JPS62262994A - D-amino acid oxidase gene - Google Patents

Abstract

PURPOSE:To provide a DNA fragment originated from Trigonopsis variabilis, coding a specific amino acid sequence and carrying a gene of D-amino acid oxidase (DAO) capable of producing DAO on an industrial scale at a low cost. CONSTITUTION:Whole DNA is extracted from Trigonopsis variabilis (T-strain) and introduced into E.coli to obtain a DNA library. A DAO capable of oxidizing cephalosporin C is extracted from T-strain and a DNA probe estimated from the amino acid sequence of the obtained DAO is synthesized. The DNA probe is hybridized with the above DNA library and a plasmid is taken out of the obtained hybrid E.coli. The base sequence of the DNA originated from T-strain is determined from the plasmid. A DNA fragment carrying a DAO gene coding the DNA amino acid sequence of formula, etc., is selected from the above DNA base sequence.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention uses Trigonopsis variabilis (Trigon
A DNA fragment carrying the gene for D-amino acid oxidase derived from S. variabilis and capable of oxidizing cephalosporin C, and this DNA
The present invention relates to a novel Escherichia coli transformed with a fragment and having the ability to produce D-amino acid oxidase.

[Conventional technology]

D-amino acid oxidase is an enzyme that catalyzes the oxidative deamination reaction of D-amino acids. This enzyme is applied to the separation of L-amino acids from DL-amino acid racemic mixtures, the preparation of keto acids from D-amino acids, and the analysis of amino acids, and is known to have industrially useful properties. Among these, D-amino acid oxidase produced by the yeast Trigonopsis variabilis not only oxidizes D-amino acids, but also oxidizes cephalosporin C, a type of antibiotic, to 7-β-(5-carboxy-5 -Oxopentanamide) is attracting attention for producing cephalosporanic acid. This compound also produces H2 at the same time.
It is known to react with O2 to produce 7-β-(4-cargoxyptanamido)cephalosporanic acid.

These compounds provide intermediate raw materials for the synthesis of cephalosporin antibiotics, which are important as pharmaceuticals (for example, Japanese Patent Publication No. 50-7158).
A method for producing 7-aminocephalosporanic acid, a more useful intermediate raw material, by using 20□ or a certain type of cephalosporin acylase has been invented (for example, Japanese Patent Publication No. 17032/1983).

[Problems to be solved by the invention] However, in order for this enzyme to be industrially advantageously used for the above purpose, it must be easily and inexpensively produced and easily linked to other reactions. However, production of D-aminodrop oxidase by the yeast requires specific induction conditions, and it is difficult to artificially suppress the production of various other enzymes present. Therefore, in order to produce useful substances by linking with other reactions, complex reaction adjustments and
In some cases, separation and purification of the enzyme to a sufficient degree is required.

[Means and actions for solving the problems] The present inventors conducted extensive research to solve the above problems, isolated the DNA carrying the D-amino acid oxidase gene from Trigonopsis variabilis, and introduced it into Escherichia coli. succeeded in cloning. Furthermore, we have found that Escherichia coli into which a recombinant DNA obtained by making necessary modifications to this DNA fragment and incorporating it into a vector can significantly produce D-amino acid oxidase. Thus, the present inventors have demonstrated the ability to produce and apply this enzyme by cloning the D-amino acid oxidase gene to easily express it, or by modifying this gene into a form that can be easily introduced into other cells. Recognizing that this could be dramatically improved, we completed the present invention.

That is, according to the present invention, a DNA carrying a D-amino acid oxidase gene derived from Trigonopsis variabilis and encoding the amino acid sequence shown in Table 1
Fragments are provided.

Further, according to the present invention, a recombinant D in which the above DNA fragment is integrated into a vector used in an E. coli host vector system is obtained.
NA is provided.

Furthermore, the present invention provides a novel E. coli transformed with a recombinant DNA incorporating the above DNA fragment.

