JPS61152286A - Dna fragment carrying genetic information to produce cephalosporin c acylase - Google Patents

Abstract

PURPOSE:A DNA fragment that originates from Pseudomonas sp., containing two DNA coding for a specific subunit and can produce a large amount of cephalosporin C acylase, when the recombinant DNA is introduced in Escherichea coil. CONSTITUTION:The title DNA fragment originating from Pseu domonas sp. and containing a DNA coding for a subunit of about 26 kdalton containing an amino-terminated aminoacid sequency of formula I and another DNA coding for another subunit of about 58 kdalton containing amino-terminated aminoacid sequence of formula II. The DNA fragment is introduced into a vector and the Escherichea coil containing the recombinant DNA is cultured to obtain a culture mixture containing a large amount of cephalosporin C acylase.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides cephalosporin acylase production genetic information (
The present invention relates to a 5 DNA fragment (hereinafter abbreviated as cephalosporin acylase gene) and a novel Escherichia coli that has the ability to produce cephalosporin acylase using this DNA fragment.

[Problems to be solved by conventional technology and invention]

Cephalosporin C is produced by a reaction that removes the acyl group bonded to the 7-position amino group (hereinafter abbreviated as dealylation).
-aminocephalosporanic acid (hereinafter abbreviated as 7A, CA), various cephalosporin antibiotics have been synthesized using this as a starting material and are in practical use as pharmaceuticals.

As a deacylation method for cephalosporin C, it is assumed that a one-step reaction performed enzymatically or by the action of microorganisms is industrially advantageous, and several methods have already been proposed (U.S. Pat. No. 3,239,394 (1966), Japanese Unexamined Patent Publication No. 52-
143289, JP-A-53-94093, JP-A-59-
4.4392). However, there are no known examples of successful industrial use of these methods. On the other hand, recently, as an industrially advantageous method, Pseud monas sp.
omonas SP, ) 5E83 (Microtechnical Laboratory No. 7
Cephalosporin C, J: p 7
A method for producing ACA was invented (Japanese Patent Application No. 14147/1986)
5).

According to this, the strain has two types of cephalosporin acylases, one of which (acylase type II) directly deacylates cephalosporin C to produce 7 ACA (in the following description, this enzyme is called cephalosporin C acylase).

Based on this, if a strain that produces only cephalosporin C acylase in significant amounts could be created, it would be dramatically advantageous industrially.
It is estimated that a method for producing ACA will be established. However, it has not been possible to obtain a strain that produces only cephalosporin C acylase in significant quantities.

[Means and effects for solving the problem] The present inventors conducted extensive research to obtain a strain that produces only cephalosporin C acylase in significant amounts, and as a result, we found that Pseudomonas 5E83 [Pseudomonas SP, 5
We isolated a DNA fragment carrying the genetic information for producing cephalic I-phosphoC acylase from E83) and succeeded in cloning it in Escherichia coli.Escherichia coli into which we introduced the recombinant DNA in which the DNA fragment was incorporated into a vector became a cephalosporinase. It was found that sporin C acylase was produced in significant amounts. The present inventors have completed the present invention based on these findings.

That is, basically, according to the present invention, it is derived from Pseudomonas sp., and (a) has the following amino group terminal amino acid sequence: H2N-Th
r-Met-Ala-Ala-Lys-Thr-Asp
-Arg-Glu -Ala-Leu-Gl n-A
l a-Al a-Leu-Pro-Pro-Leu-
DNA encoding a subunit of about 26 kilodaltons (KD) containing 8er r -Gly- and (b) the following amino group-terminal amino acid sequence: H2N-8e
r-Asn-Asn-Trp-Ala-Val-Ala
-Pro-Gly -Arg-Th r -Ala
-Th r Kokan 4 spears -G ly-Arg -Pr
o -Ile -Leu -A 1 a -G
A DNA fragment carrying genetic information for producing cephalosporin C acylase is provided.

Further, according to the present invention, Pseudomonas sp.
seudomonas sp.), and (a
) The following amino group terminal amino acid sequence: HEN-Thr-
Met-Al, a-Ala-T, ys-Thr-Asp
-Arg-Glu-Al a-Leu-Gin-Al
a-Ala-Leu-Pro-Pro-Leu-8e
DNA encoding an approximately 26 kilodalton (KD) subunit containing r -Gay- and (b) the following amino terminal amino acid sequence: H2N-8e
r-A,5n-Asn-Trp-Ala-Va
l-Al, a-Pro-Gly-Arg-Th
r -Al a-Th r 4 total -G ly-A
rg-Pro-I1e-Leu-Ala-
DN encoding an approximately 58KD subunit containing Gly −
A recombinant DNA is provided in which a DNA fragment characterized by containing A is integrated into a vector.

