JPH08205864A - Novel cephalosporin C acylase and method for producing the same - Google Patents

Abstract

(57) Abstract: A mutant CC acylase in which the amino acids at the Leu ¹⁶⁰ and / or Trp ¹⁶⁸ sites of the amino acid sequence of natural cephalosporin C acylase (CC acylase) are substituted with other amino acids, A DNA encoding the same, an expression vector containing the DNA,
A method for producing a mutant CC acylase by culturing a microorganism transformed with the expression vector and the transformant. [Effect] It is possible to provide a mutant cephalosporin C acylase having high enzyme activity and high processing efficiency, an expression system for expressing the enzyme in high yield, and a method for producing the enzyme.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel cephalosporin C acylase (hereinafter referred to as CC acylase). More specifically, it relates to a novel mutant CC acylase, a gene encoding the same, an expression vector containing the gene, a microorganism transformed with the expression vector, and a method for producing the mutant CC acylase. Furthermore, the present invention relates to a method for producing a cephalosporin compound using the culture of the above transformant or a treated product thereof.

[0002]

2. Description of the Related Art CC acylase generally uses cephalosporin C as 7-aminocephalosporanic acid (7-ACA).
It is an enzyme that hydrolyzes into. So far, three enzymes, cephalosporin C acylases SE83, N176 and V22, have been found as enzymes classified as CC acylases, and their amino acid sequences are shown in the Journal of Fer.
mentation and Bioengineering, Vol.72, p232-243 (19
91). In this document, the numbering of the amino acid sequence of CC acylase begins with the methionine group at its N-terminal site.

However, in the present specification, the numbering of the amino acid sequence of CC acylase starts from the threonine group adjacent to the methionine group at its N-terminal part. This is because the methionine at the N-terminal part of the α-subunit of mature CC acylase obtained by expressing the CC acylase gene in a prokaryote is eliminated by an enzyme (for example, aminopeptidase etc.), This is because it becomes a mature CC acylase having a threonine group.

[0004] The above-mentioned document also discloses the production of natural CC acylase by recombinant DNA technology. The expressed CC acylase is processed intracellularly to consist of α-subunit and β-subunit. It is known to be the active form. However,
E. FIG. The efficiency of the processing in E. coli is generally low.

[0005]

The object of the present invention is to provide a novel mutant CC acylase, a DNA encoding the same, an expression vector containing the DNA, a transformant into which the vector is introduced, and the transformant. It is intended to provide a method for producing a mutant CC acylase by culturing. Another object of the present invention is to provide a method for producing a cephalosporin compound using the culture of the above transformant or a treated product thereof.

[0006]

[Means for Solving the Problems] As a result of intensive studies by the present inventors to obtain a CC acylase having more desirable properties such as higher enzyme activity and higher processing efficiency, a mutant form having such desirable properties has been obtained. The present invention has been completed by successful production of CC acylase.

That is, the present invention is as follows. A mutant CC acylase in which the amino acids at the Leu ¹⁶⁰ and / or Trp ¹⁶⁸ sites of the amino acid sequence of natural CC acylase are replaced with other amino acids. Further, a DNA encoding the mutant CC acylase, an expression vector containing the DNA, a transformant into which the vector is introduced, and a method for producing the mutant CC acylase by culturing the transformant. Furthermore, a method for producing a cephalosporin compound, which comprises contacting the culture of the above transformant or a treated product thereof with the compound (II) described below.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Preferable examples of other amino acids substituted by Leu ¹⁶⁰ include alanine and the like. Preferable examples of other amino acids substituted by Trp ¹⁶⁸ include tyrosine and the like. As a preferred example of the mutant CC acylase, an amino acid sequence as a precursor form before being processed into α-subunit and β-subunit in a host cell has the formula: A ^1-159 -X ₁ -A ^{161. -167} -X ₂ -A ^169-773 (In the formula, A ^1-159 is Thr ^{1 of the} natural CC acylase.
To the Arg ¹⁵⁹ amino acid sequence. A ^161-167 is Gl of natural CC acylase
The same amino acid sequence as the amino acid sequence from y ¹⁶¹ to Val ¹⁶⁷ is shown. A ^169-773 has the same amino acid sequence as the amino acid sequences of Phe ¹⁶⁹ to Ala ⁷⁷³ of natural CC acylase. X ₁ represents Leu or another amino acid, and X ₂ represents Trp or another amino acid. However, when X ₁ is Leu, X ₂ is an amino acid other than Trp). The most preferred examples of the mutant CC acylase include X ₁ alanine, X ₂ tyrosine, and combinations thereof in the above formula.

In the present specification, for the nomenclature of individual mutant CC acylases, the following nomenclature widely used by academic societies in this field is conveniently adopted. According to this nomenclature, for example, a mutant CC acylase prepared by substituting the leucine residue at position 160 of the amino acid sequence of a natural CC acylase with alanine is named mutant CC acylase L160A. L means the one-letter code for the leucine (amino acid) residue to be replaced,
Means the substitution position of the amino acid sequence of natural CC acylase, and A means the one letter symbol of alanine (another amino acid) in which the leucine (amino acid) residue is substituted. In other words, for example, the mutant CC acylase W168Y means a mutant CC acylase in which the tryptophan residue at position 168 of the amino acid sequence of the natural CC acylase is replaced with tyrosine.

The amino acid sequence of the mutant CC acylase of the present invention having such characteristics and D encoding the same
Specific examples of the nucleotide sequence of NA are shown in the sequence listing below.
SEQ ID NO: 1 shows the nucleotide sequence of DNA encoding the precursor of mutant CC acylase L160A in plasmid pCKL160A and the amino acid sequence deduced therefrom. SEQ ID NO: 2 contains plasmid pCKW1
The nucleotide sequence of DNA encoding the precursor of mutant CC acylase W168Y in 68Y and the amino acid sequence deduced therefrom are shown.

The mutant CC acylase of the present invention can be prepared by recombinant DNA technology, polypeptide synthesis method and the like.

That is, when the recombinant DNA technology is used, the novel CC acylase is a novel CC acylase, which is obtained by culturing a host cell transformed with an expression vector containing a DNA encoding the amino acid sequence in a medium, It can be prepared by collecting CC acylase.

The details of this process will be described below. Examples of the host cell include microorganisms such as bacteria (eg, Escherichia coli, Bacillus subtilis), yeasts (eg, Saccharomyces cerevisiae), animal cell lines and cultured plant cells). Preferable microorganisms include bacteria, in particular strains belonging to the genus Escherichia (for example, E. coli JM109 ATCC 53323, E. coli HB101A).
TCC 33694, E. coli HB101-16 FERM BP-1872, E. coli
294 ATCC 31446, etc., yeast, in particular strains belonging to the genus Saccharomyces (eg Saccharomyces cerevisiae AH2
2), animal cells (eg mouse L929 cells, Chinese hamster ovary (CHO) cells, etc.) and the like.

When a bacterium, particularly E. coli, is used as a host cell, an expression vector generally comprises at least a promoter-operator region, a start codon, a DNA encoding the amino acid sequence of a novel CC acylase, a stop codon, a terminator region and a replicable unit. Composed. When yeast or animal cells are used as host cells, the expression vector preferably contains at least a promoter, a start codon, a signal peptide and a DNA encoding the amino acid sequence of the novel CC acylase, and a stop codon. Enhancer sequences, 5'and 3'untranslated regions of novel CC acylases, splicing junctions, polyadenylation sites and replicable units can also be incorporated into expression vectors.

The promoter-operator region includes a promoter, an operator and a Shine-Dalgarno (SD) sequence (for example, AAGG etc.). Preferably, the promoter-operator region is a conventionally used promoter-operator region (for example, E. c.
oli PL-promoter and trp-promoter) and the CC acylase N176 chromosomal gene promoter.

As a promoter for expressing a novel CC acylase in yeast, in the case of S. cerevisiae,
TRP1 gene, ADHI or ADHII gene,
And the acid phosphatase (pH05) gene promoter, and as a promoter for expressing a novel CC acylase in mammalian cells, SV40 early promoter or late promoter, HTLV-LTR
-Promoter, mouse metallothionein I (MMT)
-Promoter, vaccinia-promoter and the like.

Suitable start codons include the methionine codon (ATG).

The signal peptides include signal peptides of other commonly used enzymes (natural t-PA signal peptide, natural plasminogen signal peptide) and the like.

The DNA encoding the amino acid sequence of the novel CC acylase can be prepared by a conventional method.
For example, some or all DNs using a DNA synthesizer
A and / or transformants [eg,
E. FIG. coli JM109 (pCCN176-2) FERM
BP-3047] from a suitable vector (eg pC
The complete DNA sequence encoding the native CC acylase inserted into CN176-2) is added to treatment with, for example, an appropriate enzyme (eg, restriction enzyme, alkaline phosphatase, polynucleotide kinase, DNA ligase, DNA polymerase, etc.). Therefore, a conventional mutation method [eg, cassette mutation method (Tokunaga, T., Eur. J. Biochem. Vol. 15
3, p445-449 (1985)), PCR mutation method (Higuchi, R
Nucleic Acids Res. Vol. 16, p7351-7367 (1988)), Kunkel method (Kunkel, T.A. et al., Methods Enzymol.
Vol. 154, p367 (1987)), etc.] and the like.

The stop codon includes a commonly used stop codon (eg, TAG, TGA, etc.).

The terminator region includes a natural or synthetic terminator (eg, synthetic fd phage terminator).

A replication-competent unit is a DN having the ability to replicate its entire DNA sequence in a host cell.
Compound A, which includes natural plasmids, artificially modified plasmids (eg, DNA fragments prepared from natural plasmids) and synthetic plasmids. A preferred plasmid is the plasmid pBR322 for E. coli, and its artificial modifications (pBR32
DNA fragment obtained by treating 2 with an appropriate restriction enzyme), yeast 2μ plasmid and yeast chromosomal DNA in yeast, and plasmid pRSVn in mammalian cells.
eo ATCC 37198, plasmid pSV2dhfr ATCC 37145, plasmid pdBPV-MMTneo ATCC 37224, and plasmid
pSV2neo ATCC 37149 etc. are mentioned.

