Nucleic acid molecule encoding a cephalosporin acetylesterase

Details Nucleic acid molecule encoding a cephalosporin acetylesterase Nucleic acid molecule encoding a cephalosporin acetylesterase

2000-07-26

2002-10-15

Patterson, Jr., Charles L. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S051000, C435S252300, C435S320100, C536S023200

Reexamination Certificate

active

06465233

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a nucleic acid molecule which codes for the cephalosporin acetylesterase from
Bacillus subtilis
ATCC 6633 (DSM 11909), vectors and host cells which comprise such a nucleic acid molecule, a process for the recombinant preparation of cephalosporin acetylesterase from
B. subtilis
ATCC 6633 (DSM 11909) using said nucleic acid molecule, and a process for preparing 3-deacetylcephalosporin compounds.
BACKGROUND OF THE INVENTION
Cephalosporin C is cleaved by cephalosporin acetylesterase (CAE) to 3-deacetyl-cephalosporin C (see, for example, B. J. Abbott et al., Appl. Microbiol. (1975), 413-419; J. Konecny et al., Biochim. Biophys. Acta 485 (1977), 367-378). It is furthermore possible to convert the cephalosporin compound 7-aminocephalosporanic acid (7-ACA) with CAE to 7-amino-3-deacetylcephalosporanic acid (HACA). 3-Deacetylcephalosporin C and HACA are used as intermediates in preparation of semisynthetic cephalosporins (S. Tsushima et al., Chem. Pharmacol. Bull. 27 (1979), 696-702). Various cephalosporin acetylesterases from diverse
Bacillus subtilis
(
B. subtilis
hereinafter) strains have been disclosed in the literature (see T. B. Higherd et al., J. Bacteriol. 114 (1973), 1184-1192; B. J. Abbott et al., Appl. Microbiol. (1975), 413-419; A. Takimoto et al., J. Ferment. Bioeng. 77 (1994), 17-22). Another cephalosporin acetylesterase was isolated by J. Konecny et al. (see J. Konecny et al., Biochim. Biophys. Acta 485 (1977), 367-378) from the
B. subtilis
strain ATCC 6633. The last-mentioned cephalosporin acetylesterase is particularly suitable for carrying out the abovementioned conversions of cephalosporin C into 3-deacetylcephalosporin C and of 7-ACA into HACA.
It is desirable, for technical and commercial reasons, to prepare the CAE required for the said conversion by a recombinant route. In particular, it would be possible in this way to produce in a simple and cost-effective manner the amounts of CAE required for carrying out the conversion process industrially. However, it has not been possible to date to clone the nucleic acid sequence coding for the cephalosporin acetylesterase from
B. subtilis
ATCC 6633 (DSM 11909).
It is thus an object of the present invention to provide the nucleic acid sequence coding for the cephalosporin acetylesterase from
B. subtilis
ATCC 6633 (DSM 1 1909). It is a further object of the present invention to provide vectors, in particular expression vectors, which comprise this nucleic acid sequence, and host cells which comprise these vectors. It is a further object of the present invention to provide a recombinant process for preparing the said cephalosporin acetylesterase. Finally, it is an object of the present invention to provide a process for preparing 3-deacetylcephalosporin compounds using a recombinant cephalosporin acetylesterase from
B. subtilis
ATCC 6633 (DSM 11 909).
DETAILED DESCRIPTION OF THE INVENTION
It has surprisingly been possible within the scope of the present invention to establish the nucleic acid sequence codings for the cephalosporin acetylesterase from
B. subtilis
ATCC 6633 (DSM 11909).
One aspect of the present invention is thus a nucleic acid molecule which codes for the cephalosporin acetylesterase from
B. subtilis
ATCC 6633 (DSM 11909).
In this connection, the cephalosporin acetylesterase from
B. subtilis
ATCC 6633 (DSM 11909) essentially corresponds to the activity described by J. Konecny et al. (see above). In particular, J. Konecny et al. refer to a value of the Michaelis-Menten constant K
m
for the conversion of cephalosporin C into 3-deacetylcephalosporin C of about K
m
=15 mM (at 25° C.).
The cephalosporin acetylesterase from
B. subtilis
ATCC 6633 (DSM 11909) is, in particular, a polypeptide which has the amino acid sequence shown in the sequence identity No. (abbreviated to SEQ ID NO. hereinafter) 1 which is reproduced in Annex 1.
