JPS6374488A - Production of 7-aminocephalosporanic acid and derivative thereof - Google Patents

Abstract

PURPOSE:To obtain 7-aminocephalosporanic acid from cephalosporin C in one step, by reacting cephalosporin C with a specific enzyme mixture in the presence or absence of hydrogen peroxide in an aqueous medium. CONSTITUTION:The compound of formula I (R is -OCOCH3, -H or -OH) or its salt is deacylated to obtain the compound of formula II (R is same as defined above) or its salt (the compounds of formula I and formula II wherein R is -OCOCH3 are cephalosporin C and 7-aminocephalosporanic acid, respectively). The above deacylation is carried out as follows. The compound of formula I is made to react with an enzyme mixture containing 7beta-(4-carboxybutanamido) cephalosporanic acid acylase and D-amino acid oxidase produced by E.coli using a recombinant DNA technique. The reaction is carried out in an aqueous medium in the presence or absence of hydrogen peroxide.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to 7-aminocephalosporanic acid (hereinafter referred to as 7-A
CA) and its derivatives by enzymatic means.

[Conventional technology]

Cephalosporin C obtained through fermentation production is induced into 7-ACA by a reaction that removes the acyl group bonded to the 7-position amino group (hereinafter referred to as deacylation), and using this as a starting material, various cephalosporins are produced. Antibiotics have been synthesized and are now in practical use as pharmaceuticals.

Methods for deacylating cephalosporin C include chemical methods and enzymatic methods.
The method of No. 889) is known to have many industrial disadvantages, such as requiring many reaction steps, high cost of reagents used in the reaction, and large amounts of compounds being discharged as by-products of the reaction.

As an enzymatic deacylation method, glyoxylic acid is applied to the D-5-amino group of the 7β-(D-5-amino-5-carboxypentanamide) group in the 7-position side chain of cephalosporin C to chemically deacylate it. Special Publication No. 55-129 for carrying out amino reactions
10 method, or the method of Japanese Patent Publication No. 7158/1983, which involves the action of microbial enzymes to carry out the deamination reaction.
Cephalosporin C is derivatized to 7β-(5-carboxy-oxopentanamide) cephalosporanic acid.

Then, a decarboxylation reaction is carried out by the action of hydrogen peroxide to induce 7β-(4-carboxybutanamide)cephalosporanic acid (commonly known as glutarylamide cephalosporanic acid, hereinafter abbreviated as glutaryl 7-ACA). Next, Pseudomonas spp.
) Enzymes produced by bacteria (Reference: Shibuya
) et al. (1981) Agricultural Biological Chemistry
Chemistry) 45, 1561-1567)
A deacylation reaction is carried out by reacting with 7
-There is a way to induce people to ACA. Although this method can solve many of the problems of chemical deacylation, since it is a three-step reaction, there has been a desire to develop a more superior enzymatic one-step reaction method.

As a method for obtaining 7-ACA by directly deacylating cephalosporin C, Achromobacter spp.
obacter), Brevibacterium (Brevi
bacterium ) or the method of U.S. Patent No. 3239394 (1966) using bacteria of the genus Flavobacterium;
JP-A No. 5, using fungi of the genus Aspergillus or the genus Alternaria.
2-143289, the method of JP-A-53-94093 using bacteria of the genus Pseudomonas,
Japanese Patent Application Laid-open No. 59-44392 using fungi of
and the method of JP-A-61-21097 using bacteria belonging to a new species of the genus Pseudomonas. Furthermore, there is a method disclosed in JP-A-61-152286 which uses an E. coli transformant containing the cephalosporin C acylase gene cloned from a bacterium of the genus Pseudomonas.

[Problems to be solved by the invention] In general, in order to industrially utilize an enzyme reaction using a microbial culture, its processed product, or an extract, the preparation thereof must be easy and inexpensive. , or have excellent properties such as these. However, there is still no known example of success in directly deacylating cephalosporin C industrially using the above-mentioned conventional method.

[Means and effects for solving the problems] In order to solve the above problems, the present inventors have conducted research on enzymatically producing 7-ACA from cephalosporin C in one step. . During this process, Comamonas spp.
nas) The gene for 7β-(4-carboxybutanamide)cephalosporanic acid acylase (hereinafter referred to as glutaryl 7-ACA acylase), which has the ability to hydrolyze glutaryl 7-ACA, was isolated from bacteria and introduced into Escherichia coli,
Succeeded in producing this enzyme in significant quantities (Japanese Patent Application Laid-open No.
-110292). Furthermore, they isolated the D-amino acid oxidase gene, which has the ability to oxidize cephalosporin C, from yeast of the genus Trigonopsis and introduced it into Escherichia coli, successfully producing a significant amount of this enzyme (
Patent application Sho 6l-106663). Furthermore, as a result of intensive research by the present inventors, we found that a transformant of Escherichia coli containing the above D-amino acid oxidase gene and glutaryl 7-ACA
It was surprisingly found that when a culture of an E. coli transformant containing an acylase gene was simultaneously treated with cephalosporin C, 7-ACA was produced in a single step. Based on these findings, we have completed the present invention.

That is, according to the present invention, a compound represented by general formula (I) (in which R is 100OCR, -H or -○H) or a salt thereof is deacylated to obtain general formula (n) ( In the method for producing 7-ACA and its derivatives, which produces a compound represented by the formula (R has the same meaning as above) or a salt thereof, the compound represented by the formula (1) is produced in Escherichia coli by recombinant DNA technology. 7-ACA, characterized in that an enzyme mixture containing a diluted D-amino acid oxidase and glutaryl 7-ACA acylase is allowed to act in an aqueous medium in the presence or absence of hydrogen peroxide.
and a method for producing a derivative thereof.

The method of the present invention comprises a compound represented by general formula (I) or a salt thereof, and D-amino acid oxidase and glutaryl 7-A produced in Escherichia coli by recombinant DNA technology.
An enzyme mixture containing CA acylase is mixed in an aqueous medium, and a compound represented by the general formula (n) or a salt thereof is produced by the action of the enzyme of Niku et al.

Regarding the compound represented by general formula (I') or its salt used as a starting material in the method of the present invention and the compound represented by general formula (If) or its salt which is the final product obtained by the method of the present invention, general Formula % Formula % are cephalosporin C and ? respectively. - Indicates ACA. In the present invention, examples of the salts of the compounds represented by formulas (1) and (n) include sodium salts and potassium salts.

The enzyme mixture used in the present invention includes a culture of E. coli transformed with recombinant DNA containing the D-amino acid oxidase gene or a processed product thereof, and glutaryl 7-
A culture of E. coli transformed with a recombinant DNA containing the ACA acylase gene or a mixture thereof with a treated product can be used. Alternatively, a single E. coli transformant coexisting with a recombinant DNA containing a D-amino acid oxidase gene and a recombinant DNA containing a glutaryl 7-ACA acylase gene is constructed, and the culture thereof is Or its processed product can also be used, and the D-amino acid oxidase gene and glutaryl 7
- It is also possible to construct a recombinant DNA containing the AC:A acylase gene on a single vector, and use a single E. coli culture transformed with the recombinant DNA or a processed product thereof.

In addition, recombinant D containing the D-amino acid oxidase gene
To construct a single E. coli transformant coexisting with recombinant DNA containing NA and glutaryl 7-ACA acylase genes, two types of plasmids belonging to groups with different incompatibility with each other are used as vectors. , recombinant DN each containing the above two types of genes separately
A can be constructed, and a type of E. coli bacterium can be transformed with the two types of recombinant DNA obtained at the same time.