The enhancer sequence includes the enhancer sequence of SV40 (72b.p.).

As the polyadenylation site, SV
Forty polyadenylation sites are included.

The splicing junction site is SV4
0 splicing junctions are included.

The promoter, the initiation codon, the DNA encoding the amino acid sequence of the novel CC acylase, the termination codon and the terminator region can be ligated continuously and circularly to an appropriate replicable unit (plasmid),
At this time, if desired, a conventional method (for example, digestion with a restriction enzyme,
An expression vector can be obtained by using an appropriate DNA fragment (eg, a linker, another restriction site, etc.) in ligation using T4 DNA ligase.

The host cell is transformed (transfected) with the expression vector. Transformation (transfection) can be performed by a conventional method (for example, Kushner method for E. coli, calcium phosphate method for mammalian cells, microinjection, etc.) to obtain a transformant (transfectant). .

In order to produce the novel CC acylase in the process of the present invention, the thus obtained transformant containing the above expression vector is cultivated in an aqueous nutrient culture solution.

The nutrient medium includes carbon sources (eg glucose, glycerin, mannitol, fructose, lactose) and inorganic or organic nitrogen sources (eg ammonium sulfate, ammonium chloride, casein hydrolysates, yeast extract, polypeptone, bacto). Tryptone, beef extract, etc.). If desired, other nutrient sources such as inorganic salts (e.g., sodium diphosphate, potassium diphosphate, dipotassium hydrogen phosphate,
Magnesium chloride, magnesium sulfate, calcium chloride), vitamins (eg vitamin B ₁ ), antibiotics (eg ampicillin, kanamycin) etc.] may be added to the medium. For culturing mammalian cells,
Dulbecco's Mod with fetal bovine serum and antibiotics
ified Eagle's Minimum Essenntial Medium (DMEM) is often used.

Cultivation of transformants (including transfectants) is usually pH 5.5 to 8.5 (preferably pH 7 to 7.
5), 5 to 5 at 18 to 40 ° C (preferably 20 to 30 ° C)
It is carried out for 0 hours.

When the novel CC acylase thus produced is present in the culture medium, the culture medium is filtered or centrifuged to obtain a culture filtrate (supernatant). The novel CC acylase is a conventional method commonly used for purifying and isolating natural or synthetic proteins from the culture filtrate (for example, dialysis, gel filtration, affinity column chromatography using an anti-CC acylase monoclonal antibody, a suitable method). Column chromatography of the adsorbent, high performance liquid chromatography and the like).

If the novel CC acylase produced is present in the periplasm and cytoplasm of cultured transformants, the cells are collected by filtration and centrifugation and their cell walls and / or cell membranes are filtered, eg by ultrasound and / or
Alternatively, it is disrupted by treating with lysozyme to obtain cell debris and / or crushed material. The cell debris and / or the disrupted material is dissolved in an appropriate aqueous solution (eg, 8M aqueous urea solution, 6M aqueous guanidinium salt solution). New CC
Acylase can be purified from the aqueous solution by a conventional method as exemplified above.

The invention further comprises the formula:

[0034]

Embedded image

(Wherein R ¹ represents acetoxy, hydroxy or hydrogen; R ² represents an acyl carboxylate), or a salt thereof is used as a novel CC of the present invention.
A method comprising contacting with a culture of a microorganism transformed with an expression vector containing a DNA encoding an acylase or a treated product thereof:

[0036]

[Chemical 4]

The present invention provides a method for producing a compound (I) represented by the formula (wherein R ¹ has the same meaning as defined above) or a salt thereof.

As the carboxylate acyl for R ² ,
Aliphatic, aromatic or heterocyclic carboxylate acyl groups, suitable examples of which include amino groups, hydroxy groups,
A carboxy group, a C ₁ -C ₆ alkanoylamino group, a benzamido group, a thienyl group and the like, which may have one or two appropriate substituents, may be a C ₁ -C ₆ alkanoyl group. Can be mentioned.

Suitable salts of compound (I) and compound (II) include alkali metal salts (for example, sodium salt,
Potassium salt, lithium salt).

If the CC acylase activity is usually present in the transformed cells, examples of the substance obtained by treating the culture (hereinafter referred to as the treated substance) are as follows.

(1) Living cells: separated from the culture by a conventional method such as filtration or centrifugation; (2) dried cells: the living cells of the above (1) are freeze-dried or vacuum-dried. (3) Cell-free extract: live cells of (1) above or (2) above
(4) are obtained by disrupting the dried cells of (1) by a conventional method (for example, autolysis of cells using an organic solvent, crushing of cells using alumina, sea sand, etc., or treatment of cells with ultrasonic waves); Enzyme solution: Obtained by purifying or partially purifying the cell-free extract of (3) above by a conventional method (for example, column chromatography); and (5) Immobilized cells or enzymes: (1) or (2) above. Cells) or the enzyme of (4) above is immobilized by a conventional method (for example, a method using acrylamide, glass beads, an ion exchange resin, etc.).

The reaction consisting of contacting the compound (II) of the present invention with an enzyme can be carried out in an aqueous medium such as water or a buffer. That is, usually the compound (II)
Is carried out by dissolving or suspending the culture or a treated product thereof in an aqueous medium such as water or a buffer solution containing

The suitable pH of the reaction mixture, the concentration of compound (II), the reaction time and the reaction temperature may vary depending on the properties of the culture used or its treated product. Generally, the reaction is pH 6-10, preferably pH 7-9, 5-40.
It is carried out at 0 ° C, preferably 5 to 37 ° C for 0.5 to 50 hours.

The concentration of the compound (II) as a substrate in the reaction mixture can be appropriately selected from the range of 1 to 100 mg / ml.

Compound (I) thus produced
Is purified and isolated from the above reaction mixture by a conventional method.

In the following examples, plasmids, enzymes such as restriction enzymes, T4 DNA ligase, and other materials are commercially available and used by conventional methods. In particular,
Plasmid pCCN used as starting material in the examples
176-2 and the plasmid pTQiPAΔtrp,
Transformed E. coli JM109 (pCCN176-2) FERM into the Institute of Biotechnology, Institute of Biotechnology, which is an international depository.
BP-3047 and transformed E. coli HB101-16 (pTQiPAΔtr
p) Deposited as FERM BP-1870, and US Pat. No. 5,192,678 and European Patent Application Publication No. 30.
It can be easily obtained based on No. 2456. Other plasmids and the like can be easily prepared from the above pCCN176-2, pTQiPAΔtrp and their commercial products according to the description in these specifications. DNA
The procedures used for the cloning of E. coli, transformation of host cells, culture of transformants, collection of novel CC acylase from the culture, etc. are well known to those skilled in the art, or adopted from the literature. It is possible.

[0047]

EXAMPLES AND EXPERIMENTAL EXAMPLES The following examples are given for the purpose of explaining the present invention, and the present invention is not limited thereto.

Example 1 Synthesis of oligodeoxyribonucleotide (1) SO-L160A: DNA oligomer SO-L1
60A (5'-CGCGGTGATGCGCAGGGCGGGCCTGCTTATG-3 ') 3
It was synthesized using a 92 type DNA / RNA synthesizer (manufactured by Applied Biosystems). The DNA is CPG
The (supported pore glass) polymer support was liberated with 28% aqueous ammonia and then heated at 60 ° C. for 9 hours to remove all protecting groups. The reaction mixture was evaporated under reduced pressure, and the residue was mixed with TE buffer [10 mM Tris-HCl (pH
7.4) -1 mM EDTA] was dissolved in 200 μl.
The obtained crude DNA solution was subjected to reverse phase HPLC [column; COSM
OSIL C18, 4.6 mm × 150 mm (Nacalai Tesque), eluent; A: 0.1 M triethylammonium acetate buffer (pH 7.2-7.4), B: acetonitrile, gradient; start: A (100%) , End: A (60%)
+ B (40%), 25 min linear gradient, flow rate;
1.2 ml / min]. The eluate containing the target DNA oligomer was collected and evaporated under reduced pressure. T the purified DNA
It was dissolved in 200 μl of E buffer and stored at −20 ° C. until use.

(2) SO-W168Y: DNA oligomer SO-W168Y (5'-CAGCATCCGCCAAAGCTTGAAATACACC
GAACCCATAAG-3 ') was synthesized and purified by the same method as described above.