(Private T Ito) The DNA and recombinant microorganism according to the present invention can be constructed by approximately the following steps.

(1) iJ Gonodesis palabilis C'BS 409
5. Extract the total DNA from the plant, cut it with restriction enzymes, insert it into a vector, and introduce it into E. coli to create a DNA library. Trigonopsis variabilis CBS
4095 is a depository institution, Central B-Lo.
Foa Simelcultures (CentraalBu)
reau voor Schimmelculture
s), a yeast deposited in the Netherlands under deposit number 4095.

(2)) +7 Gonodesis palabilis CBS 409
D-amino acid oxidase having the ability to oxidize cephalosporin C was extracted and purified from 5, and the amino group-terminal amino acid sequence was determined.

(3) A mixture of oligonucleotides having the DNA base sequence of the gene deduced from a portion of the amino terminal amino acid sequence of D-amino acid oxidase obtained is synthesized, and this is called a DNA probe.

(4) Label the DNA probe with iP, and add the above (1)
The DNA library obtained in ) is subjected to colony hybridization, and E. coli colonies that show complementarity to the DNA probe are selected and isolated.

(5) Plasmid DNA is extracted from the selected E. coli strain, and the base sequence of the Trigonopsis variabilis CB34095-derived DNA incorporated into the vector is determined.

(6) Find an open reading frame for an amino acid sequence that correctly includes the amino terminal amino acid sequence of D-amino acid oxidase in the determined DNA base sequence, make appropriate modifications to the DNA fragment that includes this, and then construct the E. coli gene. Recombinant DNA for expression is incorporated into a vector for expression.
Create.

(7) Recombinant DNA for expression is introduced into host E. coli to create a new E. coli that produces D-amino acid oxidase.

General operations necessary for handling DNA in the above steps can be easily performed by those skilled in the art, such as molecular cloning (Molecular Cloning).
Cloning ) [: T. -r Maniatis et al., Coultospri 7 f
/% -/? -Ra? Latry (Co1d Sprin
g Harbor Laboratory (198
2) It can be easily carried out by following the experimental procedure manual. All of the enzymes and reagents used can be commercially available products, and unless otherwise specified, the purpose can be completely achieved if the usage conditions specified by the product are followed. In the above step (2), the purification of D-amino acid oxidase from Atribonobsis noguliabilis is known (for example, in the literature: Srw
(1985) Biotechnology Letters, 1-7), and the determination of the amino acid sequence of proteins is also known [for example, literature: Hunkapil et al.
(1983) Science
, 219.650-659] o The mixture of oligo9 nucleotides having the specified base sequence in step (3) above can be synthesized using a commercially available DNA synthesizer and following its operating procedures. The above process (5
) can also be determined by known methods [for example, literature: Sanger et al.
R) et al. (1977) Proe, Na.
ti, Acad, Sci, USA), 74
, 5463-5467].

When inserting the DNA fragment containing the target gene into the expression vector in step (6) above, it is necessary to delete the amino acid non-coding sequence derived from Trigonopsis variabilis CB54095, which can be an obstacle to gene expression in E. coli. There is. To delete such a specific DNA region, known methods of inducing deletion mutations using synthetic oligonucleotides (for example, literature: Zollar
ler) et al. (1983)
(Methoda Enzymol, ), 101
, 468-500). The primary expression vector may be any vector as long as it has a promoter or bosome binding site that functions in the host, but if cephalosporin C is used as a substrate, it contains a β-lactamase gene that degrades it. It is preferable to use pith or inactivated ones. The host used in step (7) above may be E. coli, which is commonly used in genetic manipulation, but since E. coli originally produces cephalosporinase that degrades cephalosporin C,
Preferably, a host lacking this gene is constructed and used. Furthermore, for simple detection of D-amino acid oxidase activity, known methods using 0-dianisidine [for example, literature: H@drick et al. (1968) Arch, Blochem , Bioph
ys, λ126, 155-1643 can be used.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to examples, but the present invention is not limited to the following examples.