The nucleic acid molecule according to the invention preferably comprises a base sequence of the SEQ ID NO. 2 depicted in Annex 2. Also preferred is a nucleic acid molecule according to the invention which has exclusively the base sequence shown in SEQ ID NO. 2. A nucleic acid molecule according to the invention is, in particular, in the form of a DNA, for example of a cDNA. However, the invention also relates to nucleic acid molecules having the said properties, for example, an RNA, for example an mRNA or a pre-mRNA.
A nucleic acid molecule according to the invention can be obtained in the following way:
(i) Chromosomal DNA is extracted from
B. subtilis
ATCC 6633 (DSM 11909), purified and prepared for subsequent amplification, for example with the aid of PCR experiments (PCR =polymerase chain reaction). Primers able specifically to hybridize with the 5′ end or with the 3′ end of the nucleic acid sequence depicted in SEQ ID NO. 2 are constructed. An example of a 5′ primer which can be used is an oligonucleotide which corresponds to nucleotides Nos 1 to 27 in the nucleic acid sequence depicted in SEQ ID NO. 2. An example of a 3′ primer which can be used is an oligonucleotide which is complementary to nucleotides Nos 93 i to 957 in SEQ ID NO. 2. It is possible and advantageous to provide one or more restriction cleavage sites in the primers for subsequent cloning. The primers are used to carry out a suitable amplification method, for example a PCR experiment. (ii) The result of the amplification, for example of the PCR, is analysed, for example by electrophoresis on an agarose gel. It is possible with the aid of the abovementioned primers to obtain DNA fragments which can be extracted from the agarose gel and cloned into suitable vectors, for example selective plasmid vectors. A vector obtained in this way can then be used to transform a suitable host cell or a suitable host organism. For example, the DNA fragments obtained in the preceding step are inserted into an
E. coli
cloning vector, and the resulting recombinant plasmid is introduced by transformation or electroporation into
E. coli
cells. To produce colonies, the transformants are plated out on agar medium with an antibiotic, which is appropriate for the plasmid vector used, as selection pressure. (iii) A few of the resulting clones are selected, and the base sequence of the DNA insert is established. For example, DNA of the recombinant plasmid is extracted from a large number of selected
E. coli
colonies and is analysed with restriction enzymes. Subsequently, as a check, the base sequence of the DNA fragment originating from
B. subtilis
is determined. For this purpose, the fragments from several independently isolated clones are sequenced in order to detect any mutations due to the process. The nucleic acid sequence which has been established, and the amino acid sequence derived therefrom, is then compared with the sequences depicted in SEQ ID NO. 2 and SEQ ID NO. 1, respectively, in order to find a nucleic acid sequence which is sought. The appropriate clone of the host cell can then be grown further, and the vector containing the nucleic acid molecule according to the invention and, finally, the nucleic acid molecule according to the invention itself can be isolated.
Particularly suitable host cells or host organisms are bacterial strains, for example,
E. coli
. It is possible and advantageous to use as
E. coli
host a strain of the
E. coli
derivative K2, for example, W3110, HB101, C600, JM87, JM103, JM105 or JM109. Examples of
E. coli
vectors which can be used for the cloning are both plasmid vectors such as pUC13, pK19, pBR322 and pAT153, and phage vectors, for example, lambda gt10. The abovementioned hosts and vectors are merely examples of many others which are commercially available and can be obtained straightforwardly.
Oligonucleotides can be synthesized using a commercially available DNA synthesizer in accordance with the manufacturer's instructions.
Determination of a nucleic acid sequence according to the invention can be carried out by the method of Sanger et at. (1977), employing

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