Furthermore, according to the present invention, it is derived from Pseudomonas sp., and (a) has the following amino group terminal amino acid sequence: H2N-Th
r-Me t-Al a-Ala-Lys-Thr
-Asp-Arg-Glu -Ala-Leu-Glr
L-Ala-Ala-Leu-Pro-Pro-Leu
DNA encoding an approximately 26 kilodalton (KD) subunit containing -8er -Gay- and (b) the following amino group-terminal amino acid sequence: H2N-8e
rH2N-8er-Asn-Asn-Trp-Ala-
Val-Ala-Pro-Gly-Ar, a-Thr sticky P own-Gly-A rg-Pro-11
Approximately 58 K containing e-Leu-Ala-Gay-
Escherichia coli into which a recombinant DNA in which a DNA fragment characterized by containing a DNA encoding a D subunit is introduced into a vector is provided.

The DNA and E. coli according to the present invention can be constructed by the following steps.

(1) Total DNA is extracted from Sheetomonas bacterium 5E83 and cut with an appropriate restriction enzyme. On the other hand, vector DN
A is cut and cleaved with a restriction enzyme whose base sequence at the cleavage site is the same as that of the restriction enzyme.

(2) A DNA fragment from the 5E83 bacterium is inserted into the cleavage site of the vector DNA, and a closed recombinant DNA is created by the action of DNA IJ gauze.

(3) Introduce the 0 recombinant DNA into host E. coli.

Then, a strain having cephalosporin C acylase activity is selected and isolated from the E. coli into which the recombinant DNA has been introduced.

(4) Plasmid DNA from selected E. coli strains
The inserted DNA fragment derived from the 5E83 bacterium is cut and reduced into fragments of various sizes by the action of various restriction enzymes, and the inserted DNA fragments are ligated after a promoter derived from E. coli to express cephalosporin C acylase activity. let In this way, the approximate position and size of the cephalosporin C acylase gene on the inserted DNA fragment is known, and the direction of gene reading is determined.

(5) 1. Cultivate the selected Escherichia coli strain, isolate the cearos I-phosphoC acylase protein from the bacterial cells, and determine the amino terminal amino acid sequence. Meanwhile, the base sequence of the DNA portion containing the cephalosporin C acylase gene will be determined. The amino acid sequence predicted from the obtained DNA base sequence is compared with the amino terminal amino acid sequence described above, and the enzyme protein synthesis initiation site is determined on the DNA base sequence. In addition to the determined enzyme protein synthesis start site, by determining the enzyme protein synthesis stop site using the DNA base sequence,
Determine the minimum DNA base sequence containing the cephalosporin C acylase gene.

All of the general operations necessary for handling DNA in the above steps are well known and familiar to those skilled in the art.
For example, Molecular Cloning [Ta
Maniati8 et al., Cold Spring Har
It can be easily carried out by following the experimental operating instructions such as those published by Bor Laboratory, Inc. (1982). All enzymes and reagents used can be commercially available products.
Unless otherwise specified, the purpose can be completely achieved by following the conditions of use specified for the product. In addition, as the E. coli used as a host, any strain that is deficient in foreign DNA restriction (1AS ability) can basically be used. To select strains with 7 ACA, test bacteria are first exposed to cephalosporin C or a better substrate, 7-β-(4-carboxybutanamide) cephalosporanic acid, which produces 7 ACA. p-
A method of developing color with trimethylaminobenzaldehyde [for example, literature: 5hibuya et al. (1981) Ag
r, Biol, Chem, 45.1561-1.56
A method can be used in which the bacterial strains selected by 73 and showing positive are allowed to act on cephalosporin C again, and the produced 7 ACA is detected by subjecting it to high performance liquid chromatography. The amino acid sequence of the amino group terminal of the enzyme protein can be determined using known methods (for example, literature: Hunkapiller
(1983) 5science, 219=650-
659), and the DNA base sequence was also determined by known methods (for example, in the literature: Maxam and G11bert (
1980) Methods in Enzymolog
y, 65,499-560).

By culturing E. coli into which recombinant DNA obtained by incorporating the DNA fragment of the present invention into a vector is introduced, a culture containing a large amount of cephalosporin C acylase can be obtained. Partially purified or substantially completely purified cephalosporin C acylase can be obtained by subjecting the culture to purification using known conventional means. The above-mentioned culture having cephalosporin C acylase activity or its partially or substantially completely purified cephalosporin C anlase can be synthesized by the general formula (1) (wherein R is 10C0CT(5,-)Tt 7A represented by general formula (n) (wherein R has the same meaning as above) by acting on cephalosporin C represented by C or a salt thereof
CA can be manufactured efficiently.

〔Example〕

Example 1. Cloning of the cephalosporin C acylase gene into Escherichia coli 1, Pseudomonas sp.
as SP,) SE83 (Bi-T Laboratory Bacteria Serial No. 7649) Pseudomonas st (Paeud
omonas SP, ) SE83 (Feikoken Bacteria No. 764
Total DNA was extracted from Marmur et al. (Reference: J, Mo1. Biol., No. 9).