〔Example〕

Example I Cloning and expression of the D-amino acid oxidase gene in Escherichia coli 1, Extraction and cleavage of total DNA from Atribonobsis .9 Riabilis Methods of Cryer et al.
12, 39-44. Academic Press, 197
5), Trigonopsis variabilis CB
Extract and purify total DNA from S4095. this DNA
Take 40μI of restriction enzyme Mb.

The mixture was reacted with 4 units of 37°C for 15 minutes. The reaction solution was extracted with a total equal volume of phenol/chloroform (1:1), and DN
After precipitating A with ethanol, 0.1x TE buffer (]OmM) Lis-salt n (p) 18.0'
).

1 mM EDTA]. Obtained DNA
The entire volume of the g solution was subjected to 0.7% agarose RLD electrophoresis, a portion containing DNA corresponding to a size of 6 to 9 kb was excised, and the DNA fragment was eluted from the gel by electroelution. The eluate was then mixed with equal volumes of phenol and phenol/chloroform. After extraction, ethanol was added to the resulting aqueous layer to precipitate the DNA, and 0.1
20μ of TE buffer at twice the temperature! Nitokashinen.

2. Cleavage of vector DNA', 0.1x TE buffer 20 μA&C
I combed it.

3. Inserting the DNA fragment into the vector The solution obtained in the above step] and the solution obtained in the above step 2 were mixed at a ratio of 3:1, and the T4/DNA IJ-ase was reacted at 15°C for 6 hours. vector and trigonopsis
A DNA fragment of S. variabilis CBS 4095 was ligated.

4. Creation of DNA library of Trigonopsis variabilis First, a se7 allosderinase-deficient mutant strain was constructed as an E. coli host. Namely, Escherichia coli (Each
eriehia aoli) MC1061 (University of Chicago 77L-:ll A Casadapa 7 (Malc
Obtained from Dr. om Ca5adaban). The properties of this bacterium are described in the Journal op Molekieler Piology (J.
Mo1. Biol, ), 138.179-207.19
80 -11111
) → Side dish →
→N-methyl-N4. ,-2) After treatment with I:1-N-nitronguanidine, colonies with increased sensitivity to Cephalos 4-phosphoC were selected. Among these, strains showing almost no cephalosporinase activity were selected.
Separate the special φ current strain and make the strain Escherichia co-IJ.
It was named MB65 and used as a host. Next, the recombinant DNA obtained in step 3 above was introduced into Escherichia coli MB65 by transformation, and 50 μg of ampicillin was added.
Collect colonies that have grown on L-pros agar medium containing C.
It was called the B54095 DNA library.

5. Determination of the amino terminal amino acid sequence of D-amino acid oxidase Tribonobusis variabilis CBS 4095 D-amino acid oxidase B'' was prepared according to the above-mentioned method of Szwajcer et al. The terminal amino acid sequence was analyzed by the method described above, and the amino acid sequence from the amino terminal to position 41 was determined as shown in Table 2.

Table 2 Ala-Lys-11e-Val-Val-11e-G
ly-Ala-Gly-Val-Ala-Gly-Le
u-Thr-Thr-Ala-La+u-Gln-Le
u-Leu-Ary-Lys-Gly-His-Glu
-Val-Thr-11e-Val-8er-Glu
-Ph @-Th r-Pro -G 1 y-As
p-L@u-8s r -I 1e-Gly-Tyr-
8:: The lower lip shows the sequence used for probe synthesis.

6. Possible NA on the gene deduced from the synthesized amino acid sequence of the DNA probe
An oligonucleotide mixture containing all the base sequences was synthesized as shown in Table 3. That is, possible oligonucleotides for each amino acid sequence are divided into two groups and synthesized to create a mixture, and these i DNA probe DAO-1
and DAO-2, and DAO-3 and DAO-4. All oligonucleotides were synthesized using an Applied Biosystem DNA synthesizer (5ynthesizer).
The test was carried out using Model 380-A f.

