Asn Ile Tyr Asp His Glu Val Thr Ile 195 GAA GCT GAT GCT TTT TTG CCT GTG GAT GAA ACC CTA ATC CCT ACA 682 Glu Ala Asp Ala Phe Leu Pro Val Asp Glu Thr Leu Ile Pro Thr 210 GGA GAA ATT GCT CCA GTG CAA GGA ACT GCA TTT GAT CTG AGG A AG 727 Gly Glu Ile Ala Pro Val Gln Gly Thr Ala Phe Asp Leu Arg Lys 225 CCA GTG GAG CTT GGA AAA CAC CTG CAG GAG TTC CAC ATC AAT GGC 772 Pro Val Glu Leu Gly Lys His Leu Gln Glu Phe His Ile Asn Gly 240 TTT GAC CAC AAT TTC TGT CTG AAG AGA TCT AAA GAA AAG CAA TTT 817 Phe Asp His Asn Phe Cys Leu Lys Arg Ser Lys Glu Lys Gln Phe 255 TGT GCA CGG GTC CAT CAT GCT GGA AGC GGG AGG GTC CTG GAA GTG 862 Cys Ala Arg Val His His Ala Gly Ser Gly Arg Val Leu Glu Val 270 TAC ACT ACC CAG CCT GGG ATC CAG TTT TAC ACG GGC AAC TTC CTG 907 Tyr Thr Thr Gln Pro Gly Ile Gln Phe Tyr Thr Gly Asn Phe Leu 285 GAT GGC ACG CTG AAG GGC AAA ACT GGA GCA GTC TAT CCC AAG CAC 952 Asp Gly Thr Leu Lys Gly Lys Thr Gly Ala Val Tyr Pro Lys His 300 TCT GGT TTC TGC CTT GAG ACC CAG AAC TGG CCT AAT GCA GTC AAT 997 Ser Gly Phe Cys Leu Glu Thr Gln Asn Trp Pro Asn Ala Val Asn 315 CAG CCC CAC TTC CCT CCT GTG CTG CTG AAG CCT GGT GAG GAG TAC 1042 Gln Pro His Phe Pro Pro Val Leu Leu Lys Pro Gly Glu Glu Tyr 330 AAC CAC ACC ACT TGG TTT GTG TTTTCT GTG GCT TAAGG AAGTGTTAAG 1090 Asn His Thr Thr Trp Phe Val Phe Ser Val Ala 341 TTATGACCTG TTTCAGGGCC AGCTGGGAGC CCCTTCAGGA ACCTGTCTCC TGTGCAGAGA 1150 TAAGATGAAG ATTTAGAAGC TTTAAAAGTG ATCCTGTGAA TTAAAATCAC ACATATGGTA 1210 GTTGTCATGA TAATCTGAAT TTCAATTTCT TTCCCAATGA CTGACTCCAG GCCAGGTCTA 1270 ATGGTCAGCT CTATTCTCTG TGTGGTGAAG ACCCAACCAG GAATATCATC ATCTAAGCCC 1330 TGACCCTAAT CCAGAAGTGG TATCCAGATC CTTGTGTTGG CTCTATCTCT CCACTCTGCT 1390 TCTTTTCACC CCTTTTTTCT TTGATTCTAC TCATTCCTTC TTTT 1434

[Brief description of drawings]

FIG. 1 shows a restriction map of M2 cDNA.

Claims (7)

[Claims] 1. A DNA carrying the genetic information of mutarotase. 2. A DNA having the restriction enzyme map shown in FIG. 1 and carrying the genetic information of mutarotase. 3. A DNA containing a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1. 4. The mutarotase is derived from pig kidney.
To the DNA according to claim 3. 5. A recombinant DNA in which a DNA carrying the genetic information of mutarotase is incorporated into a vector. 6. A transformant having the restriction enzyme map shown in FIG. 1 and introduced with recombinant DNA in which a DNA carrying the genetic information of mutarotase is incorporated into a vector. 7. A transformant is obtained by introducing into a host recombinant DNA in which a DNA carrying the genetic information of mutarotase derived from pig kidney is introduced into a host, culturing the transformant, and adding mutarotase to the culture. A method for producing mutarotase, which comprises producing the mutarotase and collecting the mutarotase from the culture.