38.227-239.1961). D
For NA cleavage, 0.3 μl of each
After adding units of RamHr and Bgl H and allowing the reaction to proceed at 37°C for 1.5 hours, add phenol/chloroform ai (1:1, V/V) equal to the reaction solution to stop the reaction. . This solution was centrifuged to remove the aqueous layer, and twice the amount of ethyl alcohol was added to it.
After standing at 20°C for 2 hours, the DNA precipitate was collected by centrifugation, dried in vacuo, and added with DNA buffer (1mM
Tris-HCL, 0.1 mM EDTA-PH8
,0).

2. Modification and cleavage of vector DNA The vector pRR, 325 for E. coli carries the gene for β-lactamase, and its product β-lactamase degrades cephalosporin compounds, so it is inconvenient for the purpose of this experiment. be. Therefore, a vector in which the β-lactamase gene was inactivated was constructed as described below, and this was named psK12. That is, 1 μg of pBR325 (Bethe
sda (manufactured by Research Laboratories) was treated with 1 unit of Pst I to completely open the ring. Next, this was treated with T4 DNA ligase to close it again, and the reaction product was transferred to Escherichia coli by transfusion.
coli) K 12 C600 (ATCC
33525) and tetracycline 10μ97m
Contains 1 bronu (1 part tryptone, 0 yeast equine)
．． 5 qA, NaCA 0.5%,) A strain that has become sensitive to angicillin is isolated from the colonies growing on the agar medium. Shira 7amide DNA was isolated from one of the strains and named pSK12. psK 1-2 of 1
Add 1 unit of Bam HI to μI and incubate for 2 hours at 37°C for complete cleavage.

3 Insertion of DNA fragment into vector 1. DNA of 5E83 bacteria cut in 0.
8 μg and pSK 120. cleaved in 2 above.
4μI in 100 pH glue gauze buffer (30mM T
ris-HCt (pH 7,5), 10 mM 2-mercaptoethanol, 0.1. (mM ATP), add 1 unit of T0n) NA ligase, and react at 15°C for 2 hours. The reaction is then stopped by heating at 70°C for 10 minutes. 4. Introduction of recombinant DNA into E. coli 3. The recombinant DNA obtained was transformed into Escherichia coli by transfusion.
chiacoli ) K 12 MM 294 (
ATCC 33625), and colonies are made to appear on a chloramphenicol 25μ9/m, loff<trL-Bronne agar medium. The colonies that appear are grown on an L-Bouronne agar medium containing tetracycline, and colonies that cannot grow are isolated as E. coli carrying the recombinant DNA.

5 Selective isolation of Escherichia coli capable of producing cephalonporin C acylase 4. The resulting tetracycline-sensitive strain was incubated with chloramphenicol at 32°C on a Brone agar medium.
After culturing the bacteria overnight, chloroform vapor was applied to the grown bacteria.
Leave to act for 5 minutes, scrape off a portion and add 100 μl of 7
-β-(4-carboxybutanamide)cephalosporanic acid CITn9/mA) in 0.1M phosphate buffer (
After suspending at pH 7.5), the mixture is reacted at 37°C for 5 hours. Then, 120 μl of reaction stop M (a mixture of 2 volumes of 100% acetic acid and 1 volume of 0.25M NaOH) was added, and an additional 4
Add 0 μl of coloring solution (p-dimethylaminobenzaldehyde added to 0.5 parts methanol).

In this reaction, E. coli that has the ability to produce acylase turns the reaction solution yellow. The thus selected large intestine 11 was treated with chloramphenicol.
ml and cultured overnight at 37°C with shaking.

The bacterial cells were collected by centrifugation, suspended in 200 μl of 0.1 M phosphate buffer (pH 7.5, +), and diluted with 10 μl of chloroform.
1 and shaken at 37°C for 30 minutes. 200 for this
Pit Cephalosboria C (15mg lrnl
(dissolved in 0.1 M phosphate buffer (pH 7, 8)) and allowed to react at 37°C for 1 hour. After removing the bacterial cells from the reaction solution, the solution was subjected to high performance liquid chromatography.
Detect and quantify CA. A μm 13ondapak CIB (manufactured by Waters, USA) is used as a column for high-performance liquid chromatography, a mixture of 98 volumes of ammonium penta-thiacetate and 2 volumes of acetonitrile is used as a mobile phase, and detection is performed at 260 nm.

As a result of the above test, one strain was found to produce 7ACA without producing by-products from cephalosporin C. Plasmid DNA was extracted from this strain and introduced into E. coli, which does not have a plasmid. All introduced E. coli strains retained the ability to produce 7 ACA from cephalosporin C. This plasmid
A] ■ was named 3, and the E. coli introduced was Escherichia coli (Fl: 5cherichia coli).
i) Named K12MM294/pAMK3. 7 from cephalosporin C of this strain.
Comparing the ACA production ability per unit weight of bacterial cells, Pseudomonas sp.
, ) was about 5 times that of SE 83.