Example 2 Preparation of expression vector of natural CC acylase N176 under control of trp promoter (1) Construction of ampicillin resistant expression vector pCC002A of natural CC acylase N176: (i) Construction of pCC001A: plasmid pCCN17
6-3 [JOURNAL OF FERMENTATION AND BIOENGINEERIN
G, Vol. 72, p235 (1991), plasmid pCCN17.
6-2 (this is a transformant E.co
li JM109 (pCCN176-2) FERM B
(Which can be obtained from P-3047) has been described] (1.0 μg).
Was digested with EcoRI and HindIII to carry the entire coding region of CC acylase N176, 2.9k.
The b fragment was isolated by agarose gel electrophoresis. On the other hand, pT which is an expression vector of mutant t-PA
QiPAΔtrp [This is a transformant E. coli HB101-16 (pTQiPAΔtrp) FERM according to a conventional method.
It can be obtained from BP-1870. Its preparation method is described in EP-A-302456] (1.0 μg) in XmaI and X
Digested with hoI and isolated the large DNA (5113 bp). Synthetic DNA oligomer CT-1 (5'-CCGGGTGTGTA
CACCAAGGTTACCAACTACCTAGACTGGATTCGTGACAACATGCGACCGT
GA-3 '), CT-2 (5'-AGCTTCACGGTCGCATGTTGTCACGAATC
CAGTCTAGGTAGTTGGTAACCTTGGTGTACACAC-3 '), TR-1
(5'-AGCTTGTCCTCGAGATCAATTAAAGGCTCCTTTTGGAGCCTTTTTT
TTTTG-3 ') and TR-2 (5'-TCGACAAAAAAAAAAGGCTCCA
AAAGGAGCCTTTAATTGATCTCGAGGACA-3 ') was phosphorylated with T4 polynucleotide kinase and ligated with T4 DNA ligase to give an XmaI / SalI DNA fragment (114 bp). The obtained DNA fragment was ligated to the above XmaI / XhoI DNA, and pTQi
PAdtrp was obtained. pTQiPAdtrp (1.0μ
g) was digested with EcoRI and HindIII. 4.3 kb carrying the resulting trp promoter, part of the t-PA coding region (Cys ⁹² to Trp ¹¹³ ), and the fd phage central terminator copy sequence.
DNA was isolated. 50 mM Tris-HCl, 10 mM
MgCl ₂ , 10 mM dithiothreitol and 1 mM
In 40 μl of ligation buffer consisting of ATP, T
In the presence of 4 DNA ligase (300 units, manufactured by Takara Shuzo), the DNA fragment of 2.9 kb and 4.3
The kb DNA fragments were mixed and allowed to ligate for 5 hours at 16 ° C. The ligation mixture was used to transform E. col
i JM109 was transformed. A desired plasmid designated as pCC001A was obtained from one of the transformants resistant to ampicillin, and its characteristics were examined by restriction enzyme mapping.

(Ii) Construction of pCC002A: The plasmid pCC001A contains a part of the t-PA gene (Cys ⁹² to T) between the trp promoter and the acylase gene.
rp ^{11 3} ) is included. To remove this region, pCC
001A (1.0 μg) was digested with ClaI and MluI and the resulting 6.1 kb DNA fragment was isolated. On the other hand, pCCN176-3 (1 μg) was added with MluI.
And Sau3AI were digested to isolate a 189 bp DNA encoding the acylase between Asp ⁷ and Arg ⁷¹ . Synthetic DNA oligomers 002a and 002b (0.5 nmol, see Table 1 below) were used as a buffer (Kination buffer; 50 mM Tris-HCl, 10 mM MgCl ₂ ) using T4 polynucleotide kinase (1.5 units, manufactured by Takara Shuzo). 10 mM DTT, 1.0 m
Phosphorylation in 10 μl of MATP) at 37 ° C. for 1 hour and the reaction mixture was heated at 55 ° C. for 20 minutes to inactivate the enzyme. The obtained mixture was mixed with 20 μl of ligation buffer in the presence of T4 DNA ligase at 15 ° C. for 3 hours with the 189 bp Sau3AI / MluI DNA to bind. To the resulting binding mixture, 6.1k
claI / MluI DNA fragment of b.
T4 DNA ligase (300
Unit) for 16 hours at 4 ° C. The resulting binding mixture was used to transform E. coli JM
109 was transformed. The desired plasmid pCC002A, which is an expression vector for CC acylase N176, was isolated from one of these transformants, and its characteristics were examined by restriction enzyme mapping.

[0052]

[Table 1]

(2) Construction of pCK002, a kanamycin resistance expression vector of CC acylase N176: DraI (Toyobo Co., Ltd.) was used as plasmid pCC002A.
Digested with. The resulting mixture was treated with phenol to remove the enzyme and precipitated with ethanol. Recovered DNA
Suspended in 20 μl of ligation buffer, mixed with phosphorylated EcoRI linker (2 μg, Pharmacia), then T4 DNA ligase (300 units)
And incubated for 16 hours at 4 ° C. The reaction mixture was extracted with phenol and precipitated with ethanol.
The recovered DNA was digested with EcoRI, and the obtained 5.6 kb DNA lacking the ampicillin resistance gene was isolated by agarose gel electrophoresis. On the other hand, the plasmid pAO97 [which contains the transformant E. coli JM
109 (pAO97) FERM BP-3772] (1 μg) was digested with EcoRI, resulting in the isolation of a 1.2 kb DNA of the kanamycin resistance gene. T4DN in 50 μl ligation buffer
1.2k using A ligase (300 units)
b. EcoRI DNA of b.
It was ligated to 6 kb EcoRI DNA. E. coli with the ligation mixture. E. coli JM109 was transformed to obtain the desired plasmid pCK002 carrying the kanamycin resistance gene as an antibiotic marker.

Example 3 Construction of pCK013 which is a high expression vector of CC acylase N176 (1) Construction of pCC013A: (i) Construction of pCC007A: Plasmid pCC001
5. A was digested with EcoRI and MluI and obtained 6.
The 4 kb DNA fragment was isolated by agarose gel electrophoresis. Synthesized DNA oligomers 007a and 007b in which the recovered DNA has been phosphorylated in advance
(0.5 μg each, see Table 2 below) was bound with T4 DNA ligase (300 units) at 16 ° C. for 5 hours. The resulting mixture was used to transform E. coli J
M109 was transformed and the desired plasmid pCC007
I got A.

(Ii) Construction of pCCNt013: Plasmid pCC007A (1.0 μg) was digested with ClaI and BamH.
After digestion with I, the resulting 6.1 kb DNA was isolated by 5% polyacrylamide gel electrophoresis. The DNA was synthesized with synthetic oligomers 013a and 013b (0.5 μg each,
Each is phosphorylated. See Table 2 below), T4
Ligation was performed using DNA ligase (300 units). E. coli with the ligation mixture. E. coli JM109 was transformed and the desired plasmid pCCNt013 was isolated from ampicillin resistant transformants.

[0056]

[Table 2]

(Iii) Construction of pCC013A: The plasmid pCCNt013 was digested with BamHI and MluI,
The resulting 6.1 kb DNA was isolated. On the other hand, pCC
002A (1.0 μg) was digested with MluI and Sau3AI to obtain a 189 bp DNA fragment. The resulting DNA was ligated to the 6.1 kb BamHI / MluI DNA fragment using T4 DNA ligase (300 units) and the ligation mixture was used to E. co
li JM109 was transformed. From one of the ampicillin-resistant transformants, AT-rich NH ₂ -terminal DN
The desired plasmid pCC013A having the A sequence (encoding an amino acid sequence identical to the amino acid sequence of native CC acylase N176) was isolated.

(2) Construction of pCK013, a kanamycin resistant expression vector of natural CC acylase N176: (i) Construction of pΔN176: plasmid pCK002
(1.0 μg) was digested with AatII (manufactured by Toyobo Co., Ltd.) and treated with T4 DNA ligase (150 units) for self-ligation of the obtained DNA. E. coli with the ligation mixture. E. coli JM109 was transformed to give the desired plasmid pΔN176 carrying a unique AatII restriction enzyme cleavage site.

(Ii) Construction of pCK013: plasmid p
ΔN176 (1.0 μg) was digested with AatII, and the linearized DNA was treated with bacterial-derived alkaline phosphatase (1 unit, manufactured by Takara Shuzo) in 100 mM Tris-HCl buffer (pH 8.0) at 42 ° C. for 1 hour. did. The dephosphorylated DNA was isolated and 2.5 kb AatIIDN from pCC013A was isolated using T4 DNA ligase.
Bound to A. E. coli with the ligation mixture. coli
JM109 was transformed to obtain a desired plasmid pCK013 carrying a kanamycin resistance gene as a marker.

Example 4 Point mutation of the DNA encoding CC acylase N176 by the Kunkel method (1) M of DNA encoding CC acylase N176
Subcloning into 13 phage: (i) Preparation of mp18p181: plasmid pCC01
The 3A digested with HpaI and SmaI, the DNA of 842bp encoding the fMet of CC acylase N176 to Pro ²⁶⁷ was isolated. The DNA was labeled with SmaI
Digested with 7250bp M13mp18, T4D
Ligation was performed in the presence of NA ligase and the ligation mixture was used to transform E. E. coli JM109 was transformed. From one of the plaques, the above acylase DNA portion is M13
The desired RF DNA mp18p181 inserted in the reverse orientation with DNA containing the phage ori was isolated and characterized by restriction enzyme mapping. The RF D
The phage solution prepared from NAmp18p181 was stored at 4 ° C until use.

(Ii) Preparation of mp18p183: CC acylase N1 derived from pCC013A was prepared in the same manner as described above.
116 encoding 76 fMet to Ala ³⁷⁴
2 bp HpaI / Eco47III DNA and Hinc
From 7250 bp M13mp18 digested with II,
RF DNAmp18p183 was prepared.

(2) Preparation of single-stranded uracil template DNA (see Kunkel, TA et al., Methods Enzyml., Vol.154, p367): (i) U-mp18p181-SS: E. coli C
A single colony of J236 (dut-, ung-, F ') (manufactured by Bio-Rad Laboratories) was cultured in 2 ml of 2XTY broth containing chloramphenicol (30 µg / ml) at 37 ° C for 16 hours. Cells (0.1
ml) was transferred to fresh 2XTY broth (50 ml) containing 30 μg / ml chloramphenicol, and cultivation was continued at 37 ° C. When the absorbance at 600 nm reached 0.3, the mp18p181 phage solution (MOI <0.2) was added to the culture, and the culture was further continued for 5 hours. This was centrifuged at 4 ° C. and 17,000 × g for 15 minutes, and then the supernatant was again centrifuged. Obtained supernatant (30
ml) at room temperature for 30 minutes, RNase (150 μg /
ml, manufactured by Sigma), and 7.5 ml of a PEG solution (3.5% NH ₄ OAc in 20% polyethylene glycol 8,000 solution) was added. Centrifuge (17,0
(00 × g, 15 minutes, 4 ° C.), the residue was treated with 300 mM Na
Cl, 100 mM Tris-HCl (pH 8.0) and 0.
The cells were suspended in 200 μl of a buffer solution containing 1 mM EDTA. The resulting solution was extracted with 200 μl phenol and 200 μl phenol / CHCl ₃ (1/1), followed by two washes with CHCl ₃ (200 μl). 7.5 M NH ₄ OAc (100 μl) and ethanol (600 μl) were added to the solution to precipitate phage DNA. The DNA was collected by centrifugation, washed with 700 μl ice-cold 90% ethanol and dried under vacuum. Purified single-stranded U-DNA (U-mp18p181-SS)
Was suspended in 20 μl of TE buffer and stored at 4 ° C. until use.