Examples of the above-mentioned D-amino acid oxidase gene include the yeast Trigonopsis parabilis (Iori on
o sis variabilis) CB S 40
95-derived derivatives, and glutaryl 7-ACA
Examples of the acylase gene include Comamonas sp. SY 77-1
(Feikoken Bibori No. 2410).

The D-amino acid oxidase gene derived from Trigonopsis parabilis and recombinant DNA containing the gene can be obtained, for example, by the method described in Japanese Patent Application No. 61-10663. That is, it can be obtained as follows.

(1) Trigonopsis palabilis CBS 409
After extracting the total DNA from 5 and cutting it with restriction enzymes, it is inserted into a vector and introduced into E. coli to create a DNA library. Trigonopsis palabilis CBS
4095 is the depositary institution Central Bureau.
Centraal B
ureau voor Schimmel-cultu
res), a yeast deposited in the Netherlands under deposit number 4095.

(2) Trigonopsis paliabris 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) For a portion of the amino terminal amino acid sequence of the D-amino acid oxidase obtained, a mixture of oligonucleotides having the DNA base sequence of the gene deduced from this is synthesized, and this is used as a DN4 probe.

(4) Label the DNA probe with ff2p and add the above (1)
Colony hybridization is performed with the DNA library obtained in ), and E. coli colonies that show complementarity with the DNIA probe are selected and isolated.

(5) Plasmid DNA is extracted from the selected E. coli strain, and the base sequence of the Trigonopsis parabilis CBS 4095-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.

In addition, glutaryl 7-AC derived from Comamonas sp.
A acylase gene and recombinant DN containing the gene
A can be obtained, for example, by the method described in JP-A-60-110292. That is, it can be obtained as follows.

1) Bacteria of the genus Comamonas 5Y-77-1
Total DNA is extracted from the bacterial cells of No. 410) and cut with restriction enzymes.

2) Cut and cleave the vector DNA with a restriction enzyme.

3) Incorporate the DNA fragment obtained in 1) into the cleavage site of the vector DNA to create a closed recombinant DNA.

4) Introduce the recombinant DNA into host E. coli.

5) Select and isolate a strain having glutaryl 7-ACA acylase activity from among the E. coli into which the recombinant DNA has been introduced.

6) Recombinant DNA obtained by extracting plasmid DNA from the selected E. coli strain and modifying it using restriction enzymes and recombinant enzymes or other vectors to increase the expression of the glutaryl 7-ACA acylase gene. Create.

7) The modified recombinant DNA is introduced into E. coli, and then a strain with an increased ability to produce glutaryl 7-ACA acylase is selected and isolated from the introduced E. coli.

In addition, preparation of the above two types of genes, recombinant D containing them
Escherichia coli transformed with NA and its recombinant D'NA can be prepared by conventional, known recombinant DNA techniques.

By culturing the E. coli obtained as described above under normal culture conditions, a culture containing significant amounts of D-amino acid oxidase or glutaryl 7-ACA acylase, or both simultaneously can be obtained.

The obtained culture may be used as it is, or the culture may be subjected to known methods such as collecting and disrupting the bacterial cells to extract the enzyme, or subjecting it to liquid chromatography, isoelectric focusing, etc. Partially purified or substantially completely purified D-amino acid oxidase and/or glutaryl 7-ACA acylase may be prepared and used by subjecting it to purification using conventional means.

Compound (n) or its salt is reacted with compound (I) or a salt thereof using a culture having both enzyme activities or a partially or substantially completely purified mixture containing both enzymes as an enzyme mixture. Salt can be efficiently generated.

This reaction is carried out in an aqueous medium, and the pH during the reaction is 7.
0 to 8.5, and the temperature is preferably maintained at 20 to 40°C. Examples of the aqueous medium used in the method of the present invention include water and buffers such as phosphate buffers.

The mechanism for producing compound (II) or a salt thereof from compound (1) or a salt thereof by the method of the present invention will be explained by taking as an example the case where R is 10COCH3 in compound (I), that is, the case where it is cephalosporin C. The explanation is as follows.

First, cephalosporin C is oxidized by the action of D-amino acid oxidase in an enzyme mixture to produce 7β-(5-carboxy-5-oxopentanamide) cephalosporanic acid. At this time, hydrogen peroxide is simultaneously generated. Due to the action of hydrogen peroxide, the generated 7β-(5-carboxy-5-oxopentanamido)cephalosporanic acid is decarboxylated and converted to glutaryl 7-ACA.

Glutaryl 7-ACA is the glutaryl 7-ACA in the enzyme mixture.
7-A is deacylated under the action of ACA acylase.
CA [A compound represented by the general formula (n) in which R is -○COCH3 is produced.

When ordinary Escherichia coli is used as Escherichia coli to prepare the enzyme mixture used in the above reaction system, catalase is mixed into the enzyme mixture because it has catalase activity that decomposes hydrogen peroxide. Therefore, when the enzyme mixture is used, it decomposes the hydrogen peroxide produced in the above reaction process and hinders the progress of the reaction. In order to solve this problem, the enzyme mixture-1[i- may be allowed to act in an aqueous medium in the presence of hydrogen peroxide. As for the method of making hydrogen peroxide present, it is preferable to add the necessary amount to keep the concentration as low as possible, as high concentrations of hydrogen peroxide will decompose cephalosporin compounds. Replenishment is recommended.

Furthermore, when a mutant strain of Escherichia coli lacking catalase activity is induced and a transformant using this as a host is used, the enzyme mixture can be made to act in the absence of hydrogen peroxide.

The above-mentioned catalase-deficient mutant strain can be induced by a conventionally known method, such as a method using a mutagenic agent such as N-methyl-N''-nitrosoguanidine.

The compound (Ir) or its salt produced as described above can be prepared by a known conventional method, such as column chromatography.
It can be separated and purified from the reaction solution using the isoelectric focusing method or the like.

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.

In the examples, a high performance liquid column chromatograph was used for quantitative determination of compound (II) or its salt. The column was Micro Bondapak C18 (
Waters, Inc.), and a mixed solution of 98 volumes of 5% ammonium acetate and 2 volumes of acetonitrile was used as the mobile phase. Compound (II) or its salt was detected at 260 mm.

Furthermore, in the Examples, the production rate of 7-ACA or its derivatives was determined by a linear equation. The amount of 7-ACA or its derivatives is determined by dissolving it in water, subjecting it to the liquid chromatography, quantifying the amount of 7-ACA or its derivatives, and determining the content (%) of 7-ACA or its derivatives in the powder.
) was obtained by calculating.

〔Example〕

Example 1 This example is a method using cultures obtained by separately culturing a transformant containing the D-amino acid oxidase gene and a transformant containing the glutaryl 7-ACA acylase gene.

[1] Yeast Trigonopsis palabilis (Irigo
The recombinant pEDAO1 containing the D-amino acid oxidase gene cloned from Escherichia coli (E.
hia codan) MB65/pEDAO1 (Special application Showa 6l)
-10663) was prepared by the following method.