7. Selection and isolation of D-amino acid oxidase clone candidates from the DNA library. Using each of the DNA probes obtained in step 6 above, use the method of Inglia et al. [Reference: Nucleic Acids Res.
+9+1627-1642.1982] using T4 polynucleotide kinase and γ-P-ATP. Next, the E. coli obtained in step 4, which is the DNA library, was treated with ampicillin at 50 μm//d.
Grows as a colony on L-Pross agar medium containing D
I used AO-2 and high-resolution technology. The order Hyprididae is 6 times concentrated SSC (0.15M NaCt).
.

0, ol'5M Rizya sodium + pH 7-0), 0
．． 5 (4/Nidet P-40 (Sigma, USA), D
AO-1 or DAO-2 approx. 2 x 105 cpm/
+- for 1.5 hours at 44°C, then twice at room temperature with 6x SSC and then twice at room temperature with 6x SSC.
44 results using SC, DAO-1 or DAO-2
Many colonies were found to be positive for hybridasesin. Therefore, 40 strains were taken from the positive colonies, and after liquid culture, Birnboim
[Reference: Nucleitsu Acid Research (Nu
Cleic Acids Res, ), 7.1513-
Plasmid DNA was prepared using No. 1523.19791. Then, after denaturing these DNAs by a conventional method,
It was placed on a nitrocellulose filter and covered with a DNA probe for hybridization. Hypridise carbonate is 6 times concentrated 5SC, 110 times concentrated Denhardt solution (0.02% Ficoll, 0%
．． 02% polyvinylpyrrolidone, 0.02% bovine serum albumin) and a labeled DNA probe.
4℃ (for DAO-1 and DAO-2)'' or 4℃
1 hour at 0°C (for DAO-3 and DAO-4), then once at room temperature with 6x concentration of SSC, then at 44°C (for DAO-1 and DAO-2) or at 40°C. (For DAO-3 and DAO-4) After one wash, 6 strains were found that were positive for DAO-2 and also positive for DAO-3 or DAO-4. It was done. The plasmid DNA from these six strains was cut with various restriction enzymes, and after performing low-glue electrophoresis, the labeled DNA probe and SADEN hybridization were performed. ) Journal of Molecular Biology (J, Mo1. Biol,
).

98.503-517). As a result, Eeo
DAO is added to the approximately 0.6 kb DNA fragment generated by RI cleavage.
-2 and DAO-31 were found to be plasmid DNAs that were strongly positive for hybridization of FiDAO-4. This plasmid was named pDAOc2-12 and D-
It was selected as a candidate for amino acid oxidase clone.

8. Identification of D-amino acid oxidase clone and determination of DNA base sequence The DNA base sequence of the 0.6 kb DNA fragment generated from plasmid pDAOc2-12 by EcoRI digestion was determined according to the method of Sanger et al. . As a result, as shown in Table 4, the amino acid sequence at the amino terminal of the 7'hD-amino acid oxidase obtained in step 5 above was completed, sandwiching the intron-like sequences present in eukaryotic genes. A nucleotide sequence was found that encodes an amino acid sequence matching that of D-amino acid oxidase, and it was revealed that this fragment contains a part of the D-amino acid oxidase gene. Regarding plasmid pDAOc2-12, a restriction enzyme cleavage map shown in the WrJ1 diagram was created based on the results of restriction enzyme cleavage. Based on the above results, we determined the DNA base sequence of the region below ffl in the gene reading direction from the already determined base sequence, and found that it encodes a protein consisting of 355 amino acids shown in Table 1 above. It has been found that there is a base sequence (in Figure 2, the intron-like sequence shown in Table 4 has been deleted).

It was thus estimated that the structural gene for D-amino acid oxidase was completely contained in the DNA fragment derived from Trigonopsis variabilis CB84095 in plasmid pDAOc 2-12.