(Ii) U-mp18p183-SS: U which is another single-stranded U-DNA used in the Kunkel mutation method.
-Mp18p183-SS was also prepared in the same manner as described above.

(3) RF DNA (mp18p181L160) encoding mutant CC acylase L160A
Preparation of A): (i) Phosphorylation of oligodeoxyribonucleotides: D
NA oligomer SO-L160A (1.84 μl, about 1
67 pmol) using T4 polynucleotide kinase (4.5 units, manufactured by Takara Shuzo), 50 mM Tris-HCl (pH 7.8), 10 mM MgCl ₂ , 20 m
Phosphorylation was carried out for 1 hour at 37 ° C. in 30 μl of a ligation buffer consisting of M dithiothreitol (DTT), 1 mM ATP and 50 μl / ml bovine serum albumin (BSA).

(Ii) Annealing reaction: 10 mM Tris-HCl (pH 8.0), 6 mM MgCl ₂ and 40 m
The above phosphorylated oligomer (1 μl, about 5.6 pmol) was added to template U-mp18p181-SS (0.1 pmol, about 2) in 9 μl of a buffer solution containing M NaCl.
50 ng). The mixture was heated at 70 ° C. for 5 minutes, then cooled at 30 ° C. over 40 minutes and left at 0 ° C.

(Iii) Double-stranded DNA in vitro
Synthesis of: The obtained annealing solution (10 μl) was added to a synthesis buffer [200 mM Tris-HCl (pH 8.0) -40
mMMgCl ₂ ] 1 μl, 5 mM dNTP (dAT
P, dCTP, dGTP and dTTP) 1 μl, T4
DNA ligase (300 units, Takara Shuzo) and T
Mixed with 4 DNA polymerase (10 units, Pharmacia). The mixture is placed on ice for 5 minutes at 25 ° C. for 5 minutes.
The cells were sequentially incubated for 30 minutes at 37 ° C. for 90 minutes. 10 mM Tris-HCl (pH 8.0) and 10 m
The reaction was stopped by adding 90 μl of a buffer consisting of M EDTA.

(Iv) E. Transformation of E. coli JM109: A portion (3 μl) of the above reaction mixture was subjected to Sigesada method [Si
gesada, K., Cell Engineering 2, p616-626 (1983)]. E. coli JM109 was added to competent cells (200 μl) and the cells were incubated on ice for 30 minutes. E. FIG. A single colony of E. coli JM109 was cultured in L broth (2 ml) for 16 hours.
The culture (0.1 ml) was transferred to fresh L broth (2 ml) and further cultured for 2 hours to obtain an indicator cell. The indicator cells (200 μl) were added to the transformed cells (200 μl) and they were heated to 55 ° C.
H-Top agar (1% bactotryptone, preheated with
0.8% NaCl, 0.8% agar) 3 ml.
Spreading the cell-agar mixture on H-plates (1% bactotryptone, 0.5% NaCl, 1.5% agar),
The plate was incubated at 37 ° C for 16 hours.

(V) RF DN obtained from the transformant
Characteristics of A: E. E. coli JM109 single colonies were treated with 2XTY broth (1.6% bactotryptone, 1%
It was cultured in 2 ml of yeast extract, 0.5% NaCl) for 16 hours. The culture (0.1 ml) was transferred to fresh 2XTY broth (2 ml) and further cultured for 2 hours. The plaques of the plate prepared in step (iv) were picked up with a bamboo toothpick (15 cm) and immediately transferred to the above-cultured 2XTY broth. Culturing was continued at 37 ° C. for 5 to 6 hours. 1 ml of the culture at room temperature for 15 seconds for 10,0
Cells were collected by centrifugation at 00 rpm and the supernatant (phage solution used for bulk preparation of RF DNA in subsequent experiments) was stored at 4 ° C until use. The cells were treated with GTE buffer [50 mM glucose, 25 mM Tris-HCl (pH 8.0), 25 mM EDTA] 100 μm.
1 and suspended in an alkali-SDS solution (0.2N NaO
H., 1% SDS) gently mixed with 200 μl and high salt buffer (3M K) using a Vortex ^™ mixer.
Mix vigorously with 150 μl of OAc, 3M AcOH). The obtained mixed solution is kept at room temperature for 5 minutes and 10,000 rp
Centrifuge at m. The supernatant (400 μl) was treated with 300 μl of isopropyl alcohol to give 10,000 rp
Centrifuge for 5 minutes at m. The upper layer (300 μl) was separated and mixed with 600 μl of ethanol. After centrifuging it (10,000 rpm, 5 minutes), the precipitate was washed with 500 μl of ice-cold 70% ethanol, dried under vacuum, and added to 50 μl of TE buffer containing 100 μg / ml of RNase (manufactured by Sigma). Suspend and RF DNA mp
18p181L160A was obtained. A portion (10 μl) of the RF DNA solution was used for restriction endonuclease mapping with FspI. This is because the DNA oligomer SO-L160A systematically introduces an FspI site into its sequence to investigate mutations.

(4) RF DNA (mp18p183W168) encoding mutant CC acylase W168Y
Preparation of Y: Mutant RF DNA (mp18p183W
168Y) and mp18p18 in the same manner as above.
Prepared from 3 single-stranded U-DNA (as template) and oligomer SO-W168Y.

Example 5 Construction of Expression Vector (1) Construction of pCKL160A: mp18p181L1
60A was digested with MluI and BstBI and 582b
p DNA was isolated. On the other hand, pCK013 is BstB
Digested with I and MluI to give large DNA (about 6.2k
b) was isolated. The obtained DNA was ligated with the 582 bp DNA fragment using T4 DNA ligase to obtain a desired plasmid pCKL160A carrying a kanamycin resistance gene as a marker.

(2) Construction of pCKW168Y: mp18
p183W168Y was digested with MluI and BstBI. A small DNA fragment (582 bp) was isolated,
PCK digested with MluI and BstBI
013 large DNA (about 6.2 kb), ligation buffer [50 mM Tris-HCl (pH 7.8), 10 m
M MgCl ₂ , 20 mM dithiothreitol, 1 mM
ATP, 50 μg / ml BSA] was added in 20 μl. E. coli with the ligation mixture. coli JM10
9 was transformed. The desired plasmid pCKW168Y was isolated from one of these transformants and characterized by restriction endonuclease digestion.

Example 6 Nucleotide Sequence Determination The nucleotide sequences of pCKL160A and pCKW168Y were determined using Dye Deoxy using a 373A type DNA sequencer (manufactured by Applied Biosystems).
Determined directly by the ^TM method. DNA oligomer P4
08 (CCCTGCCTGTCGAATACGGA, coding chain: nucleotide No. 408-427) and P661 (ATCGCC
TTCAGCAGCGCATC, reverse chain: nucleotide No. 671-6
90) was used as the primer.

Example 7 Recombinant E. coli for culture col
i Each expression vector obtained in Example 5 (pCKL16
0A, pCKW168Y) and E. c
It was introduced into oli JM109. Expression vector (E.c.
oli JM109 / pCKL160A or E. co
E. coli JM109 / pCKW168Y).
50 μg of a single colony of E. coli JM109
/ 3 in L broth (5 ml) containing kanamycin
It was cultured at 7 ° C. for 16 hours. The obtained culture (0.8 m
l) was mixed with 80% glycerol water (0.2 ml),
Stored at -80 ° C until use.

Example 8 Recombinant E. Culture of E. coli (1) E. coli coli JM109 / pCKL160A
Culture: E. coli JM109 / pCKL160A
(1 ml) glycerol stock, 50 μg / ml
Add to 100 ml of L broth containing kanamycin,
The mixture was incubated at 30 ° C. for 8 hours. Part of the culture
(3.75 ml) with 1% glycerol and 25 μg /
N-3 broth containing ml kanamycin (component: 5.0
% Soybean Hydrolyzate, 0.608% Na₂HPO_Four, 0.7% K
H₂PO_Four, 0.7% K ₂HPO_Four, 0.12% (NH_Four)₂
SO_Four, 0.02% NH_FourCl, 0.0011% FeSO _Four, 0.00
11% CaCl₂, 0.00028% MnSO_Four・ NH₂O, 0.
00028% AlCl₃・ 6H₂O, 0.00011% CoCl₂
・ 6H₂O, 0.000055% ZnSO_Four・ 7H₂O, 0.0000
55% NaMoO_Four・ 2H₂O, 0.000028% CuSO_Four・
7H₂O, 0.000014% H₃BO_Four, 0.096% MgS
O_Four) Added to 25 ml. The mixture was stirred at 20-22 ° C.
Incubate and indole act 16 hours after the start of culture.
Lyric acid (final concentration 20 μg / ml) was added to 16 and 24
Glycerol (1.0% final concentration) was added over time. Three
After 0 hours, the absorption of the culture at 600 nm reached 15.0.
The cells were centrifuged (4 ° C., 7,000 rp).
m, 10 minutes) and crushed by sonication
Was. After centrifugation, collect the supernatant and store at 4 ° C to
Measures enzyme activity and GL-7ACA acylase activity
And purified mutant CC acylase L16
Used to prepare 0A.

(2) E. coli JM109 / pCK
Culture of W168Y: E. coli JM109 / pCK
W168Y was also cultured in the same manner as above.