6. Restriction enzyme map of recombinant DNA The DNA of cilasmid pAMK3 was isolated from Escherichia coli, cut with various restriction enzymes, and its structure was analyzed. As a result, a restriction enzyme map as shown in FIG. 1 was obtained. This figure revealed that in pAMK3, a DNA fragment of about 6 Kb was inserted into the BamHI site of psK12.

Example 2. Determining the position of the cephalosporin C acylase gene on the DNA fragment and the direction of gene reading After cutting 4 μI of plasmid pAMK 3 with Sma I and then cutting with Hind Ill, the reaction product was subjected to agarose-r99) Suguru electrophoresis [ Low melting point
agarose(RetheSda Re5earc
A DNA fragment corresponding to 3.0 KbK was cut out from the agarose scale, and phenol and phenol-chloroform-c [produced by D.H.
Collect NA. This DNA is then treated with DNA polymerase rKlenow fragment and four types of deoxynucleoside triphosphates (ATP, GTP).

By acting on CTP, TTP), Hi
The sticky ends generated by ndnTK are changed to blunt ends.

Meanwhile, add 0.5 pH of vector pSK 12 to BamHI
The resulting Ram HT sticky end is similarly converted to a blunt end. The two types of D thus obtained
NA is mixed and T4 ligase is activated to bind and close the ring. Next, I added this to Escherichia coli (Escher
ichia colt) K 12 MM 294, and colonies highly resistant to chloramphenicol were selected. The obtained colonies are examined for the presence or absence of cephalosporin C acylase activity, and the cilasmids possessed by these colonies are separated and analyzed using restriction enzymes.

As a result of carrying out the above experiment, two types of novel cilasmids shown in FIGS. 2 and 3 were obtained.

The resulting plasmids were incubated at pH 81 and pH 81, respectively.
' was named. The two types of cilasmids obtained were Hin
They had the same structure except that the fragments generated by d m -Sma I were inserted in opposite directions. Among these, Hind! for the tetracycline-resistant promoter on vector pSK12! DN in the direction of Tl→Sma I
All E. coli introduced with plasmid pH81 into which the A fragment was inserted showed cephalosporin C acylase activity, while all E. coli into which cilasmid pH81' into which Sma I − + Hind ■ was inserted showed cephalosporin C acylase activity. It did not show phosphorus C acylase activity.

From the above results, the following facts were revealed.

(1) The cephalosporin C acylase gene is fused with HindlTl and Sma on the cloned DNA fragment.
limited between the I sites.

(2) The reading direction of the gene is HindlTl→S
ma I, and gene expression is dependent on the promoter on the promoter.

Example 3. Amino terminal amino acid sequence of cephalosporin C acylase protein and gene DNA base sequence 1, amino terminal amino acid sequence determination of Escherichia coli (Bscherjchia coli)
i) After culturing K12MM 294/pAMK 3 and collecting the bacterial cells and treating them with an ultrasonic disrupter, the centrifuged supernatant was precipitated with saturated ammonium sulfate at 60% DE.
The mixture was passed through an AE Cephalosporin C acylase A30 column (manufactured by Pharmacia) and eluted by concentration gradient elution with NaCl to collect a cephalosporin C acylase active fraction. Both parts were then subjected to chromatofocusing, and the pH was adjusted to 4.
, 5 was collected. The enzyme protein thus obtained was subjected to SDS polyacrylamide gel electrophoresis (1
0 Tigre), approximately 26 KD and approximately 58 KD.
It was found that cephalosporin C acylase consists of two subunits. The above two subunits were extracted from Dal, and the amino terminal amino acid sequences of each were determined. As a result, the 26 KD subunit is: H2N-Thr-Met-Ala-Ala-Lys
-Thr-Asp-Arg-Glu-Al 5-Le
u-Gl n=A ] a-Ala ”-Leu-P
ro-Pro-Leu-8e r-Gl'y-t58
The KD subunits are: H2N-6e r -As n-As n-Trp-A
l a'-Va 1 = A1 a'-Pro'-Gl
y-Arg-Thr-Ala-Thr-G
Iy-Arg-Pro-I Ie-Leu-At
It was found that it has the amino group-terminal amino acid sequence of a-Gly-.

2. DNA base sequencing DNA base sequences were read from various restriction enzyme sites on the inserted DNA fragment. Among these, the sequence near Mlul-■ ACG-ATG-GCG-GCC-AAG-ACC-
GAT-CGC-GAG-GCC-CTG-CAG-G
CG-GCG-CTG-CCG-CCG-CTT-TC
C-GGC(7Thr-Met-Ala-Al
a-Lys-Thr-Asp-Arg-Glu-
Al a -Leu-Gl n-Ala -Al
a -Le o-Pro-Pro 〒Le u-8e
r-Gly-) encoded an amino acid sequence that completely matched the amino terminal amino acid sequence of the approximately 26 KD subunit.