9. Modification of the D-amino acid oxidase gene The portion of the DNA base sequence that is an obstacle to the expression of the cloned D-amino acid oxidase gene in E. coli was added to Trigonopsis variabilis in the plasmid pDAOc2-12. The DNA fragment derived from CB84095 was deleted according to the steps shown in Figure 2. Each step is detailed below.

(1) Cut plasmid pDAOc2-1240Ill/ with EeoRI and Hindl to obtain approximately 0.4
After purifying the 5 kb fragment, its 0.8 μF K dA
Final concentrations of TP, dGTP, dcTP, and TTP were each 0.
．． 33mM, DNA polymerase Klenow (Kle
now) Add 5 units of I @ 10mM) Lithium salt Q& (pH 7,5), 10nIMMgCt2.1mM
Dithiothreitol, 50 mM NaCl reaction solution 3
30°C in 0 aZ.

The reaction was allowed to proceed for 20 minutes. A DNA fragment with blunt ends was purified by this, and approximately 0.2 μI of VCBam was added to the DNA fragment.
f(Z linker (0,01750,D) and T4DNA
Add 1 unit of ligase, 50mM) Lis-HCl (-7,
5), 10mM MgCl2.0.5mM ATP
, 20μ of 5mM dithiothreitol reaction solution! The reaction was carried out overnight at 15°C. After the reaction, purify the DNA fragment,
Cut both ends with Eco RI and BamHI, and EcoR
It was recovered as an I-BamHI fragment. On the other hand, Phage M]
After cutting the double-recorded DNA of 3mp18 with EeoRI and BimHI, the above F:coRI-BamHI fragment was added to it and ligated with T4 DNA IJ gauze.
Phage M13M2-12-7 incorporating a portion of the D-amino acid oxidase gene was constructed.

(2) In order to delete the intron-like sequence, as shown in Figure 6, oligonucleotides complementary to the sequence with W added before and after the intron-like sequence were synthesized, and DAOE
It was named 1. DAOEI 25 pmole and M13
10 pmole of primer Ml (manufactured by Zenshuzo Co., Ltd.) was phosphorylated with T4 polynucleotide kinase, and meshing (M
essing ) method [Reference 2: Methods Enzymol, 1
01+ 20-78+19833) Jl Yoshiku Furano M13M2-12-7 single stranded DNA approximately 0
．． 5 pmole was added, heated at 95° C. for 5 minutes, and then allowed to cool to room temperature. Then add T 4 DNA ligase (2 units) and 7 mM Tris-HCl (p
H7,5), Mgcz2. NaCt and ]4- dithiothreitol reaction solution 50μ! Inside 37℃, 30
Allowed to react for minutes. 0.5M PIT) TA5μ! After stopping the reaction, the meshing method (
According to Escherichia coli (ibid.)
Erichia 'doli) JM105 was subjected to transfection fractionation using the reaction solution, and the phage-introduced bacteria were extracted as plaques (4')i. Plaque hybridization method [Reference: Benton et al. (1977) Science (Sc.
i@nce), 196.180-1821 by P
Label L next DAOE]! :Hyperidization was performed and a positive plaque was found. Escherichia coli JM105 was infected again with the phages in the positive plaques, and plaques showing hybridization with DAOE 1 were purified and isolated in the same manner as above.After that, the phages present in the plaques were infected with the conventional method. liquid culture,
Be sure to separate the single-stranded DNA. Analysis of the base sequence of the single-stranded DNA obtained revealed that Trigonopsis variabilis C had a base sequence lacking an intron-like sequence as expected.
Since it was found that the phage contained a DNA fragment derived from BS 4095, this phage was named M13ME].