Example 9 Purification and properties of mutant CC acylase (1) Purification of mutant CC acylase (i) Mutant CC acylase L160A: 20 m culture
from E.I. coli JM109 / pCKL1
Ammonium sulfate (4.18 g, final concentration: 35% saturated) was added to the crushed product of 60 A (20 ml), and the mixture was stirred at room temperature for 20 minutes. The resulting mixture was centrifuged (20 ° C., 15,000 rpm, 20 minutes), and the supernatants were collected and treated with ammonium sulfate (5.56 g, final concentration: 75% saturated). Centrifuge (20 ℃, 15,000
The precipitate was collected at 25 rpm for 20 min.
It was dissolved in 5 ml of M Tris-hydrochloric acid (pH 8.0). The resulting solution was dialyzed twice at 4 ° C. against 4 L each of 25 mM Tris-HCl buffer. The dialyzed product obtained is Mille
x-HV ^™ (0.45 μm, manufactured by Millipore), and high performance liquid chromatography [column; TSK-gel ^™]
DEAE-5PW (manufactured by Tosoh, 7.5 mm x 75 m)
m), eluate; A: 25 mM Tris-HCl (pH 8.
0), B: 0.5 M NaCl-25 mM Tris-HCl (pH 8.0), gradient; start: A (80%) +
B (20%), end (after 30 minutes): A (50%) + B
(50%), flow rate; 1.0 ml / min]. The main peak eluted with NaCl at a concentration of about 0.16 M was collected and dialyzed against 4 L of Tris-hydrochloric acid (pH 9.0) to obtain pure mutant CC acylase L160A.

Purified mutant CC acylase was purified by reverse phase HP
LC [column; COSMOSIL, 4.6 mm x 50 m
m (manufactured by Nacalai Tesque), eluate; linear gradient of 60% in 0.05% trifluoroacetic acid from acetonitrile water at an initial concentration of 36% for 20 minutes] and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-P)
AGE). The purity of the acylase thus obtained was about 95% by HPLC and SDS-PAGE.

(Ii) Mutant CC acylase W168Y:
The mutant CC acylase W168Y was purified and analyzed by the same method as described above.

(2) Properties of mutant CC acylase: (i) GL-7ACA acylase activity: GL-7ACA solution pre-incubated at 37 ° C. for 10 minutes [0.1
10 mg / ml, p in 5M Tris-HCl (pH 8.7)
H was readjusted to 8.7 with 1N NaOH] 500μ
100 μl of sample acylase was added to 1 μl and the mixture was incubated at 37 ° C. for 5 minutes. The reaction was stopped by the addition of 550 μl of 5% acetic acid and the resulting supernatant was used to measure 7ACA production. HPLC conditions; column: LiChrospher 10
0 RP-18 (4.0 mm x 250 mm (E. Mer
k), eluent: 2.09% citric acid and 0.09%
9.5% (v / v) acetonitrile water containing 1-hexanesulfonic acid, flow rate; 1.0 ml / min, injection rate; 1
0 μl, detector 254 nm 1 unit was defined as the activity capable of producing 1.0 μmol of 7ACA from GL-7ACA at 37 ° C. for 1 minute.

(Ii) CC acylase activity: 10 at 37 ° C.
CC acylase solution pre-incubated for 10 minutes [10
0.1 mg / ml containing cephalosporin C sodium salt.
15M Tris-HCl (pH 8.7) solution, pH is 1N N
100 μl of sample acylase was added to 500 μl, which was readjusted to 8.7 with aOH, and the mixture was mixed at 37 ° C. for 3 times.
Incubated for 0 minutes. Reaction is 5% acetic acid 550μ
It is stopped by adding 1 l, and the resulting supernatant is added to 7ACA.
Used to measure production. HPLC conditions; column: LiChrospher 10
0 RP-18 (4.0 mm x 250 mm (E. Mer
k), eluent: 2.09% citric acid and 0.09%
9.5% (v / v) acetonitrile water containing 1-hexanesulfonic acid, flow rate; 1.0 ml / min, injection rate; 1
0 μl, detector 254 nm 1 unit, cephalosporin C for 1 minute at 37 ° C.
It was defined as the activity capable of producing 1.0 μmol of 7ACA from the sodium salt. The results are shown in Table 3.

[0081]

[Table 3]

(Iii) Keto-CC acylase activity: 37 ° C.
305 μl of CC acylase solution pre-incubated for 10 minutes with 0.15 M phosphate buffer (pH 7.5) solution containing 10 mg / ml cephalosporin C sodium salt, pH was readjusted to 7.5 with 1N NaOH
To this was added 24 units of D-amino acid oxidase and 20 μl of catalase (final volume 500 μl). The mixture was vigorously stirred at ambient temperature for 5 minutes. After confirming the complete disappearance of CC acylase by HPLC analysis, the reaction mixture was treated with the substrate, 7-β- (5-carboxy-5-oxo-1-
Pentanoyl) amino-cephalosporanic acid (keto-C
Used for C). Sample acylase 1 was added to 500 μl of keto-CC solution pre-incubated at 37 ° C. for 10 minutes.
00 μl was added and the mixture was incubated at 37 ° C. for 30 minutes. The reaction was stopped by adding 550 μl of 5% acetic acid. Centrifugation of the resulting mixture (normal temperature, 1
After 10,000 rpm for 5 minutes), the supernatant was used to measure 7ACA production. HPLC conditions; column: LiChrospher 10
0 RP-18 (4.0 mm x 250 mm (E. Mer
k), eluent: 2.09% citric acid and 0.09%
9.5% (v / v) acetonitrile water containing 1-hexanesulfonic acid, flow rate; 1.0 ml / min, injection rate; 1
0 μl, detector 254 nm 1 unit from Keto-CC for 1 min at 37 ° C. for 1 min.
It was defined as the activity capable of producing 0 μmol of 7ACA. Table 4 shows the results.

[0083]

[Table 4]

(3) Determination of amino terminal sequence of mutant CC acylase: (i) Isolation of α and β subunits of mutant CC acylase L160A: Reversed phase HPLC of mutant CC acylase L160A [column: COSMOSIL 5C4]
(4.6 mm × 50 mm, manufactured by Nacalai Tesque), eluent: 0.05% trifluoroacetic acid, acetonitrile water initial concentration 36% to 60% linear gradient in 20 minutes,
Flow rate: 1.0 ml / min]. In the HPLC analysis, the purified acylase was dissociated into two-component peptides, α and β subunits, which were about 51% each.
And eluted with about 48% strength acetonitrile.
Each eluate was collected and used as a sample for amino terminal analysis.

(Ii) Determination of amino terminal sequence: the α
The amino-terminal sequence of the (β) subunit was determined by a gas phase sequencer 470A (manufactured by Applied Biosystems), and it was confirmed that it coincided with the expected one.

Example 10 (1) Large-scale purification of mutant CC acylase L160A:
E. FIG. E. coli JM109 / pCKL160A was cultured in a 5 L jar fermenter by the same method as described above. Cells obtained from 2 L of culture were
The suspension was suspended in 1.5 L of Tris-hydrochloric acid (pH 8.0) and crushed with Manton-Golin (500 kg / cm, 7 times). Centrifuge (4 ° C, 7,000 rpm, 30 minutes)
After that, the supernatant was added to Polymin P (final concentration 0.01%).
Processed in. The mixture was centrifuged at 7,000 rpm for 30 minutes and the supernatant was dialyzed against water (yield 3.8
g). The acylase (2.3 g) was subjected to QAE-Toyopearl 550C column chromatography [conditions: column: 5.0 cm × 13 cm (260 ml), starting equilibrium solution: 20 mM Tris-HCl (pH 8.0), eluent: 0.
5M NaCl-20 mM Tris-HCl (pH 8.0),
Flow rate: 10 ml / min], 0.92 g
The purified mutant CC acylase L160A was obtained. The specific activity of the purified mutant CC acylase L160A is shown in Example 9.
It was 3.51 unit / mg, which was the same as that of CC acylase measured according to the method described in (3).

(3) Large-scale purification of mutant CC acylase W168Y: E. coli JM109 / pCKW168
Y was cultured in a 5 L jar fermenter by the same method as described above. Cells obtained from 2 L of culture were suspended in 1.5 L of 20 mM Tris-hydrochloric acid (pH 8.0) and Manton-Gorlin (500 kg / cm, 7
It was crushed by Centrifuge (4 ℃, 7,000rp
m, 30 minutes), the supernatant was treated with Polymin P (final concentration 0.01%). 7,000 rp of the mixture
The mixture was centrifuged at m for 30 minutes, and the supernatant was dialyzed against water (yield 8.3 g). QAE with the acylase (1.0 g)
-Toyopearl 550C column chromatography [conditions: column: 5.0 cm x 13 cm (260 m
l), starting equilibration solution: 20 mM Tris-HCl (pH 8.
0), eluate: 0.5 M NaCl-20 mM Tris-HCl (pH 8.0), flow rate: 10 ml / min], and 0.9 g of purified mutant CC acylase W168.
I got Y. The specific activity of the purified mutant CC acylase W168Y was 4.06 units / mg, which was the same as CC acylase measured according to the method described in Example 9 (3).

[0088]

INDUSTRIAL APPLICABILITY According to the present invention, a mutant cephalosporin C having high enzyme activity and high processing efficiency
It is possible to provide an acylase, an expression system for expressing the enzyme in high yield, and a method for producing the enzyme.