Discarded Sac l-■ Neighboring array knee AGC-AAC-AAC-TGG-GCG-GTC-
GCG-CCG-GGC-CGC-ACG-GCG-A
CG-GGC-CGG-CCC-ATC-CTC-GC
G-GGC(-8e r -As n-As n-Tr
p-A I a-Va l -Ala-P ro-G
l y-Arg-Th r-Ala-Thr-Gly
-Arg-Pro-11e-Leu-Ala-Gly-
) encoded an amino acid sequence that completely matched the amino terminal amino acid sequence of the approximately 58 KD subunit.

There is a protein synthesis initiation codon (ATG) immediately before the sequence encoding the amino terminal f1m of the approximately 26KD subunit, and when a triplet codon is assembled from this position,
The amino acid sequence shown in Table 1 was obtained, and the following findings were made.

(1) The amino terminal amino acid sequences of the approximately 26 KD and approximately 58 KD subunits are correctly arranged on a continuous amino acid sequence.

(2) The DNA base sequence encoding the amino terminal amino acid sequence of the approximately 26 KD subunit and the approximately 58 KD subunit.
There is a protein synthesis stop codon (TGA) between the DNA base sequences encoding the terminal amino and amino acid sequences of the subunits.
, TAA, TAG) are not present, and the H4nd m-Sma I fragment limited in Example 2 is
The first protein synthesis stop codon (TGA) near the I site
appears.

(3) The DNA base sequence from the amino terminal sequence of the approximately 26 KD subunit to just before the amino terminal sequence of the approximately 58 KD subunit coats the approximately 26 KD protein, and the DNA base sequence from the amino terminal sequence of the approximately 58 KD subunit coats the protein. It encodes a protein of about 58 KD up to the synthesis stop codon.

Based on the above facts, it was determined that the DNA sequence shown in Table 1 includes the minimum position II of the cephalosporin C acylase gene. Of the underlined parts in Table 1, ■ corresponds to the amino terminal amino acid sequence of subunit 7 of approximately 26 KD, and ■ corresponds to approximately 58 KD.
This indicates that the portion corresponds to the amino terminal amino acid sequence of the KD subunit.

(Hereinafter referred to as "Effect of the Invention") [Effects of the Invention] As detailed in the Examples, the present inventors have developed a clear DNA sequence containing genetic information corresponding to two subunits of cephalosporin C acylase on a continuous DNA base sequence. By obtaining the determined DNA fragment, we were able to provide the prerequisites for dramatically superior production of cephalosporin C acylase.

It is a known fact that cephalosporin C acylase production ability can be increased and regulated by optionally binding a strong promoter in front of the enzymatic protein synthesis initiation site of the above DNA fragment (for example, literature: Pribr4ow (1979)).
# Biological Regulation a
ndDevelopment, VOl, 1#231-
277, Plenum Press), and its remarkable effects are seen in Example 1. The new E. coli into which the recombinant PNA thus obtained was introduced is Pseudomonas sp., the original cephalosporin C acylase producing bacterium.
eudomonas SP,) SE83 in the following points.

(1) Cephalosporin C acylase activity increases dramatically.

(2) Conventional 5E83 bacteria possess multiple enzymes that act on cephalosporin C, and therefore act on cephalosporin C to produce compounds other than 7 ACA as by-products. However, the new E. coli produces only cephalosporin C acylase and therefore directly converts 7AC from cephalosporin C.
Only A can be efficiently generated.

[Brief explanation of drawings]