(3) Next, in order to delete the amino acid non-coding region derived from Trigonopsis variabilis CB54095, which is also upstream of the D-amino acid oxidase protein synthesis initiation site (ATG), this region was A polynucleotide complementary to the directly linked sequence was synthesized and named DAOE2. Then, from the single-stranded DNA of DAOE2 and phage M13ME], the above step (
Following the same steps as described in 2), construct a phage carrying a part of the D-amino acid oxidase gene in which in addition to the deletion of the intron-like sequence, the amino acid non-coding region upstream of ATG has also been deleted, This is M13M
It was named E12.

10. Construction of expression vector pAKKMl, which is the starting plasmid for construction of the expression vector used in this example, is derived from the well-known document Matsuda (Mats
(1985) Journal of Pacteriology (J, 13acterio1.), 163.122
2-1228 shows a method for its construction, and it has a special feature in which the β-lactamase gene is inactivated. As shown in Figure 3, pAKKMl was first treated with EcoRI and BamHI.
From this, a DNA fragment of approximately 5 kb was isolated and purified. On the other hand, the 95 bp plasmid containing the UV5 promoter was cut with EcoRI and BamHI by cutting the plasmid pRZ4022C (obtained from Dr. W. Reznikoff, University of Wisconsin, Dubry, USA) with EcoRI and BamHI.
fragments were prepared. Then, the above two fragments were converted into T 4 DNA.
Connect using IJ gauze and create the expression vector pEX.
002 was obtained. In addition, the plasmid pR24 used above
022 is the method of Gilbert (G11bsrt) et al. [Reference: Proc. Natl.
d, Sei, USA) + 7(L 3581-3
584, 1973].
A 95 bp ALu I fragment containing 1%-tar was isolated and E e o RI - B am HI of pBR322.
It is created by inserting one substitution at the site.

11. Expression of D-amino acid oxidase gene Figure 4r
The steps for constructing a recombinant for D-amino acid oxidase gene expression are shown. First, Phage M13ME] 2 of 2 'A
Approximately 0 is obtained by cutting DNA with EcoRI and BamHI.
．． I) Isolation of NA fragment encoding the amino terminal portion of 3 kb QD-amino acid oxidase. EcoRI and Sal I were then added to pDAOc 2-12.
By cleavage, a DNA fragment of approximately 1.2 kb encoding the structural gene portion of D-amino acid oxidase, excluding the portion contained in M13ME12, was isolated. Furthermore, after cutting the expression vector pKXOO2 with BamRI and XhoI, it was mixed with the above two fragments and T4DNA
The binding reaction was carried out with IJ gauze. The reaction at- was used to transform Escherichia coli MB 65, and colonies growing on an agar medium containing 40 μl/- of kanamycin were selected.

As a result of detecting the D-amino acid oxidase activity of the obtained colonies using D-methionine as a substrate and O-dianisidine (refer to the method reference above),
A large number of positive colonies were obtained. Plasmid DNA was extracted from one of these strains and named pEDAo], and the structure shown in FIG. 4 was confirmed by restriction enzyme digestion. In addition, Escherichia coli harboring this recombinant was transformed into Escherichia coli.
It was named JMB65/pEDAO1.

12. Oxidation of D-methionine and cephalosporin C by Escherichia coli carrying the recombinant Escherichia coli MB65/pEDAo 1 was inoculated into L-pros 100- containing 40 μy/- of kanamycin, and photographed at 37°C for 24 hours. It was cultured. The culture solution was collected by centrifugation and diluted with 7-100mM birophosphate buffer (
p) 18.0), disrupted the cells with an ultrasonic disruptor, centrifuged the cells, and used the resulting supernatant as an enzyme solution. Meanwhile, for comparison, Trigonopsis variabilis CBS 4
095, yeast extract 1 part, malt extract 1.5%, DL-
Inoculate the medium 100- containing 0.2% methionine,
The cells were photographed and cultured at 0''C for 48 hours. Bacteria were then collected in the same manner as above and treated with a cell disruptor (Braun, West Germany), and the cell disrupted supernatant was used as an enzyme solution. D-amino acid oxidase reaction The reaction was carried out at 37°C in a 31Rt reaction solution consisting of 27mM D-methionine, 80mM pyrophosphate buffer (pH 8°O), and enzyme solution, and an oxygen electrode [Yellow Springs Instruments (Yellow
Enzyme activity was quantified by measuring 0 □ consumption using a Spring Instruments Inc. (USA) model 513]. 1ttrn per minute of reaction
Define the enzyme activity that consumes 02 of ol* as 1 unit, and express the specific activity as the number of units of p per 1 da of protein in the enzyme solution. As a result, Escherichia coli MB65/pEDAO
The specific activity of 1 was 1.6. In contrast, the specific activity of Trigonopsis variabilis CBS 4095 i0.1
7 and 9.