1. Extraction and cleavage of total DNA from Trigonopsis palabilis using the method of Cryer et al. [Reference: Methods in Cell Biology
Biology) Volume 12, 39-44. Academic Press, 1975
) according to Trigonopsis palabilis CBS
Total DNA was extracted and purified from 4095. this DNA
Take 40 μg and add 4 units of restriction enzyme MboI at 37°C for 1 hour.
The reaction was allowed to proceed for 5 minutes. The reaction solution was extracted with an equal volume of phenol/chloroform (1:1), the DNA was precipitated with ethanol, and then added to a 0.1x TE buffer (10mM Tris-HCl (pH 8.0), 1mM EDTA). I combed it. The entire amount of the obtained DNA solution was subjected to 0.7% agarose gel electrophoresis, a portion containing DNA corresponding to a size of 6 to 9 kb was cut out from the gel, and the DNA fragment was eluted from the gel by electroelution method. . The eluate was then sequentially extracted with equal amounts of phenol and phenol/chloroform, and ethanol was added to the resulting aqueous layer to precipitate the DNA, which was then dissolved in 20 µΩ of 0.1x TE buffer.

2. Cleavage of vector DNA 30 μg of vector pUc18 was completely cleaved with Bam HI, and the obtained DNA fragment was injected with 0.1 times concentration of T.
It was dissolved in 2oμQ of Efi buffer.

3. Inserting the DNA fragment into the vector Mix the solution obtained in step 1 above and the solution obtained in step (2) above at a ratio of 3:1, and react with T4 DNA ligase at 15°C for 6 hours. , Vector and Trigonopsis
The DNA fragment of P. parabilis CBS 4095 was ligated.

4. Creation of DNA library of Trigonopsis palabilis First, a cephalosporinase-deficient mutant strain was constructed as an E. coli host. Namely, Escherichia coli (Esch
Erichia Codan) MC1061 (Malcom Casadab, University of Chicago, USA)
an) Obtained from Dr. The properties of water locks are described in the Journal of Molecular Biology (J, Mol.

Colonies with increased sensitivity to cephalosporin C were selected after treatment with N-methyl-N'-nitrosoguanidine (as described in Biol. Biol., 138, 179-207.1980). . Among these, a strain showing almost no cephalosporinase activity was selected and isolated, and this strain was named Escherichia coli MB65 and used as a host. This mutant strain, Escherichia coli MB65, has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, as Fiber Science and Technology Research Institute No. 8900. Next, the recombinant DNA obtained in step 3 above was introduced into transformed N. coli MB65, and L-broth agar medium containing 50 μg/mΩ of ampicillin [Batato tryptone (manufactured by Difco)] 1 %, yeast extract 0.5%, NaCl 0.5%, agar 1°5%]
The colonies grown on the tube were collected and called the Trigonopsis parabilis CBS 4095 DNA library.

5. Determination of the amino terminal amino acid sequence of D-amino acid oxidase D-amino acid oxidase was purified from Trigonopsis parabilis CBS 4095 according to the method of Szvajcsr et al. described above. The amino acid sequence of the amino terminal of the purified enzyme protein was analyzed by the method described above, and the amino acid sequence from the amino terminal to position 41 was determined. The obtained N-terminal amino acid sequence is expressed by the following formula.

Ala-Lys-11e-Val-Val-11e-G
ly-Ala-Gly-Val-Ala-Gly-Le
u-Thr-Thr-Ala-Leu-Gln-Leu
-Leu-Ar -L 5-Gl Once 1s-Glu-
Val-Thr-11e-Val-3er-Glu-P
he-Th r-Pro-G 1y-A 5p-Leu
-5er-I 1e-Gly-Tyr- Note: The underlined part indicates the sequence used for probe synthesis.

6. Synthesis of DNA probe Select the two underlined sequences in the above formula from the amino acid sequence obtained in step 5 above, and synthesize all possible NA base sequences on the gene deduced from these amino acid sequences. An oligonucleotide mixture containing the following was synthesized as shown in Table 1. That is, two groups of possible oligonucleotides are synthesized for each amino acid sequence to create a mixture, and these are used as DNA probes DAO-1, DAo, and 2.
, and DAo-3 and DAO-4. All oligonucleotide synthesis was performed by Applied Biosystems (
Applied Biosystem) DNA synthesizer (Synthesizer) model 380-
This was done using A.

7. D-amino acid oxy-Zj from DNA library
- ~24- Selection/isolation of candidates for tase clones The DNA probes obtained in step 6 above were used using the method of Inglia et al. [Reference: Nucleic Acid Research, Inc.]
9.

1627-1642.1982) using T4 polynucleotide kinase and γ-''P-ATP.Next, the E. coli obtained in step 4, which is a DNA library, was treated with 50 μg of ampicillin/
Colonies were grown on an L-broth agar medium containing
atman) 541 F paper, lysed with lysozyme, denatured the DNA with alkali, neutralized with hydrochloric acid, and then hybridized with DAo-1 and DAO-2. Hybridization was carried out using 6x concentrated SSC (0,15M NaCl, 0,015M
Sodium citrate, pH 7.0), 0.5% Nonidet P-40 (Sigma, USA), DAo-1 or DAO-2 at 44 °C using approximately 2 X 10” cprn/rn Q. Do this for 1 and a half hours, then apply 6 times the concentration of S.
The paper was washed twice at room temperature with SC and once at 44°C with 6x SSC. After this, the swamp paper was dried, and autoradiography was performed (photosensitive conditions: 80°C,
3 hours). As a result, DAO-1 or DAO
A large number of colonies positive for hybridization to -2 were found. Therefore, 40 strains were taken from the positive colonies, and after liquid culture, Birnboim (Birnboi)
m) et al. [Reference: Nucleitsquacid Research (
Nucleic Ac1dsRes,)+1.1513
-1523.1979), plasmid DNA was prepared. Next, these DNAs were denatured using a conventional method, and then spotted on a nitrocellulose filter.
hybridized with the probe. Hybridization was carried out using 6x 5SC, 110x Denhardt's solution (0,02% 7,1':I).
-JL/, 0.02% polyvinylpyrrolidone, 0.02
% bovine serum albumin) and labeled DNA probe at 44 °C (for DAO-1 and DAO-2) or 40 °C (for DAO-3 and DAO-4).
-27 = 1 hour, 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). ) was washed once. After that, the filter was subjected to autoradiography (photosensitive conditions: 80°C, 3 hours), and as a result, it showed positive hybridization to DAO-1 or DAO-2, and DAO-3 or DAO-2.
Six strains were also found to be positive for 4. Plasmid DNA from these six strains was cut with various restriction enzymes, subjected to agarose gel electrophoresis, and then subjected to Southern hybridization with a labeled DNA probe [For the method, refer to Southern (197, 5).
Journal of Molecular Biology (J, Mo1
．． Biol, ), 98, 503-517). As a result, DAO-2 and DAO-3 or DAO-4 were found in the approximately 0.6 kb DNA fragment generated by EcoRI digestion.
Plasmid D showing strong hybridization positivity
NA was found. This plasmid was converted into pDAOc2-1
It was named as D-amino acid oxidase clone candidate.

8. Identification and DN of D-amino acid oxidase clones
Determination of A nucleotide sequence A 0.6 kb DNA fragment generated from plasmid pDAOc2-12 by Eco RI digestion was analyzed according to the method of Sanger et al.
The base sequence was determined. As a result, as shown in Figure 2, the amino acid sequence at the amino terminal of the D-amino acid oxidase obtained in step 5 was completely combined with the intron-like sequences present in eukaryotic genes. A nucleotide sequence encoding a matching amino acid sequence was found, 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 FIG. 3 was created based on the results of restriction enzyme cleavage.