Figure 1 shows Escherichia coli.
aCo 1 i ) Restriction enzyme map of the recombinant plasmid isolated as K12 MM294/pAMK3. FIGS. 2 and 3 are restriction enzyme maps of plasmids pH81 and pH81' constructed in Example 2, respectively. 3 shown in the figure. The detailed map within OKb is identical to the corresponding part of pAMK3. In addition, the part indicated by the hour symbol in the figure indicates the position of the tetracycline resistance promoter on vector psK12. pH51 p) 151' Procedural amendment-t (voluntary) January 9, 1985 Manabu Shiga, Commissioner of the Japan Patent Office 1, Indication of the case Kuγ-ko''・(Jlo,. Patent filed on December 27, 1988) Petition 2, Name of invention DN carrying genetic information for cephalosporin C acylase production
Fragment A 3, Relationship with the case of the person making the amendment. Patent applicant address: 1-2-6 Dojimahama, Kita-ku, Osaka-shi, Osaka Prefecture. Claims column and detailed description of the invention column 5 of the Ministry of Specification, Contents of the amendment as shown in the attached sheet [Patent application filed on December 27, 1982 (4
)] The statement in the description is amended as follows. ■Amend the claims in the specification as follows. (1) Pseudomonas sp.
onas 'SP,''), and (a> the following amino acid sequence at the end of the amino curtain: H2N-'
rhr-Met-Ala-Ala-'Lys-Thr-
Asp”Arg-Glu”Ala-Leu-Gln-A
la-Ala-Leu-Pro-Pro-Leu=Se
DNA encoding the entire subunit of t-Gly (approximately 26 kilodaltons (K, T)) and the following amino group-terminal amino acid sequence: 2N-8er-Asn-Asn-Tr'p-Ala-V
al-ALa-Pro41y-rg-Thr-Ala-
Thr-Gly-Arg-Pro-11e-Leu'A
A DNA fragment carrying genetic information for producing cephalosporin C acylase, characterized in that it contains DNA encoding the entire subunit of approximately 58 KD containing 1a-. (2) Thr-MET-Ala-Ala-Lys-
Thr-Aap-Arg-Glu-Ala-Leu-G
ln-Ala-Ala-Leu-Pro-Pro-L
eu-So r-Gly-8er-Leu-8et-1
1e-Pro-Gly-Leu-8sr-Ala-Pr
o-Val-Ar g-Va 1-Gl n-Ar g
-Asp-Gly-Tr p-Gly-I le -
Pro-H4,s-115-11e-Lys-Ala
-8er-Gly-Glu-Ala-Asp-Ala-
Tyr-Ar a-Leu-Gly-Phe-Va
1-Hls-Ala-Gln-Asp-Arg
-Leu-Phe-Gln-MET-Glu-Leu-
Thr-Arg-Arg-Lys-Ala-Leu-G
ly-Arg-Ala-Ala-Glu-Trp-Le
u-Gly-Ala-Glu-Ala-Ala-Glu
-Ala-Asp-11e-Leu-Val-Arg-
Arg-Leu-Gly-MET-Glu-Lys-V
al-Cys-Arg-Arg-Asp-Phe-Gl
u-Ala-Leu-Gl, y-Ala-Glu-A
la-Ll'Y8-As p-MET-Le u-Ar
g-Ala-Tyr-Va1-Ala-Gl
,y-Val-Asn-Ala-Phe-Le
u-Ala-8e r-Gly-Ala-Pro-
Le u-Pro -I le -Glu-Tyr-
Gly-Leu-Leu-Gly-Ala-Gl
u-Pro-Glu-Pro-Tr p-Glu-Pr
o -Trp-His-8er-11e-Ala-
Val-MET-Arg-Arg-Leu-Gly-L
eu-Leu-MET-Gly-8er-Val-Tr
p-Phe-Lys-Leu-Trp-Arg-MET
-Leu-Ala-Leu-Pro-Val-Val-
Gly-Ala-Ala-Asn-Ala-Leu-T
, ys-Leu-Arg-Tyr-Asp-Asp-G
ly-Gly-Gln-Asp-Leu-Leu-Cy
s-11e-Pro-Pro-Gly-Val-Glu
-Ala-Glu-Ar g -TJ e u -G1
u -A l a -AS p -L e u -A
1 a -A l a -L e u -A r g
-P r O-A1. a-Va 1-Asp-Ala
-Leu-Leu-Lys -Ala-Mli;'
I'-Gly-G:Ly-Asp-Ala-8er-A
sp-Ala-Ala-Gly-()ly-Gly-8
er-Asn-Asn-Trp-Ala-Val-Al
a-Pro-01y-Arg-Thr-Ala-Thr
-Gly-Arg-Pro-11e-Leu-Ala-
Gly-Asp-Pro-Hls-Arg-Val-P
he-Glu-11e-Pro-Gly-MET-Ty
r-Ala-Gin-Hls-Hls-Leu-Ala
-Cys-Asp-Arg-Phe-Asp-MET-
Ile-Gly-Leu-Thr-Val-Pro-G
ly-Val-Pro-(')1y-Phe-Pro-
Hls-Phe-Ala-Hls-Asn-Gly-L
ys-Val-Ala-Tyr-Cys-Val-Th
r-Hls-Ala-Phe-MET-Asp-11e
-Hls-Asp-Leu-Tyr-Leu, -Glu
-Gln-Phe-Ala-Glu-Asp-flly
-Arg-Thr-Ala-Arg-Phe-()ly
-Asn-Glu-Phe-Glu-Pro-Val-
Ala-Trp-Arg-Arg-Asp-Arg-
Ile-Ala-Val-Arg-Gly-Gly-A
la-Asp-Arg-Glu-Phe-Asp-Xl
e-Val-Glu-Thr-Arg-Hls-Gly
-Pro-Val-11e-1, a-Gly-Asp-
Pro-Leu-Glu-Gly-Ala-Ala-L
eu-Thr-Leu-Arg-8er-Val-Gl
n-Phe-Ala-Glu-Thr-Asp-Le
u-8er-Phe-Asp-Cys-Leu-Thr
-Arg-MET-Pro-Gly-Ala-8er-
Tbr-Val-Ala-Gln-Leu-Tyr-A
sp-Ala-Thr-Arg-Gly-'T'rp-
Gly-Leu-(1e-Asp-Hls-Asn”L
eu-Val-Ala-Gly-Asp-Val-Al
a-Gly-8er-11e-()ly-Hls-Le
u-Val-Arg-Ala-Ar g-Val-Pr
o-8sr-Arg-Pro-Arg-Glu-Asn
-G'Ly-Trp-Leu-Pro-Van-Pro
-Gly-Trp-8er-Gly-Glu-Hls-
Glu-Trp-Arg-Gly-Trp-Ile-P
r o-Hls-1Glu-Ala-MET-Pro
-Ar g-Va 1-I 1e-Asp-Pro
-Pro-Gly-Gly-Le u-II e
-Va 1-Thr -Ala = As 1l-A8n
-Ar g-Val-Val-Ala=Asp-Asp
-Hls-Pro-Asp-Tyr-Leu-Cys-
Thr -Asp-Cys-)] is -Pr
o-Pr o-Tyr-Arg-Aha-Glu-A
r g-I 1 a -MET-Glu-Ar g-
Leu-Va 1-Ala-8e r-Pro-Al
a-Phe-Ala-Va 1-Asp-As
p-Ala-Ala-Ala-I1e-His
-Ala-Asp-Thr-Leu-Ber-Pro
-Hls-Va 1-Gl-y-Leu-Leu-Ar
g, -Ala-Ar g-Leu-Glu-Ala-L
eu-Gly-I 1 e-Gln-Gly-8e
r-Leu-Pro-Ala-C)lu-Glu-Le
u-Arg-Gln-Thr-1, +eu-11e-A
la-T rp-Asp -Gly-Ar g-MET
-Asp-Ala-Gly-Ser-Gln-Al