[0089]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 2332 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Sequence features Characteristic symbols: CDS Location: 11. ． 2329 Method for determining feature: E Feature symbol: peptide Location: 11. ． 2329 Method for determining characteristic: S Character for expressing characteristic: mutation Location: 160 Method for determining characteristic: S sequence CGATAAA ATG ACT ATG GCA GCT AAT ACG GAT CGC GCG GTC TTG CAG GCG 49 Met Thr Met Ala Ala Asn Thr Asp Arg Ala Val Leu Gln Ala -1 1 5 10 GCG CTG CCG CCG CTT TCC GGC AGC CTC CCC ATT CCC GGA TTG AGC GCG 97 Ala Leu Pro Pro Leu Ser Gly Ser Leu Pro Ile Pro Gly Leu Ser Ala 15 20 25 TCG GTC CGC GTC CGG CGC GAT GCC TGG GGC ATC CCG CAT ATC AAG GCC 145 Ser Val Arg Val Arg Arg Asp Ala Trp Gly Ile Pro His Ile Lys Ala 30 35 40 45 TCG GGC GAG GCC GAT GCC TAT CGG GCG CTG GGC TTC GTC CAT TCG CAG 193 Ser Gly Glu Ala Asp Ala Tyr Arg Ala Leu Gly Phe Val His Ser Gln 50 55 60 GAC CGT CTT TTC CAG ATG GAG CTG ACG CGT CGC AAG GCG CTG GGA CGC 241 Asp Arg Leu Phe Gln Met Glu Leu Thr Arg Arg Lys Ala Leu Gly Arg 65 70 75 GCG GCC GAA TGG CTG GGC GCC GAG GCC GCC GAG GCC GAT ATC CTC GTG 289 Ala Ala Glu Trp Leu Gly Ala Glu Ala Ala Glu Ala Asp Ile Leu Val 80 85 9 0 CGC CGG CTC GGA ATG GAA AAA GTC TGC CGG CGC GAC TTC GAG GCC TTG 337 Arg Arg Leu Gly Met Glu Lys Val Cys Arg Arg Asp Phe Glu Ala Leu 95 100 105 GGC GTC GAG GCG AAG GAC ATG CTG CGG GCT TAT GTC GCC GGC GTG AAC 385 Gly Val Glu Ala Lys Asp Met Leu Arg Ala Tyr Val Ala Gly Val Asn 110 115 120 125 GCA TTC CTG GCT TCC GGT GCT CCC CTG CCT GTC GAA TAC GGA TTG CTC 433 Ala Phe Leu Ala Ser Gly Ala Pro Leu Pro Val Glu Tyr Gly Leu Leu 130 135 140 GGA GCA GAG CCG GAG CCC TGG GAG CCT TGG CAC AGC ATC GCG GTG ATG 481 Gly Ala Glu Pro Glu Pro Trp Glu Pro Trp His Ser Ile Ala Val Met 145 150 155 CGC AGG GCG GGC CTG CTT ATG GGT TCG GTG TGG TTC AAG CTC TGG CGG 529 Arg Arg Ala Gly Leu Leu Met Gly Ser Val Trp Phe Lys Leu Trp Arg 160 165 170 ATG CTG GCG CTG CCG GTG GTC GGA GCC GCG AAT GCG CTG AAG CTG CGC 577 Met Leu Ala Leu Pro Val Val Gly Ala Ala Asn Ala Leu Lys Leu Arg 175 180 185 TAT GAC GAT GGC GGC CGG GAT TTG CTC TGC ATC CCG CCG GGC GCC GAA 625 Tyr Asp Asp Gly Gly Arg Asp Leu Leu Cys Ile Pro Pro Gly Ala Glu 190 195 200 205 GCC GAT CGG CTC GAG GCG GAT CTC GCG ACC CTG CGG CCC GCG GTC GAT 673 Ala Asp Arg Leu Glu Ala Asp Leu Ala Thr Leu Arg Pro Ala Val Asp 210 215 220 GCG CTG CTG AAG GCG ATG GGC GGC GAT GCC TCC GAT GCT GCC GGC GGC 721 Ala Leu Leu Lys Ala Met Gly Gly Asp Ala Ser Asp Ala Ala Gly Gly 225 230 235 GGC AGC AAC AAC TGG GCG GTC GCT CCG GGC CGC ACG GCG ACC GGC AGG 769 Gly Ser Asn Asn Trp Ala Val Ala Pro Gly Arg Thr Ala Thr Gly Arg 240 245 250 CCG ATC CTC GCG GGC GAT CCG CAT CGC GTC TTC GAA ATC CCG GGC ATG 817 Pro Ile Leu Ala Gly Asp Pro His Arg Val Phe Glu Ile Pro Gly Met 255 260 265 TAT GCG CAG CAT CAT CTG GCC TGC GAC CGG TTC GAC ATG ATC GGC CTG 865 Tyr Ala Gln His His Leu Ala Cys Asp Arg Phe Asp Met Ile Gly Leu 270 275 280 285 ACC GTG CCG GGC GTG CCG GGC TTC CCG CAC TTC GCG CAT AAC GGC AAG 913 Thr Val Pro Gly Val Pro Gly Phe Pro His Phe Ala His Asn Gly Lys 290 295 300 GTC GCC TAT TGC GTC ACC CAT GCC TTC ATG GAC ATC CAC GAT CTC TAT 961 Val Ala Tyr Cys Val Thr His Ala Phe Met Asp Ile His Asp Leu Tyr 305 310 315 CTC GAG CAG TTC GCG GGG GAG GGC CGC ACT GCG CGG TTC GGC AAC GAT 1009 Leu Glu Gln Phe Ala Gly Glu Gly Arg Thr Ala Arg Phe Gly Asn Asp 320 325 330 TTC GAG CCC GTC GCC TGG AGC CGG GAC CGT ATC GCG GTC CGG GGT GGC 1057 Phe Glu Pro Val Ala Trp Ser Arg Asp Arg Ile Ala Val Arg Gly Gly 335 340 345 GCC GAT CGC GAG TTC GAT ATC GTC GAG ACG CGC CAT GGC CCG GTT ATC 1105 Ala Asp Arg Glu Phe Asp Ile Val Glu Thr Arg His Gly Pro Val Ile 350 355 360 365 GCG GGC GAT CCG CGC GAT GGC GCA GCG CTC ACG CTG CGT TCG GTC CAG 1153 Ala Gly Asp Pro Arg Asp Gly Ala Ala Leu Thr Leu Arg Ser Val Gln 370 375 380 TTC GCC GAG ACC GAT CTG TCC TTC GAC TGC CTG ACG CGG ATG CCG GGC 1201 Phe Ala Glu Thr Asp Leu Ser Phe Asp Cys Leu Thr Arg Met Pro Gly 385 390 395 GCA TCG ACC GTG GCC CAG CTC TAC GAC GCG ACG CGC GGC TGG GGC CTG 1249 Ala Ser Thr Val Ala Gln Leu Tyr Asp Ala Thr Arg Gly Trp Gly Leu 400 405 410 ATC GAC CAT AAC CTC GTC GCC GGG GAT GTC GCG GGC TCG ATC GGC CAT 1297 Ile Asp His Asn Leu Val Ala Gly Asp Val Ala Gly Ser Ile Gly His 415 420 425 CTG GTC CGC GCC CGC GTT CCG TCC CGT CCG CGC GAA AAC GGC TGG CTG 1345 Leu Val Arg Ala Arg Val Pro Ser Arg Pro Arg Glu Asn Gly Trp Leu 430 435 440 445 CCG GTG CCG GGC TGG TCC GGC GAG CAT GAA TGG CGG GGC TGG ATT CCG 1393 Pro Val Pro Gly Trp Ser Gly Glu His Glu Trp Arg Gly Trp Ile Pro 450 455 460 CAC GAG GCG ATG CCG CGC GTG ATC GAT CCG CCG GGC GGC ATC ATC GTC 1441 His Glu Ala Met Pro Arg Val Ile Asp Pro Pro Gly Gly Ile Ile Val 465 470 475 ACG GCG AAT AAT CGC GTC GTG GCC GAT GAC CAT CCC GAT TAT CTC TGC 1489 Thr Ala Asn Asn Arg Val Val Ala Asp Asp His Pro Asp Tyr Leu Cys 480 485 490 ACC GAT TGC CAT CCG CCC TAC CGC GCC GAG CGC ATC ATG AAG CGC CTG 1537 Thr Asp Cys His Pro Pro Tyr Arg Ala Glu Arg Ile Met Lys Arg Leu 495 500 505 GTC GCC AAT CCG GCT TTC GCC GTC GAC GAT GCC GCC GCG ATC CAT GCC 1585 Val Ala Asn Pro Ala Phe Ala Val Asp Asp Ala Ala Ala Ile His Ala 510 515 520 525 GAT ACG CTG TCG CCC CAT GTC GGG TTG CTG CGC CGG AGG CTC GAG GCG 1633 Asp Thr Leu Ser Pro His Val Gly Leu Leu Arg Arg Arg Leu Glu Ala 530 535 540 CTT GGA GCC CGC GAC GAC TCC GCG GCC GAA GGG CTG AGG CAG ATG CTC 1681 Leu Gly Ala Arg Asp Asp Ser Ala Ala Glu Gly Leu Arg Gln Met Leu 545 550 555 GTC GCC TGG GAC GGC CGC ATG GAT GCG GCT TCG GAG GTC GCG TCT GCC 1729 Val Ala Trp Asp Gly Arg Met Asp Ala Ala Ser Glu Val Ala Ser Ala 560 565 570 TAC AAT GCG TTC CGC AGG GCG CTG ACG CGG CTG GTG ACG GAC CGC AGC 1777 Tyr Asn Ala Phe Arg Arg Ala Leu Thr Arg Leu Val Thr Asp Arg Ser 575 580 585 GGG CTG GAG CAG GCG ATA TCG CAT CCC TTC GCG GCT GTC GCG CCG GGC 1825 Gly Leu Glu Gln Ala Ile Ser His Pro Phe Ala Ala Val Ala Pro Gly 590 595 600 605 GTC TCA CCG CAA GGC CAG GTC TGG TGG GCC GTG CCG ACC CTG CTG CGC 1873 Val Ser Pro Gln Gly Gln Val Trp Trp Ala Val Pro Thr Leu Leu Arg 610 615 620 GAC GAC GAT GCC GGA ATG CTG AAG GGC TGG AGC TGG GAC CAG GCC TTG 1921 Asp Asp Asp Ala Gly Met Leu Lys Gly Trp Ser Trp Asp Gln Ala Leu 625 630 635 TCT GAG GCC CTC TCG GTC GCG TCG CAG AAC CTG ACC GGG CG A AGC TGG 1969 Ser Glu Ala Leu Ser Val Ala Ser Gln Asn Leu Thr Gly Arg Ser Trp 640 645 650 GGC GAA GAG CAT CGG CCG CGC TTC ACG CAT CCG CTT GCC ACG CAA TTC 2017 Gly Glu Glu His Arg Pro Arg Phe Thr His Pro Leu Ala Thr Gln Phe 655 660 665 CCG GCC TGG GCG GGG CTG CTG AAT CCG GCT TCC CGT CCG ATC GGT GGC 2065 Pro Ala Trp Ala Gly Leu Leu Asn Pro Ala Ser Arg Pro Ile Gly Gly 670 675 680 685 GAT GGC GAT ACC GTG CTG GCG AAC GGG CTC GTC CCG TCA GCC GGG CCG 2113 Asp Gly Asp Thr Val Leu Ala Asn Gly Leu Val Pro Ser Ala Gly Pro 690 695 700 CAG GCG ACC TAT GGT GCC CTG TCG CGC TAC GTC TTC GAT GTC GGC AAT 2161 Gln Ala Thr Tyr Gly Ala Leu Ser Arg Tyr Val Phe Asp Val Gly Asn 705 710 715 TGG GAC AAT AGC CGC TGG GTC GTC TTC CAC GGC GCC TCC GGG CAT CCG 2209 Trp Asp Asn Ser Arg Trp Val Val Phe His Gly Ala Ser Gly His Pro 720 725 730 GCC AGC GCC CAT TAT GCC GAT CAG AAT GCG CCC TGG AGC GAC TGT GCG 2257 Ala Ser Ala His Tyr Ala Asp Gln Asn Ala Pro Trp Ser Asp Cys Ala 735 740 745 ATG GTG CCG ATG CTC TAT AGC TGG GAC AGG ATC GCG GCA GAG GCC GTG 2305 Met Val Pro Met Leu Tyr Ser Trp Asp Arg Ile Ala Ala Glu Ala Val 750 755 760 765 ACG TCG CAG GAA CTC GTC CCG GCC TGA 2332 Thr Ser Gln Glu Leu Val Pro Ala 770