Next, an enzyme solution of Escherichia coli MB65/pEDAO was allowed to act on cephalosporin C. The reaction was carried out using cephalosporin C22, 3mM, potassium phosphate buffer (
The reaction was carried out at 37° C. for 90 minutes in a reaction solution consisting of 100 mM (pH 7.0) and 200 μl of enzyme solution, and the reaction solution was subjected to high performance liquid column chromatography, and the product was purified. Column for high performance liquid force ram chromatogram is Coamosil 5C,
8 (manufactured by Gyudon Kagaku Co., Ltd.), a solution consisting of ammonium penta-thiacetate and 1.4% acetonitrile was used as the mobile phase, and detection was performed at 280 nm. As a result, cephalosporin C was not detected, and 7-β-(5-carboxy-5-oxobentaneado)cephalosporanic acid 1
2.5mM and 11.8mM of 7-β-(4-cal-xybutanamide)cephalosporanic acid were produced.

〔Effect of the invention〕

As detailed in the Examples, the present inventors cloned the D-amino acid oxidase gene that oxidizes cephalosporin C, and obtained a DNA fragment modified to a form that can be expressed in E. coli. This provided the prerequisites for the production of excellent D-amino acid oxidase and its widespread application. It is also possible to increase and regulate the enzyme protein production ability by linking an arbitrary promoter and a ribosome binding site in front of the protein synthesis initiation site of the above-mentioned DNA fragment by a known conventional method. In addition, the modified DNA fragments described above have a form that can be easily expressed in other cells such as yeast, Bacillus subtilis, etc., as long as there is an appropriate expression vector in addition to E. coli, and they can be easily expressed in cells such as yeast, Bacillus subtilis, etc. It provides an extremely powerful means for linking and beneficially utilizing the function of D-amino acid oxidase.

[Brief explanation of drawings]

FIG. 1 shows the restriction enzyme cleavage map of plasmid pDAOc 2-12. FIG. 2 shows an outline of the process for deleting the diamino acid non-coding region from a D-amino acid oxidase clone. FIG. 3 shows an outline of the process for constructing a vector for expressing D-amino acid oxidase. In the figure, symbols Crnr and Kmr. Aprti respectively chloramphenicol, kanamycin,
PlacUV5 indicates the tacUV5 promoter, and PlacUV5 indicates the ampicillin resistance gene. FIG. 4 shows an outline of the process for constructing a recombinant for D-amino acid oxidase gene expression. In the figure, the thick line indicates the amino acid sequence coding region of D-amino acid oxidase (indicated by the abbreviation DA'O). Patent applicant: Asahi Kasei Industries, Ltd. Figure 3 Figure 4

Claims (3)

[Claims] (1) A DNA fragment derived from Trigonopsis variabilis and carrying the gene for D-amino acid oxidase, which encodes the amino acid sequence represented by [Amino acid sequence exists]. (2) A recombinant DNA obtained by incorporating the DNA fragment according to claim 1 into a vector used in an E. coli host vector system. (3) Escherichia coli transformed with a recombinant DNA incorporating the DNA fragment according to claim 1.