a-Ala-Be r -Ala-Tyr-As n
-Ala-Phe -Ar g-Ar g-Ala-L
eu-Thr-Arg-Leu-Va1-Thr-
Ala-Arg-8or-Gly-Leu-G
lu -Gln-Ala-11e-Ala-Hls-P
ro-Phe-Ala-Ala-Val-Pro-Pr
o-Gly-Val-Be r-Pro-Gln-G
ay-Gin-Van-Trp-Trp-Ala-Va
l-Pro-Thr-Leu-Leu-Arg-Asn
-Asp-Asp-Ala-Gly-MET-Leu
-Lys -Gly-Trp-8e r -T rp-
Asp-Glu-Ala-Leu-8er-Glu-
Ala-Leu-8er-Val-A1a=Thr-G
ln-Asn-Leu-Th r -Gly-Ar g
-Gly-Trp-Gly-()lu-Glu-18i
s -Ar g -Pro-Ar g-Phe-'r
hr-H15-Pro-Leu-8er-Ala-Gi
n -Phe -Pr o -Ala-Tr p-A
la-Ala-Le u-Leu-Asn-Pr
o-Val -8e t -Ar g-Pro -I
l e "Gly-Gly-Asp-Gly-As
p -Th r -Va 1-Leu-Ala-As
n-Gly-Le u-Va 1-Pr o-8e r
-Ala-Gly-Pro-[)1u-Ala-T
h r -Tyr -Gly-Ala-Le u-8e
r-Ar g -Tyr -Va 1-Phe -A
sp-Va 1-G 1y-As n-Tr p-As
p-ASrl-Ser-Ar g-Trp-V
a 1-Val-Phe-Hls-Gly-Ala-8
er-Gly-His-Pro-Ala-8or-Pr
o-Hls-Tyr-Ala-Asp-Gln-Asn
-Ala-Pro-Trp-8er-Asp-Cys
-Ala-MIBT-'Va1-Pro-M
gT-Leu-Tyr-Ber-Trp-As
p-Arg-I1e-Ala-Ala-G
lu-Al a-Va 1-Thr-8e r-Gln
2. The DNA fragment according to claim 1, which encodes the amino acid sequence represented by -Glu-Leu-Val-PrO-Ala. (3) Pseudomonas sp.
nasSF, ), and (a) the following amino group-terminal amino acid sequence: H2N-Th
r-Met-Ala-Ala-Lys-Thr-Asp
-Arg-Glu-Ala-Leu-Gln-Ala-
Ala-Leu-Pro-Pro-Leu-8er-G
DNA encoding an approximately 26 kilodalton (KD) subunit containing ay- and (b) the following amino-terminal amino acid sequence: H2N-8e
r-Asn-Asn-Trp-Ala-Val-Ala
-Pro-GlH2N-8er-Asn-Asn-Tr
p-Ala-Val-Ala-Pro-Gly-Arl
58 containing y-, D encoding the subunit of D
A recombinant DNA in which a B DNA fragment is incorporated into a vector (4) Pseudomonas x sp.
onasSP, ) and (a) the following amino group-terminal amino acid sequence: H2N-Th
r-Met-Ala-Ala-Lys-Thr-Asp
-Arg-Glu-Ala-Leu-Gln-Ala-
Ala-Leu-Pro-Pro-Leu-8a-d-l
y- DNA encoding the approximately 26 kilodalton (KD) subunit and (b) the following amino terminal amino acid sequence: HN-8er
-Asn-Asn-Trp-Ala-Val-Ala-
Pro-Gly-Arg-Thr-A, 1a-Thr-
Gly-Arg-Pro-11e-Leu-Ala-. 1) encoding an approximately 58KD subunit containing Gly-
Escherichia coli into which a recombinant DNA'e is introduced into which a DNA fragment characterized by being contained in NA' is incorporated into a vector. +1. The column for the detailed description of the invention in the specification shall be amended as follows. r*661 in lines 4 to 6 of Table 1 on page 31 of the specification
72° *GTT,
GAT, GCC,...GCC), CCC, GGC'
, GGC, GGC, AGC" are corrected as follows. "*661 GTT, GAT, GCC, CTC, CT(), AAA
, ()CG, AT(), 0()C, ()GC, GA
CVal-As p-A1. a-Le u-Leu-
Lys-Ala-MET-Gly-Gly-As
p720*, GCC, TCC, GAT, GCG, GCC, GGC,
GGC, ()GC, AGC procedure correction band (method) % formula % 1, Indication of the case 1982 Patent Application No. 274.108 2, Name of the invention r) responsible for cephalosporin C acylase production genetic information
NA Fragment 3, Relationship with the case of the person making the amendment Patent applicant address: 1-2-6-4 Dojimahama, Kita-ku, Osaka-shi, Osaka 530, Date of amendment order: April 30, 1985 5, Details subject to amendment Column 6 of Detailed Explanation of the Invention in the Specification (Japanese Patent Application No. 59-2741.08) is amended as follows, as shown in the appendix. (1) rMol, ecul on page 14, lines 13-15,
ar C], on-jng... (1,982) J [Molecular cloning
njng) (T. ManiaUs et al., Cold Spring Harbor Laboratory (Cold 5pri, ngtlarbor)
Laboratory) Inc. (1,982)]. (2) rshjbuya et al. on page 15, lines 9-10...
・1567J as “Shibuya et al., (1,
981) Agr, Paiol, Sam, (Agr,
Rjo], Chem,), vol. 45, 1561-156
Corrected to page 7. (3) rHunkapil on page 15, lines 15-16]
er et al... 6594 as ``Banka Pillar (Ilan
(1,983), Science, Vol. 219, pp. 650-659. (4) rMaxam on page 15, lines 17-18...
560” [Maxam and Gilbert
i (bert) (1980), Methods in Enzymolo
gy) Vol. 65, pp. 499-560. (5) Pages 1 and 7, lines 14-15, Marmur et al., 961. ) J as “Marmar”
[Reference Nijiei, Mol, Paiol, (J, M
o1. Biol, ), Vol. 38, pp. 227-239 19
1961]”. (6) r (Bethesd) on page 18, lines 13-14
a-・= manufactured by Bethesda Research Laboratory
Corrected to ``Made by Orjes Inc.''. (7) rLow meltjng on page 23, lines 1-2
・・Produced by Bethesda Research Laboratory (Bethesda Research Laboratory)
Manufactured by Laboratories). (8) Page 37, No. 1] - ~ rPrjbnow in lines 1-3
-Press” to “Prj,bnow”
(1979), Biological Regulation and Development
on and Development)
, Volume 1, pp. 231-277, Plenum Press.
m Press)”. 12 = 51rl-