Based on the above results, we analyzed the DNA in the downstream region in the gene reading direction from the already determined base sequence.
When the base sequence was determined, it was found that there was a base sequence encoding a protein consisting of 355 amino acids shown in Figure 1 (in Figure 1, the intron-like sequence shown in Figure 2 was deleted). ). Thus, the DNA fragment derived from Trigonopsis paliabris CBS 4095 in plasmid pDAOc2-12 contains D-
It was estimated that the structural gene for amino acid oxidase was completely contained.

9. Modification of the D-amino acid oxidase gene A DNA base sequence portion that may be an obstacle to the expression of the cloned D-amino acid oxidase gene in E. coli was extracted from the fourth DNA fragment derived from Trigonopsis parabilis CB5 4095 in the plasmid pDAOc2-12. It was deleted according to the steps shown in the figure. Each step will be explained in detail below.

(1) 40μg of BooyXmid pDAOc2-12
After cleaving with c o RI and Hin d III and purifying the obtained fragment of approximately 0.45 kb, the 0.45 kb fragment was purified.
dATP, dGTP, dcTP, and TTP were added to 8 μg at a final concentration of 0.33 mM each, and DNA polymerase Klenow (Kl
add 5 units of enov) fragment and add 5 units of 10mM Tris-HCl (
pH 7.5), 10mM MgC1□, 1mM dithioleitol, 50mM NaCl in a 30μΩ reaction solution.
℃ for 20 minutes. From this, a DNA fragment with both ends blunted was purified, and about 0.2 pg of B a
mHI linker (0,01750, D,) and T4
Add 1 unit of DNAIJ gauze and add 50mM Tris-
Hydrochloric acid (pH 7.5), 10mM Mg CI, 0.
5mM ATP, 5mM dithiothreitol reaction solution 20
The reaction was carried out overnight at 15°C. After the reaction, the DNA fragment was purified and Ec.

Cut both ends with RI and BamHI, and
It was recovered as a BamHI fragment. On the other hand, phage M13
Double-stranded DNA of m p 1-8 tr-E co
After cutting with RI and BamHI, this was treated with the above E c
o Add RI-B a m HI fragment, T4DN
Phage M13M2-12-7 that incorporates a part of the D-amino acid oxidase gene by ligation with A ligase
was created.

(2) In order to delete the intron-like sequence, an oligonucleotide complementary to the sequence directly connected before and after the intron-like sequence as shown in FIG. 4 was synthesized and named DAOE 1. 25 pmole of DAOEI and 10 pmole of M13 primer Ml (manufactured by Zenshuzo Co., Ltd.) were phosphorylated with T4 polynucleotide kinase, and meshing (Mes
sing) method [Reference: Methods in Enzymology (Methods Enzymol, ), slow onset, 20
-78.1983) Phage M1 prepared according to
3M2-12-7 single-stranded DNA #0.5 pmole was added, heated at 95° C. for 5 minutes, and then left to stand until the temperature reached room temperature.

Next, dATP, dGTP, dCTP, TTP (
0.4mM each), ATP (0.4mM), DNA polymerase Klenow fragment (5 units), T4
Add DNA ligase (2 units) and add 7mM Tris-
Hydrochloric acid (pH 7,5), MgCl2, NaCl and 14
m M dithiothreitol in a 50 μΩ reaction solution.
The reaction was carried out at 7°C for 30 minutes. 0.5M EDTA 5μQ
After stopping the reaction, Escherichia-Coli (E.
scherichia coli) J M 1=31
- 〇5 was transfected with the reaction solution, and the phage-introduced bacteria were detected as plaques. Phage that appeared
Plaques were hybridized with 32p-labeled DAOE 1 by the plaque hybridization method (Reference: Benton et al. (1977) Science, 196, 180-182), and plaques that showed positive results were found. Phages in positive plaques were re-inoculated with Escherichia coli JM10.
After the plaques showing hybridization with DAOEl were purified and separated in the same manner as above, the phages present in the plaques were cultured in liquid by a conventional method, and the single-stranded DNA was isolated. Obtained single stranded DNA
Analysis of the nucleotide sequence revealed that the phage contained a DNA fragment derived from Trigonopsis parabilis CB54095, which had a nucleotide sequence lacking an intron-like sequence as expected, so this phage was named M13MHI.

(3) Next, in order to delete the amino acid non-coding region derived from Trigonopsis parabilis CB54095 upstream of the D-amino acid oxidase protein synthesis initiation site (ATG), we removed the region before and after this region as shown in Figure 4. A polynucleotide complementary to the directly linked sequence was synthesized and named DAOE2. Next, from the single-stranded DNA of DAOE2 and phage M13ME1, in addition to the deletion of the intron-like sequence, the amino acid non-coding region upstream of ATG was also deleted according to the same process as described in step (2) above. A phage carrying a part of the D-amino acid oxidase gene was constructed, and this was transformed into Ml 3ME1.
It was named 2.

10. Construction of expression vector The starting plasmid pAKKM1 for constructing the expression vector used in this example is based on the well-known literature Matsuda et al. (1985) Journal of Bacteriol. 16
3.1222-1228 shows its construction method, and it has the characteristic that the β-lactamase gene is inactivated.

As shown in FIG. 5, pAKKMl was first converted into Eco
Cut with RI and BamHI to obtain approximately 5kb of DNA.
The fragments were isolated and purified. On the other hand, plasmid pRZ4022 containing the fiacUV5 promoter (LRezniko, Rice Consin University, USA)
ff) obtained from Dr. Eco RI and BamHI
A 95 bP fragment containing the QaCUv5 promoter was prepared by cutting with QaCUv5 promoter. Then, the above two fragments were T4D
Combine using NA ligase to create expression vector pEX
Obtained OO2. In addition, the plasmid pR240 used above
22 is the method of Gilbert et al. [Reference:
Proceedings of the National Academy of Sciences USA (Proc, Natl, Acad, Sc
i, USA), 70.3581-3584.1973]
95bP including nacUV5 promoter according to
Prepare the A L u I fragment of pBR322 and
o RI - B am Created by insertion into the HI site.

11. Expression of D-amino acid oxidase gene Figure 6 shows the steps for constructing a recombinant for expressing the D-amino acid oxidase gene. First, the double-stranded DNA of phage M13ME12
Approximately 0.3 by EcoRI and BamHI cleavage from A
A DNA fragment encoding the amino-terminal portion of kb D-amino acid oxidase was isolated. Then pDAO
Approximately 1 excluding the part included in M13ME12 from c2-12 by E co RI and 5alI cleavage
．． A DNA cleavage encoding a 2 kb structural gene portion of D-amino acid oxidase was isolated. Further, the expression vector pEXOO2 was cut with B am HI and Xho I, mixed with the above two fragments, and a ligation reaction was performed with T4 DNA ligase. Escherichia coli MB65 was transformed using the reaction solution, and colonies growing on L-broth agar medium containing 40 μg/mfl of kanamycin were selected. For the obtained colonies, D-amino acid oxidase activity was detected using D-methionine as a substrate and ○-dianisidine according to Hedrick et al. (1968) Archives of Biochemistry and Biophysics (Arch.
, Biochem, Biophys, ), 126,15
As a result, a large number of positive colonies 5D-1 were obtained. Plasmid DNA was extracted from one of these strains and named pEDAO1-, and the structure shown in FIG. 6 was confirmed by restriction enzyme cleavage. In addition, Escherichia coli MB65/p harboring this recombinant
It was named EDAO1.