SEQ ID NO: 2 Sequence length: 2332 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Sequence features Characteristic symbols: CDS Location: 11. ． 2329 Method for determining feature: E Feature symbol: peptide Location: 11. ． 2329 Method for determining characteristic: S Character for expressing characteristic: mutation Location: 168 Method for determining characteristic: S sequence CGATAAA ATG ACT ATG GCA GCT AAT ACG GAT CGC GCG GTC TTG CAG GCG 49 Met Thr Met Ala Ala Asn Thr Asp Arg Ala Val Leu Gln Ala -1 1 5 10 GCG CTG CCG CCG CTT TCC GGC AGC CTC CCC ATT CCC GGA TTG AGC GCG 97 Ala Leu Pro Pro Leu Ser Gly Ser Leu Pro Ile Pro Gly Leu Ser Ala 15 20 25 TCG GTC CGC GTC CGG CGC GAT GCC TGG GGC ATC CCG CAT ATC AAG GCC 145 Ser Val Arg Val Arg Arg Asp Ala Trp Gly Ile Pro His Ile Lys Ala 30 35 40 45 TCG GGC GAG GCC GAT GCC TAT CGG GCG CTG GGC TTC GTC CAT TCG CAG 193 Ser Gly Glu Ala Asp Ala Tyr Arg Ala Leu Gly Phe Val His Ser Gln 50 55 60 GAC CGT CTT TTC CAG ATG GAG CTG ACG CGT CGC AAG GCG CTG GGA CGC 241 Asp Arg Leu Phe Gln Met Glu Leu Thr Arg Arg Lys Ala Leu Gly Arg 65 70 75 GCG GCC GAA TGG CTG GGC GCC GAG GCC GCC GAG GCC GAT ATC CTC GTG 289 Ala Ala Glu Trp Leu Gly Ala Glu Ala Ala Glu Ala Asp Ile Leu Val 80 85 9 0 CGC CGG CTC GGA ATG GAA AAA GTC TGC CGG CGC GAC TTC GAG GCC TTG 337 Arg Arg Leu Gly Met Glu Lys Val Cys Arg Arg Asp Phe Glu Ala Leu 95 100 105 GGC GTC GAG GCG AAG GAC ATG CTG CGG GCT TAT GTC GCC GGC GTG AAC 385 Gly Val Glu Ala Lys Asp Met Leu Arg Ala Tyr Val Ala Gly Val Asn 110 115 120 125 GCA TTC CTG GCT TCC GGT GCT CCC CTG CCT GTC GAA TAC GGA TTG CTC 433 Ala Phe Leu Ala Ser Gly Ala Pro Leu Pro Val Glu Tyr Gly Leu Leu 130 135 140 GGA GCA GAG CCG GAG CCC TGG GAG CCT TGG CAC AGC ATC GCG GTG ATG 481 Gly Ala Glu Pro Glu Pro Trp Glu Pro Trp His Ser Ile Ala Val Met 145 150 155 CGC CGG CTG GGC CTG CTT ATG GGT TCG GTG TAT TTC AAG CTT TGG CGG 529 Arg Arg Leu Gly Leu Leu Met Gly Ser Val Tyr Phe Lys Leu Trp Arg 160 165 170 ATG CTG GCG CTG CCG GTG GTC GGA GCC GCG AAT GCG CTG AAG CTG CGC 577 Met Leu Ala Leu Pro Val Val Gly Ala Ala Asn Ala Leu Lys Leu Arg 175 180 185 TAT GAC GAT GGC GGC CGG GAT TTG CTC TGC ATC CCG CCG GGC GCC GAA 625 Tyr Asp Asp Gly Gly Arg Asp Leu Leu Cys Ile Pro Pro Gly Ala Glu 190 195 200 205 GCC GAT CGG CTC GAG GCG GAT CTC GCG ACC CTG CGG CCC GCG GTC GAT 673 Ala Asp Arg Leu Glu Ala Asp Leu Ala Thr Leu Arg Pro Ala Val Asp 210 215 220 GCG CTG CTG AAG GCG ATG GGC GGC GAT GCC TCC GAT GCT GCC GGC GGC 721 Ala Leu Leu Lys Ala Met Gly Gly Asp Ala Ser Asp Ala Ala Gly Gly 225 230 235 GGC AGC AAC AAC TGG GCG GTC GCT CCG GGC CGC ACG GCG ACC GGC AGG 769 Gly Ser Asn Asn Trp Ala Val Ala Pro Gly Arg Thr Ala Thr Gly Arg 240 245 250 CCG ATC CTC GCG GGC GAT CCG CAT CGC GTC TTC GAA ATC CCG GGC ATG 817 Pro Ile Leu Ala Gly Asp Pro His Arg Val Phe Glu Ile Pro Gly Met 255 260 265 TAT GCG CAG CAT CAT CTG GCC TGC GAC CGG TTC GAC ATG ATC GGC CTG 865 Tyr Ala Gln His His Leu Ala Cys Asp Arg Phe Asp Met Ile Gly Leu 270 275 280 285 ACC GTG CCG GGC GTG CCG GGC TTC CCG CAC TTC GCG CAT AAC GGC AAG 913 Thr Val Pro Gly Val Pro Gly Phe Pro His Phe Ala His Asn Gly Lys 290 295 300 GTC GCC TAT TGC GTC ACC CAT GCC TTC ATG GAC ATC CAC GAT CTC TAT 961 Val Ala Tyr Cys Val Thr His Ala Phe Met Asp Ile His Asp Leu Tyr 305 310 315 CTC GAG CAG TTC GCG GGG GAG GGC CGC ACT GCG CGG TTC GGC AAC GAT 1009 Leu Glu Gln Phe Ala Gly Glu Gly Arg Thr Ala Arg Phe Gly Asn Asp 320 325 330 TTC GAG CCC GTC GCC TGG AGC CGG GAC CGT ATC GCG GTC CGG GGT GGC 1057 Phe Glu Pro Val Ala Trp Ser Arg Asp Arg Ile Ala Val Arg Gly Gly 335 340 345 GCC GAT CGC GAG TTC GAT ATC GTC GAG ACG CGC CAT GGC CCG GTT ATC 1105 Ala Asp Arg Glu Phe Asp Ile Val Glu Thr Arg His Gly Pro Val Ile 350 355 360 365 GCG GGC GAT CCG CGC GAT GGC GCA GCG CTC ACG CTG CGT TCG GTC CAG 1153 Ala Gly Asp Pro Arg Asp Gly Ala Ala Leu Thr Leu Arg Ser Val Gln 370 375 380 TTC GCC GAG ACC GAT CTG TCC TTC GAC TGC CTG ACG CGG ATG CCG GGC 1201 Phe Ala Glu Thr Asp Leu Ser Phe Asp Cys Leu Thr Arg Met Pro Gly 385 390 395 GCA TCG ACC GTG GCC CAG CTC TAC GAC GCG ACG CGC GGC TGG GGC CTG 1249 Ala Ser Thr Val Ala Gln Leu Tyr Asp Ala Thr Arg Gly Trp Gly Leu 400 405 410 ATC GAC CAT AAC CTC GTC GCC GGG GAT GTC GCG GGC TCG ATC GGC CAT 1297 Ile Asp His Asn Leu Val Ala Gly Asp Val Ala Gly Ser Ile Gly His 415 420 425 CTG GTC CGC GCC CGC GTT CCG TCC CGT CCG CGC GAA AAC GGC TGG CTG 1345 Leu Val Arg Ala Arg Val Pro Ser Arg Pro Arg Glu Asn Gly Trp Leu 430 435 440 445 CCG GTG CCG GGC TGG TCC GGC GAG CAT GAA TGG CGG GGC TGG ATT CCG 1393 Pro Val Pro Gly Trp Ser Gly Glu His Glu Trp Arg Gly Trp Ile Pro 450 455 460 CAC GAG GCG ATG CCG CGC GTG ATC GAT CCG CCG GGC GGC ATC ATC GTC 1441 His Glu Ala Met Pro Arg Val Ile Asp Pro Pro Gly Gly Ile Ile Val 465 470 475 ACG GCG AAT AAT CGC GTC GTG GCC GAT GAC CAT CCC GAT TAT CTC TGC 1489 Thr Ala Asn Asn Arg Val Val Ala Asp Asp His Pro Asp Tyr Leu Cys 480 485 490 ACC GAT TGC CAT CCG CCC TAC CGC GCC GAG CGC ATC ATG AAG CGC CTG 1537 Thr Asp Cys His Pro Pro Tyr Arg Ala Glu Arg Ile Met Lys Arg Leu 495 500 505 GTC GCC AAT CCG GCT TTC GCC GTC GAC GAT GCC GCC GCG ATC CAT GCC 1585 Val Ala Asn Pro Ala Phe Ala Val Asp Asp Ala Ala Ala Ile His Ala 510 515 520 525 GAT ACG CTG TCG CCC CAT GTC GGG TTG CTG CGC CGG AGG CTC GAG GCG 1633 Asp Thr Leu Ser Pro His Val Gly Leu Leu Arg Arg Arg Leu Glu Ala 530 535 540 CTT GGA GCC CGC GAC GAC TCC GCG GCC GAA GGG CTG AGG CAG ATG CTC 1681 Leu Gly Ala Arg Asp Asp Ser Ala Ala Glu Gly Leu Arg Gln Met Leu 545 550 555 GTC GCC TGG GAC GGC CGC ATG GAT GCG GCT TCG GAG GTC GCG TCT GCC 1729 Val Ala Trp Asp Gly Arg Met Asp Ala Ala Ser Glu Val Ala Ser Ala 560 565 570 TAC AAT GCG TTC CGC AGG GCG CTG ACG CGG CTG GTG ACG GAC CGC AGC 1777 Tyr Asn Ala Phe Arg Arg Ala Leu Thr Arg Leu Val Thr Asp Arg Ser 575 580 585 GGG CTG GAG CAG GCG ATA TCG CAT CCC TTC GCG GCT GTC GCG CCG GGC 1825 Gly Leu Glu Gln Ala Ile Ser His Pro Phe Ala Ala Val Ala Pro Gly 590 595 600 605 GTC TCA CCG CAA GGC CAG GTC TGG TGG GCC GTG CCG ACC CTG CTG CGC 1873 Val Ser Pro Gln Gly Gln Val Trp Trp Ala Val Pro Thr Leu Leu Arg 610 615 620 GAC GAC GAT GCC GGA ATG CTG AAG GGC TGG AGC TGG GAC CAG GCC TTG 1921 Asp Asp Asp Ala Gly Met Leu Lys Gly Trp Ser Trp Asp Gln Ala Leu 625 630 635 TCT GAG GCC CTC TCG GTC GCG TCG CAG AAC CTG ACC GGG CG A AGC TGG 1969 Ser Glu Ala Leu Ser Val Ala Ser Gln Asn Leu Thr Gly Arg Ser Trp 640 645 650 GGC GAA GAG CAT CGG CCG CGC TTC ACG CAT CCG CTT GCC ACG CAA TTC 2017 Gly Glu Glu His Arg Pro Arg Phe Thr His Pro Leu Ala Thr Gln Phe 655 660 665 CCG GCC TGG GCG GGG CTG CTG AAT CCG GCT TCC CGT CCG ATC GGT GGC 2065 Pro Ala Trp Ala Gly Leu Leu Asn Pro Ala Ser Arg Pro Ile Gly Gly 670 675 680 685 GAT GGC GAT ACC GTG CTG GCG AAC GGG CTC GTC CCG TCA GCC GGG CCG 2113 Asp Gly Asp Thr Val Leu Ala Asn Gly Leu Val Pro Ser Ala Gly Pro 690 695 700 CAG GCG ACC TAT GGT GCC CTG TCG CGC TAC GTC TTC GAT GTC GGC AAT 2161 Gln Ala Thr Tyr Gly Ala Leu Ser Arg Tyr Val Phe Asp Val Gly Asn 705 710 715 TGG GAC AAT AGC CGC TGG GTC GTC TTC CAC GGC GCC TCC GGG CAT CCG 2209 Trp Asp Asn Ser Arg Trp Val Val Phe His Gly Ala Ser Gly His Pro 720 725 730 GCC AGC GCC CAT TAT GCC GAT CAG AAT GCG CCC TGG AGC GAC TGT GCG 2257 Ala Ser Ala His Tyr Ala Asp Gln Asn Ala Pro Trp Ser Asp Cys Ala 735 740 745 ATG GTG CCG ATG CTC TAT AGC TGG GAC AGG ATC GCG GCA GAG GCC GTG 2305 Met Val Pro Met Leu Tyr Ser Trp Asp Arg Ile Ala Ala Glu Ala Val 750 755 760 765 ACG TCG CAG GAA CTC GTC CCG GCC TGA 2332 Thr Ser Gln Glu Leu Val Pro Ala 770