Claims (4)

[Claims] (1) Pseudomonas sp.
asSP. ), and (a) DNA encoding a subunit of approximately 26 kilodaltons (KD) containing the following amino-terminal amino acid sequence: [Amino acid sequence is available] and (b) the following amino-terminal amino acid sequence: Sequence: [Amino acid sequence is available] DN encoding a subunit of approximately 58KD containing
A DNA fragment carrying genetic information for producing cephalosporin C acylase, characterized by containing A. (2) The DNA fragment according to claim 1, which encodes the amino acid sequence represented by [there is an amino acid sequence]. (3) Pseudomonas sp.
asSP. ), and (a) a DNA encoding an approximately 26 kilodalton (KD) subunit containing the following amino-terminal amino acid sequence: [Amino acid sequence exists] and (b) the following amino-terminal amino acid sequence: : [There is an amino acid sequence] DN encoding a subunit of approximately 58KD containing
A recombinant DNA obtained by incorporating a DNA fragment characterized by containing A into a vector. (4) Pseudomonas sp.
asSP. ), and (a) a DNA encoding an approximately 26 kilodalton (KD) subunit containing the following amino-terminal amino acid sequence: [Amino acid sequence exists] and (b) the following amino-terminal amino acid sequence: : [There is an amino acid sequence] DN encoding a subunit of approximately 58KD containing
Escherichia coli into which a recombinant DNA in which a DNA fragment characterized by containing A is inserted into a vector is introduced.