This bacterium was inoculated into 100 mQ of L-broth (L-broth agar medium with agar removed) containing 40 μg/m 11 of kanamycin, and cultured with shaking at 32° C. for 24 hours. The culture solution was centrifuged to collect bacteria, and this was transferred to a 10 m Q
of 0.1 M pyrophosphate buffer (pH 8,0). The cells were crushed by ultrasonication, and the supernatant after centrifugation was used as a D-amino acid oxidase enzyme solution.

[2] Comamonas e sp.
p,) Recombinant pAKKB1001 (Japanese Unexamined Patent Application Publication No. 1989-1991) containing the glutaryl 7-ACA acylase gene cloned from SY-77-1 (Feikoken Bibori No. 2410)
-110292) was prepared by the following method.

1) Bacteria of the genus Comamonas 5Y-77-1
Preparation of total DNA and its cleavage from nutriendo broth (beef extract 0.3%, peptone 0.5%)
) Comamonas genus bacteria 5Y-77-1 (Feikoken Bacteria No. 2410) is inoculated into 100 mQ, cultured overnight at 30°C, harvested, and suspended in 8 mΩ TE buffer. Lysozyme was added to this to a final concentration of 1 mg/mQ, and the mixture was allowed to react at 37°C for 1 hour. Next, pronase E was added to this to a final concentration of 200 μg/mQ.
Subsequently, sodium dodecyl sulfate was added to give a final concentration of 1%, and the mixture was allowed to react at 37°C for 1 hour. After the reaction is complete, add an equal volume of TNE buffer (50mM Tri 5-H) to the reaction solution.
CI, 5mM EDTA, 100mM NaCl; p
Add phenol saturated with H8,0) and after mixing 1
Centrifuge at 0.000 rpm for 10 minutes and collect the aqueous layer.

Add an equal volume of phenol/chloroform mixture (1
:1. V/V) is added, the aqueous layer is collected after centrifugation in the same manner, and an equal volume of chloroborm is added, and the aqueous layer is collected after centrifugation in the same manner. Ribonufrease A is added to the collected aqueous solution to a final concentration of 40 μg/m +2, and the reaction is allowed to proceed at 37° C. for 1 hour. After the reaction is complete, N a C
l and polyethylene glycol 6000 at a final concentration of IM.
and 10%, kept at 4°C for 4 hours, centrifuged at 5000 rpm for 5 minutes, and dissolved the resulting precipitate in 0.1x TE buffer. Sodium acetate was added to the solution obtained in this manner to a final concentration of 300 mM, 2 times the volume of ethanol was added, and the mixture was heated at -20'C for 4 hours.
After holding for a time, 10. Centrifuge at OOOrpm for 20 minutes to collect the DNA precipitate. Transfer this precipitate to 0.1 times the concentration of TE.
Dissolve it in a buffer solution and use it for the subsequent cleavage reaction. For DNA cleavage, 0.3 units of Pst per 1 μg of DNA.
I and 10mM Tri 5-HCI (pH 7,4
), 10mM MgSO4゜50mM NaCl, 1m
The reaction is carried out in a 30 μΩ buffer of M dithiothreitol at 37° C. for 1.5 hours, followed by heating at 70° C. for 10 minutes to stop the reaction.

2) Cleavage of vector pBR322 Add 1 unit of PstI to 1 μg of pBR322,
The reaction was carried out at 37° C. for 2 hours in the same buffer solution 3011μ as in 1), and the reaction was stopped in the same manner as in 1).

3) Recombination reaction 0.8 μg of cleaved Comamonas bacteria 5Y-77-
IDNA, 0.4 μg cleaved PBR322,30
mM Tri 5-HCI (pH 7°5), 10mM
MgC1z, 10mM 2-mercaptoethanol,
100mM ATP, containing 1 unit T4 ligase
React in μQ reaction solution at 22°C for 2 hours. Thereafter, the reaction is stopped by heating at 70° C. for 10 minutes.

4) Introduction of the recombinant plasmid into E. coli The DNAm solution obtained by the recombination reaction in 3) was introduced into Escherichia coli with 12C600r-m-(ATCC33525
) was transformed with tetracycline 10 μg/m
Colonies growing on L-broth agar medium containing Q were isolated. These colonies were treated with 50 μg of ampicillin.
The cells were transferred onto an L-broth agar medium containing /mQ to test for growth, and colonies showing ampicillin sensitivity were isolated as E. coli carrying recombinant DNA.

5) Selective isolation of Escherichia coli having the ability to produce glutaryl 7-ACA acylase The ampicillin-sensitive strain obtained as described above was cultured overnight at 32°C on an L-broth agar medium containing tetracycline, and one of the grown bacteria was cultured. Scrape off 100μ
Glutaryl 7-ACA of Q (1 mg/m Q)
After suspending in 0.1M phosphate buffer (pH 7.0) containing , the mixture was reacted at 37°C for 5 hours. Then add 120μQ of reaction stop solution (2 volumes of 100% acetic acid and 0.25M NaOH)
Add 1 volume of coloring solution (1 volume of mixed solution) and add 40μΩ coloring solution (p-
Add a solution of dimethylaminobenzaldehyde (0.5% in methanol). In this reaction, E. coli having the ability to produce glutaryl 7-ACA acylase turns the reaction solution yellow. The new Escherichia coli obtained in this way was introduced into Escherichia coli using 12C600 (pAKKloo).
It was named l).

4O-6) Isolation and analysis of Escherichia coli plasmid DNA The above Escherichia coli was inoculated into L-broth containing IQOmQ tetracycline, cultured overnight at 37°C, collected by centrifugation, and incubated with 15% sucrose, 50mM Tris-HCl (PH8, 5)
, 50 m M E D T A and 1 m g
/ mΩ of a solution consisting of lysozyme, and then maintained at room temperature for 1 hour.

Next, add Triton solution (0.1% Trit
onX-100, 50mM Tris-HCI, 50m
Add 2 mQ of MEDTA; pH 8,5) and hold at 37°C for 30 minutes. This solution was then heated at 5°C for 30.
Centrifuge at OOOrpm for 30 minutes and collect the supernatant. This is subjected to ethidium bromide-cesium chloride equilibrium density gradient centrifugation (at 15° C., 40.00 rpm, 48 hours) to separate and purify the plasmid DNA. This was cut with PstI in the same manner as in 2), and the length of the fragments was measured by agarose electrophoresis using 1% agarose. In addition to the 4.3 Kb fragment corresponding to the vector DNA, the length of the fragment was 3.6 Kb.
A fragment of b was found at Kb, -2.6 Kb, 0.9.

This indicates that the recombinant DNA isolated from the new E. coli
A has the structure shown in the restriction enzyme map in FIG. 7, and the recombinant DNA has 7.A in pBR322. IKb's DN
It was found that the A fragment was incorporated.