[Brief description of drawings]

FIG. 1 is a diagram showing the steps for constructing pTQiPAdtrp.

FIG. 2 is a diagram showing a construction process of pCC001A.

FIG. 3 is a diagram showing a construction process of pCC002A.

FIG. 4 is a diagram showing a construction process of pCK002.

FIG. 5 shows the construction steps of pCC007A and pCCNt013.

FIG. 6 is a diagram showing a construction process of pCC013A.

FIG. 7 shows the steps for constructing pΔN176 and pCK013.

FIG. 8 is a diagram showing a construction process of mp18p181 and mp18p183.

FIG. 9: mp18p181L160A and pCKL1
It is a figure which shows the construction process of 60A.

FIG. 10: mp18p183W168Y and pCKW
It is a figure showing a construction process of 168Y.

FIG. 11: DN encoding precursor of mutant CC acylase L160A in plasmid pCKL160A
It is a figure which shows the nucleotide sequence of A (1-840) and the amino acid sequence estimated from it.

FIG. 12: DN encoding precursor of mutant CC acylase L160A in plasmid pCKL160A
It is a figure which shows the nucleotide sequence (841-1680) of A and the amino acid sequence estimated from it.

FIG. 13: DN encoding precursor of mutant CC acylase L160A in plasmid pCKL160A
It is a figure which shows the nucleotide sequence (1681-2332) of A and the amino acid sequence estimated from it.

FIG. 14: DN encoding precursor of mutant CC acylase W168Y in plasmid pCKW168Y
It is a figure which shows the nucleotide sequence of A (1-840) and the amino acid sequence estimated from it.

FIG. 15: DN encoding precursor of mutant CC acylase W168Y in plasmid pCKW168Y
It is a figure which shows the nucleotide sequence (841-1680) of A and the amino acid sequence estimated from it.

FIG. 16: DN encoding precursor of mutant CC acylase W168Y in plasmid pCKW168Y
It is a figure which shows the nucleotide sequence (1681-2332) of A and the amino acid sequence estimated from it.

FIG. 17 is a diagram showing a restriction enzyme map of plasmid pCKL160A.

FIG. 18 is a diagram showing a restriction enzyme map of plasmid pCKW168Y.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12R 1:19) (72) Inventor Nagashi Yamada 3-19-8 Akashiadai, Mita City, Hyogo Prefecture

Claims (8)

[Claims] 1. A mutant cephalosporin C acylase in which the amino acids at the Leu ¹⁶⁰ and / or Trp ¹⁶⁸ sites of the amino acid sequence of natural cephalosporin C acylase are substituted with other amino acids. 2. A precursor amino acid sequence before being processed into α-subunit and β-subunit in a host cell has the formula: A ^1-159 -X ₁ -A ^161-167 -X _2- A ^169-773 (In the formula, A ^1-159 represents the same amino acid sequence as Thr ¹ to Arg ¹⁵⁹ of the natural cephalosporin C acylase. A ^161-167 represents the natural cephalosporin. The amino acid sequence identical to the amino acid sequence of Gly ¹⁶¹ to Val ¹⁶⁷ of C acylase is shown.
^169-773 is P of natural cephalosporin C acylase.
The same amino acid sequence as that of he ¹⁶⁹ to Ala ⁷⁷³ is shown. X ₁ represents Leu or another amino acid, and X ₂ represents Trp or another amino acid. However, when X ₁ is Leu, X ₂ is an amino acid other than Trp), The mutant cephalosporin C acylase according to claim 1. 3. X ₁ is Ala and / or X ₂ is Ty
The mutant cephalosporin C acylase according to claim 2, which is r. 4. A DNA encoding the cephalosporin C acylase according to claim 1. 5. An expression vector containing the DNA according to claim 4. 6. A host cell transformed with the expression vector according to claim 5. 7. The mutant cephalosporin C acylase according to claim 1, wherein the host cell according to claim 6 is cultured in a medium, and the mutant cephalosporin C acylase is collected from the resulting culture. Manufacturing method. 8. The formula: (In the formula, R ¹ is acetoxy, hydroxy or hydrogen,
R ² represents an acyl carboxylate) (I
I) or a salt thereof is contacted with a culture of the transformant according to claim 6 or a treated product thereof, and the compound has the formula: (Wherein R ¹ has the same meaning as defined above), and a method for producing the compound (I) or a salt thereof.