7) Confirmation of reaction product The above novel E. coli was inoculated into 5 m fl of L-broth containing tetracycline, cultured overnight at 32°C, collected by centrifugation, and inoculated with 1 mΩ glutaryl 7-ACA (25 m g
/ m j2 ) in 0.1M phosphate buffer. This is reacted at 37° C. for 6 hours, centrifuged to collect the supernatant, and subjected to high performance liquid chromatography. Linchrosorb S for high performance liquid chromatography
Using a column (150 x 4 mm) packed with 1100, elution was carried out using 0.1 M phosphate buffer (pH 7,5) as the solvent.
Detection is done by measuring the absorbance of From the above reaction solution, 7-A
An ultraviolet absorbing substance completely matching CA was detected, and 7-
It was confirmed that ACA was generated.

8) Glutaryl 7-ACA by modification of recombinant DNA
Isolated recombinant DNA of E. coli with increased acylase production ability
glutaryl 7-ACA by increasing the expression of the glutaryl 7-ACA acylase producing gene present in A.
In order to increase the production of acylase, the recombinant DNA was modified according to the steps described below.

a) Escherichia coli with 12C600 (pAKKlo
Plasmid DNA isolated from oI) as in 6)
is partially cleaved with B a m HI as follows. 0.5 units of Ba per μg of plasmid DNA
mHI was added, and the mixture was reacted for 1 hour at 37°C in 30 μΩ of the DNA cleavage buffer described in 1), and then phenol saturated with 1 equal volume of TNE buffer was added to stop the reaction. Next, after removing the phenol by centrifugation, an equal volume of ethyl ether is added and mixed to remove the ethyl ether. The DNA in this solution is precipitated with ethanol, and the precipitate is dissolved in 0.1x TE buffer.

b) Transfer vector pBR325 to the method described in 2) 4j
- Therefore, after cleavage with PstI and ring-closing again using the method described in 3), 12C600r-m-C was added to Escherichia coli.
ATCC 33525), and strains that have become sensitive to ampicillin are selectively isolated from colonies that grow on L-broth agar medium containing tetracycline. From the ampicillin-susceptible strain thus obtained, plasmid DNA
By acquiring A, ampicillin resistance gene (β
- A novel vector in which the lactamase-producing gene) is inactivated is obtained.

C) Transform the above novel vector plasmid into B a m H
■ Cleavage.

d) The DNA obtained in a) and the new cleaved vector plasmid were recombined by the method described in 3), and Escherichia coli K 12 C600r-m- (ATCC33
525), then 20 μg of chloramphenicol
Colonies growing on L-broth agar medium containing Q/mQ are selectively isolated.

e) Select and isolate a strain capable of producing glutaryl 7-ACA acylase from the selected strains by the method described in 5). Next, the glutaryl 7-ACA acylase activity of the strains capable of producing glutaryl 7-AC:A acylase is measured, and the strain with the highest activity is selected and isolated. Glutaryl 7-
To measure ACA acylase activity, bacterial cells cultured overnight in 5 mQ of L-broth containing chloramphenicol were incubated with glutaryl 7-ACA (25 mg/mQ).
) 0.1M phosphate buffer (pH 7.0) 2m
The 7-ACA produced was suspended in R and reacted for 15 minutes at 37°C. Immediately after the reaction, the cells were removed by centrifugation. A certain amount of the reaction solution was subjected to high-performance liquid chromatography.
Quantify.

A novel strain of Escherichia coli having a high ability to produce glutaryl 7-ACA acylase thus obtained was named Escherichia coli 12C600 (pAKKB 1001). When comparing the specific activity of the new glutaryl 7-ACA acylase production ability of Escherichia coli, we found that the activity of Comamonas 5Y-77-1 (Feikoken Bacteria Serial No. 2410) was 1.
When 12C600 (pA
KK1001) was 3, whereas Escherichia coli 12C600 (PAKKBlooI) was 15.

9) Isolation and analysis of plasmid DNA from E. coli with increased ability to produce glutaryl 7-ACA acylase Plasmid DNA was isolated from the new E. coli obtained in 8) by the method described in 6), cut with BamHI, and subjected to agarose gel. When the length of the DNA fragment was measured by electrophoresis, it was found that 8
5,9K corresponding to the new vector obtained in b) of
In addition to the b fragment, 4.8 Kb and 0.7 Kb fragments were found. Furthermore, based on the results of cutting the plasmid DNA with PstI and analyzing it by agarose gel electrophoresis,
A restriction enzyme map of the recombinant DNA shown in FIG. 8 was created.

Therefore, 12C600 (pA
In the recombinant DNA isolated from KKBloool),
It was found that a 5.5 Kb DNA fragment, which is a part of the recombinant-47 DNA isolated from Escherichia coli 12C600 (pAKKlool), was incorporated into a new vector DNA obtained by modifying pBR325.

The obtained plasmid PAKB100I was modified according to the steps shown in FIG. 9, and a transformant was obtained and used for enzyme production. This process will be explained in detail below.

(2) Plasmid pAKKB1001 was cleaved with restriction enzyme HpaI, then partially cleaved with restriction enzyme PvulI, and then religated with T4 DNA ligase. Escherichia coli MM294 [American Type Culture Collection]
1ture Co11ection) ATCC No.
33678] and grow on L-broth agar plates containing 25 μg/m Q of chloramphenicol but 10 μg/m tetracycline.
Colonies that could not grow on medium containing mQ (referred to as tetracycline sensitive) were selected. This colony was cultured, plasmid DNA was isolated, and pAK
It was named KB1003. The modified recombinant retains the glutaryl 7-ACA acylase gene and has miniaturized features.

■The DNA generated by cutting the plasmid pUC9 (available from American Type Culture Collection as ATCC No. 37252) with the restriction enzyme Haem
A fragment of approximately 0.2 Kb in size was separated from the A fragment by agarose gel electrophoresis and purified. On the other hand, plasmid p
After cleavage of AKKB1003 with the restriction enzyme Hindm and subsequent partial cleavage with Haem, the DNA
Polymerase KLenov fragment and dAT
The cut ends were made blunt by the action of P, dGTP, dCTP and TTP. This fragment was mixed with the fragment isolated from pUC9 described above, and a ligation reaction was performed using T4 DNA ligase. Escherichia coli MM294 was transformed with this, and 25 μg/mQ of chloramphenicol and 5-
Colonies growing on L-broth agar plates containing 20 μg/m Q of bromo-4-chloro-3-indolyl-β-D-galactoside and exhibiting a blue color were selected. These colonies were treated with glutaryl 7-ACA, and glutaryl 7-A was produced using the method described in 5) above for coloring 7-ACA with p-dimethylaminobenzaldehyde.
CA acylase activity was detected. Select colonies with enzyme activity, isolate plasmid DNA from them,
It was named pAKKlacool.

In order to make this recombinant even simpler, pAK
Klac 1001 with restriction enzyme Hind
The fragment was cut with m and SmaI, treated with DNA polymerase Flenow fragment in the same manner as above to obtain a fragment with blunt ends, and then religated with T4 DNA ligase.

With this, Escherichia coli MB65 (Feikoken Bacteria 89th
oO), and colonies resistant to chloramphenicol and having glutaryl 7-ACA acylase activity were selected. Plasmid DNA was isolated from this colony and named PAKKΔSH1, and the resulting transformant was transformed into Escherichia coli M B 65/p.
It was named AKKΔSHI. Recombinant pAKKΔS
H1 is characterized by containing the glutaryl 7-ACA acylase gene, which is fused with the promoter-containing portion of the β-galactosidase gene of Escherichia coli and whose expression can be regulated.

Escherichia coli MB65/p thus obtained
A K KΔSHI with 25 μg of chloramphenicol
A glutaryl 7-ACA acylase enzyme solution was prepared by culturing in L-broth containing /mn and treating in the same manner as in (1) above.

[3] Mix 1 omQ of D-amino acid oxidase enzyme solution, 10 mQ of glutaryl 7-ACA acylase enzyme solution, and 30 mα of 0.1M phosphate buffer (pH 8,0), and add 714 mg of cephalosporin C (purity 70%). was added, and the reaction was carried out at 37°C with shaking. 30%
0.01 ml of hydrogen peroxide solution was added at 15 minutes, 30 minutes, and 45 minutes after the start of the reaction, and 0.01 ml each was added at 70 minutes, 85 minutes, and 100 minutes, and the reaction was carried out for 120 minutes. The production rate of 7-ACA in the reaction-completed liquid was 55.7%.

The reaction solution was diluted 3 times with water and 100 mQ of DEAE
It was passed through a column of Sephadex A-25 (C1-type, manufactured by Pharmacia). Washing with 120 mQ water followed by passage through 0.05M saline eluted 7-ACA. 7-ACA fractions were collected, adjusted to pH 7.0, concentrated under reduced pressure, and passed through a 100 mQ column of Diaion HP-20 (manufactured by Mitsubishi Kasei) that had been washed in advance with 0.1 M phosphate buffer (pH 7.0). After washing the column thoroughly with deionized water, the column was eluted with 50% methanol water. The 7-ACA elution fractions were collected, concentrated under reduced pressure, adjusted to pH 3.0, and left in a cool place, whereupon 7-ACA precipitated. The precipitate was collected and dried under vacuum to obtain 160 mg of 7-ACA.

The purity was 80%.

Example 2 500 mg of deacetyl cephalosporin C (purity 65%) was added to the same reaction system as in Example 1, and the mixture was heated at 37°C for 120°C.
A minute reaction was carried out. The production rate of 7-amitubacetylcephalosboranic acid in the reaction-completed solution was 52%.

Example 3 This example relates to a method using a transformant in which a recombinant containing the D-amino acid oxidase gene and a recombinant containing the glutaryl 7-ACA acylase gene coexist.

(1) Both pEDA○1 and pAKKΔSHI are p
This is a recombinant constructed using a vector originating from BR322. Therefore, plasmid pAcY0184 (
ATCC from American Type Culture Collection
No. 37033), the glutaryl? -A
A DNA fragment containing the CA acylase gene was integrated.

That is, first, pAcYc184 was cleaved with Hind'lII. On the other hand, add SHI to pAKK using the restriction enzyme PvulI.
and Ndel, and the generated approximately 3 Kb DNA fragment was separated by agarose gel electrophoresis and purified.
The polymerase Flenow fragment was treated in the same manner as described above to obtain a fragment with blunt ends. Hin d ■ clinker is added to this by the action of TDNA ligase, and Hin d ■ clinker is added to this.
After treatment with dm, bi- was mixed with the above pACYC184, and both were ligated with T4 DNA ligase. Now Escherichia Cori MB6
5 was transformed to be resistant to chloramphenicol, sensitive to tetracycline, and glutaryl 7-
Colonies with ACA acylase activity were selected. This colony was cultured and plasmid DNA was isolated, which was named pACYCacl. p A CY Ca
Escherichia Colli M B 65/p E in c 1
Transform D A O1 and add 40 μg of kanamycin.
/ mΩ and chloramphenicol 25 μg / m Q
Colonies growing on L-broth agar plates containing Escherichia coli M B 65 /
It was named pEDA O1/pA CYCacl.

(2) Escherichia Colli M B 65 / p E
D A 01/pAcYcacl with 40μ of kanamycin
g/mQ and chloramphenicol 25μg/mQ
An enzyme solution was prepared in the same manner as in Example 1 by culturing in L-broth containing . Enzyme solution 1mF! 70 mg of cephalosborin C (purity 70%) was added to 0.1 M phosphate buffer (pH 8,0) 4rnQ, and the reaction was carried out in the same manner as in Example 1 for 120 minutes. 7-ACA in the reaction completed solution
The production rate was 40.6%.

Example 4 This example is a method using a culture of a transformant carrying a recombinant in which the D-amino acid oxidase gene and the glutaryl 7-ACA acylase gene coexist.

(1) The process of constructing a recombinant in which both of the above genes coexist is shown in FIG. 11. That is, pAKKΔSHI was cut with PvuU and NdeI, and both ends of the generated DNA fragment were cut with D
NA polymerase Flenow fragment was used in the same manner as above to make blunt ends.

From this, a fragment of approximately 3 Kb in size was separated by agarose gel electrophoresis and purified. Meanwhile, pEDAOI is Sm
This was cleaved with al, and after mixing this with the above fragment, the two were ligated together by the action of T4 DNA ligase. This was used to transform Escherichia coli MB65, and colonies growing on L-broth agar plates containing 40 μg/ml of kanamycin and exhibiting glutaryl 7-ACA acylase activity were selected. Plasmid DNA was isolated from this colony, named pDOacl, and a transformant carrying this was transformed into Escherichia coli MB65/pD.
It was named Oac 1.

(2) Escherichia Colli M B 65 / p D
Oa cl was cultured in L-broth containing 40 μg/μg of kanamycin, and an enzyme solution was prepared in the same manner as in Example 1. When this was allowed to act on cephalosporin C in the same manner as in Example 3, the production rate of 7-ACA in the completed reaction solution was 1.
It was 6%.

Example 5 Escherichia coli MB65 was treated with the mutagen N-methyl-
After treatment with N'-nitro N-nitrosoguanidine,
Loewen's method (Reference: Journal of Bacteriology (J, Bacteriol) 15
2.622-626.1984), a mutant strain lacking catalase activity was isolated and named Escherichia coli MBC1013. This mutant strain is 6pEDAO1, which has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, as Microtechnology Research Institute-〇D-Bacteria Collection No. 8901.
and Escherichia coli M independently in PAKKΔSHI
BC1013 was transformed, each of the resulting transformants was cultured, and an enzyme solution was prepared in the same manner as in Example 1. Add cephalosporin c to 1 mQ each of enzyme solution from each transformant, 3 mQ of 0.1 M phosphate buffer (pH 8,0).
(purity 70%) was added, and the reaction was carried out for 120 minutes while shaking at 37° C. without adding hydrogen peroxide solution. The production rate of 7-ACA in the reaction-completed solution was 59%.

Example 6 Escherichia coli MBC1013 was co-transformed with pEDAol and pAcYcacl and transformed with kanamycin 40 μg/ml and chloramphenicol 25 μg/ml.
Colonies growing on L-broth agar plates containing mQ were isolated as transformants. This transformant was treated with 40 μg/mQ of kanamycin and 2 chloramphenicol.
An enzyme solution was prepared in the same manner as in Example 1 by culturing in L-broth containing 5 μg/m Q. Enzyme solution 1mQ, 0
．． 70 mg of cephalosporin C (purity 70%) was added to 1M phosphate buffer (pH 8, 0), and the reaction was carried out for 120 minutes with shaking at 37° C. without adding hydrogen peroxide. The production rate of 7-ACA in the reaction finished solution was 42.6
%Met.

〔Effect of the invention〕

The present inventors used a D-amino acid oxidase gene and a glutaryl 7-ACA acylase gene cloned into E. coli.

(1) Coexistence of cultures or processed products of transformants carrying both genes separately; (2) Coexistence of cultures or processed products of transformants containing both genes separately; or , (3) W
7-ACA is highly efficiently converted from cephalosporin C and its derivatives in a single step using either a culture of a single transformant or a treated product of a single recombinant in which the J gene coexists. and its derivatives can be produced. In addition, in order for the above reaction to proceed, hydrogen peroxide needs to be supplied when ordinary Taiyoga is used as a host, but as is clear from the example, when a mutant strain of E. coli lacking the ability to produce catalase is used as a host, If used, there is no need to replenish hydrogen peroxide, and the reaction process can be made easier. In this way, from cephalosporin C to 7
- A radically superior method for producing ACA compared to conventional methods is provided.

[Brief explanation of the drawing]

FIG. 1 shows the amino acid sequence of D-amino acid oxidase derived from Trigonopsis parabilis and the base sequence of the D-amino acid oxidase gene encoding it. In the figure, the upper row shows the base sequence, and the lower row shows the amino acid sequence. Figure 2 shows Eco from plasmid pDAOc2-12.
A part of the base sequence of the approximately 0.6 Kb fragment generated by RI cleavage is shown. In the figure, the upper row shows the base sequence, the lower row shows the corresponding amino acid sequence, and the dotted line shows the intron-like sequence. FIG. 3 shows a restriction enzyme cleavage map of plasmid pDAOc2-12. FIG. 4 shows an outline of deletion of the amino acid non-coding region from the D-amino acid oxidase clone. FIG. 5 shows an outline of the process for constructing a vector for expressing D-amino acid oxidase. Symbol Cmr in the figure. K mr and A pr are respectively chloramphenicol,
Resistance genes for kanamycin and ampicillin are
lacUV5 indicates lacUV5 promoter. FIG. 6 shows an outline of the process for constructing a recombinant for expressing the D-amino acid oxidase gene. In the figure, the minus part indicates the amino acid sequence coding region of D-amino acid oxidase (indicated by the abbreviation DA◯). Figure 7 shows 12C600 (pAKK) in Escherichia coli.
This is a restriction enzyme map of a recombinant plasmid isolated from (lool). Figure 8 shows 12C600 (pAKK) in Escherichia coli.
This is a restriction enzyme map of a recombinant plasmid isolated from Blool. FIG. 9 shows the steps for modifying recombinant pAKKB1001. In the figure, only restriction enzyme cleavage sites that are involved in the process or serve as markers are indicated by the following abbreviations. E. EcoRI. Pv. P v u II, H, Hin d m. B, B
amHI. He, Hae III. Hp, HpaI. N, NdeI. Sm, SmaI. Also, in the figure, Cm r, T cr, A p
r represents a resistance gene to chloramphenicol, tetracycline, and ampicillin, respectively, and a6yl represents a glutaryl 7-ACA acylase gene. Figure 10 shows glutaryl 7 in plasmid pAcYc184.
- Recombinant pAcY incorporating the ACA acylase gene
The process of creating cacl is shown. Figure 11 shows the D-amino acid oxidase gene (DA in the figure).
A recombinant PEDAOI containing the glutaryl 7-ACA acylase gene (indicated by the abbreviation O)
The creation process of D Oac 1 is shown. In the figure, Kmr indicates the kanamycin resistance gene. bo - Figure 1 * 841 GAC, CGA, TTC, CCG, GAA, CTG, A
CC, AAA, GAT, GGC, CCT. Asp-Arg-Phe-Pro-C1u-Leu-T
hr-Lys-Asp-Gly-Pro-*90] CAC, CGT, CCT, GGT, AGA, GAG, G
GC, GGT, CCC, CGA, GTA. Hlm-Arg-Pro-Gly-Arg-C1u-G
ly-Gly-Pro-Arg-Val-* 96] TTT, GTT, GTC, CAT, AAC, TAT, G
GT, GCC, GCC, GGT, GCT. Pha-Val-Val-T(In-Asn-Tyr-
Gly-Ata-Ala-Gly-Ala-*1021 GAT, GAA, GCT, GTT, TCT, TAC, G
TC, GAA, AGA, GCT, CTT. Asp-C1u-Ala-Val-8or-Tyr-V
al-Glu-Arg-Ala-Leu- (Part 3) 900* Mouth TT, GAC, ATT, GTG, CGC, GAA, T
GC, GTT, GGCLeu-Asp-11e-Val
-Arg-Glu-Cys-Val-Gly960* CAA, TTA, GAG, AAC, ATC, CCC, G
GC, GTT, GGCClu-Leu-Glu-Ly
s-11e-Pro-Gly-Val-Gly]020
* GGT, TAC, CAA, TCC, TCT, TAC,
GGC, ATG, GCTCly-Tyr-C1n-3
s r-8er-Tyr-Gly-Me t-Al p
cACT, CGT, CCA, AAC, CTT, TAfl
. Thr-Arg-Pro-Agn-Leu-Lucky**-Figure 6

Claims (6)

[Claims] (1) General formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) (In the formula, R is -OCOCH_3, -H, or -OH) Deacylated compound or its salt General formula (II) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(II) (In the formula, R has the same meaning as above) of 7-aminocephalosporanic acid and its derivatives that produce the compound or its salt. In the manufacturing method, the formula (I
) D-amino acid oxidase and 7β-(
7- characterized in that an enzyme mixture containing 4-carboxybutanamide) cephalosporanic acid acylase is allowed to act in an aqueous medium in the presence or absence of hydrogen peroxide.
A method for producing aminocephalosporanic acid and its derivatives. (2) The enzyme mixture is a culture of E. coli transformed with a recombinant DNA containing a D-amino acid oxidase gene, or a treated product thereof, and a combination containing a 7β-(4-carboxybutanamide)cephalosporanic acid acylase gene. The production method according to claim (1), which is a mixture with a culture of E. coli transformed with the recombinant DNA or a processed product thereof. (3) The enzyme mixture is a single E. coli strain transformed with recombinant DNA containing the D-amino acid oxidase gene and recombinant DNA containing the 7β-(4-carboxybutanamide) cephalosporanic acid acylase gene. The production method according to claim 1, wherein the production method is a culture or a processed product thereof. (4) A single E. coli culture in which the enzyme mixture has been transformed with a single recombinant DNA containing both a D-amino acid oxidase gene and a 7β-(4-carboxybutanamide) cephalosporanic acid acylase gene. or a processed product thereof
The manufacturing method described in section 1). (5) The enzyme mixture contains D-amino acid oxidase produced by Escherichia coli that does not lack catalase production ability and 7β-
A patent for an enzyme mixture containing (4-carboxybutanamide) cephalosporanic acid acylase, characterized in that the enzyme mixture is allowed to act on a compound represented by general formula (I) or a salt thereof in the presence of hydrogen peroxide. A manufacturing method according to any one of claims (1) to (4). (6) The enzyme mixture contains D-amino acid oxidase produced by E. coli lacking catalase production ability and 7β-(
A patent for an enzyme mixture containing 4-carboxybutanamide) cephalosporanic acid acylase, characterized in that the enzyme mixture is allowed to act on a compound represented by general formula (I) or a salt thereof in the absence of hydrogen peroxide. A manufacturing method according to any one of claims (1) to (4).