Glutaryl cephalosporin amidase from Pseudomonas diminuta BS-203

Details Glutaryl cephalosporin amidase from Pseudomonas diminuta BS-203 Glutaryl cephalosporin amidase from Pseudomonas diminuta BS-203

2001-09-21

2003-03-04

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C536S023200, C435S320100, C435S252300

Reexamination Certificate

active

06528300

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a novel glutaryl cephalosporin amidase (glutaryl 7-ACA amidase) enzyme from
Pseudomonas diminuta
BS-203, which catalyzes the hydrolysis of 7-(4-carboxybutanamido)-3-acetoxymethyl-3-cephem-4-carboxylic acid (glutaryl 7-ACA) to yield 7-aminocephalosporanic acid (7-ACA) and glutaric acid. The invention also relates to nucleic acids having sequences which encode the glutaryl 7-ACA enzyme, including the nucleic acid sequence of the glutaryl 7-ACA amidase gene from
P. diminuta
BS-203, and to nucleic acid sequences derived therefrom. The invention also relates to vectors and host cells containing these nucleic acid sequences, and methods of producing glutaryl 7-ACA amidase with these vectors and host cells. The invention also relates to a method of preparing 7-ACA by using glutaryl 7-ACA amidase from
P. diminuta
BS-203.
BACKGROUND OF THE INVENTION
3-Acetoxymethyl 7-amino-3-cephem-4-carboxylic acid (7-aminocephalosporanic acid, 7-ACA) is the starting material for the synthesis of many semi-synthetic cephalosporin antibiotics. 7-ACA can be generated from cephalosporin C (a readily available fermentation product) by a two-step enzymatic process (Scheme 1), using first a D-amino acid oxidase in conjunction with oxidative decarboxylation to produce glutaryl 7-ACA (Step A), and then using a glutaryl 7-ACA acylase to remove the glutaryl group to produce 7-ACA (Step B).
Steps A and B can also be carried out by chemical processes. The chemical processes, which involve the use of large quantities of organic solvents and toxic chemicals, have safety and environmental disadvantages. By using enzymatic processes, on the other hand, 7-ACA is obtained under mild conditions in an aqueous solvent system. Enzymes that catalyze the hydrolysis of glutaryl 7-ACA to 7-ACA (Step B) have been readily available, but enzymes that efficiently catalyze direct hydrolysis of cephalosporin C to 7-ACA (Step C) have not. Consequently, a two-step process has been generally employed, where step A is carried out chemically or enzymatically, and step B is carried out enzymatically. See for example Cambiaghi et al., U.S. Pat. No. 5,424,196, and references therein.
Step A is usually carried out with D-amino acid transaminases (also known as D-amino acid oxidase, EC-1.4.3.3) (Aretz et al., U.S. Pat. No. 4,745,061), in order to oxidize the amino group of the D-amino acid side chain to a keto group, followed by treatment with hydrogen peroxide to effect decarboxylation and provide glutaryl 7-ACA.
Much effort has gone into the development of efficient methods for carrying out Step B. Crawford et al., in U.S. Pat. No. 5,104,800, provide a brief summary of earlier work in the field of 7-ACA synthesis. Matsuda et al, in U.S. Pat. No. 3,960,662, describe glutaryl 7-ACA amidase activity from cultures of Comamonas sp. and
Pseudomonas ovalis.
Workers at Fujisawa Pharmaceutical Co. (Aramori et al., U.S. Pat. No. 5,310,659 and EP 0482844; Aramori et al., 1991,
J. Bacteriol.
173:7848-7855) describe a glutaryl 7-ACA acylase isolated from
Bacillus
(
Brevibacillus
)
laterosporus.
Chu et al. (U.S. Pat. No. 5,766,871) describe a glutaryl 7-ACA acylase isolated from
Pseudomonas nitroreducens.
Battistel et al., in EP 0525861, describe glutaryl 7-ACA acylases from various Pseudomonas, Bacillus, and Achromobacter species.
A number of enzymes capable of directly catalyzing hydrolysis of cephalosporin C to 7-ACA (Scheme 1, Step C) have been reported. For example, workers at Asahi Chemical (Ichikawa et al., U.S. Pat. No. 4,774,179; Matsuda et al.,
J. Bact.,
1987, 169:5815-5820 and 5821-5826) disclosed strain SE-495 of
Pseudomonas diminuta,
and strain SE83 of a closely related Pseudomonas species, both of which produce enzymes capable of effecting the direct conversion of cephalosporin C into 7-ACA. Lein, in U.S. Pat. No. 4,981,789 and EP 0283218, reported a cephalosporin C amidase from
Athrobacter viscous.
Lein, in EP 0322032, reported a cephalosporin C amidase from
Bacillus megaterium,
as did Crawford et al., in U.S. Pat. No. 5,104,800 (and divisional U.S. Pat. No. 5,229,247) and EP 0405846. Iwami et al., in U.S. Pat. No. 5,192,678 (and divisional U.S. Pat. No. 5,320,948) and EP 0475652 later disclosed a cephalosporin C acylase from
Pseudomonas diminuta
N-1 76 which is capable of carrying out Step C directly, but which is more adept at catalyzing the conversion of glutaryl 7-ACA into 7-ACA. Such enzymes have not yet been shown to be economically viable for production of 7-ACA.
Preparation of recombinant host cells expressing various glutaryl 7ACA amidases has been described by numerous workers. See for example M. Ishiye and M. Niwa,
Biochim. Biophys. Acta,
1992, 1132:233-239; Croux et al., EP 0469919; Aramori et al., U.S. Pat. No. 5,310,659; Iwami et al., U.S. Pat. No. 5,192,678; and Honda et al.,
Biosci. Biotechnol. Biochem.,
1997, 61:948-955, all of which are incorporated herein by reference.
In view of the value of 7-ACA as a pharmaceutical intermediate, there exists a need for improved 7-ACA amidases that provide superior results in terms of factors such as enzyme cost, reaction rate and yield, and enzyme stability.
SUMMARY OF THE INVENTION
The invention provides a novel glutaryl 7-ACA amidase isolated from the bacterial strain
Pseudomonas diminuta
BS-203, and derivative, subunits, and fragments thereof. The invention also provides oligonucleotides, i.e. DNA and RNA molecules, comprising a nucleic acid sequence encoding the glutaryl 7-ACA amidase of the invention, as well as derivatives, fragments and partial sequences thereof, and polynucleotides complementary to the DNA molecules of the invention. The present invention also relates to vectors and host cells comprising the polynucleotides of the invention.
The invention also provides homologous proteins, preferably having at least 80% homology to the
Pseudomonas diminuta
BS-203 glutaryl 7-ACA amidase. Proteins having up to 113 conservative amino acid substitutions, and/or up to 20 amino acid additions or deletions, are contemplated as being part of the invention. Nucleic acid sequences encoding such homologous proteins are also contemplated as being part of the invention.
The invention further relates to methods of obtaining the
Pseudomonas diminuta
BS-203 amidase by culturing
P. diminuta
BS-203 in a suitable medium, and recovering a protein fraction having glutaryl 7-ACA amidase activity. The invention also relates to methods of using the nucleic acids, vectors, and host cells of the invention to produce the glutaryl 7-ACA amidase of the invention.
The invention further relates to a process for obtaining 7-aminocephalosporanic acid (7-ACA) from 7-&bgr;-(4-carboxybutanamido)cephalosporanic acid (glutaryl 7-ACA) and other 7-&bgr;-(acylamido)cephalosporanic acids, by contacting such substrates with a glutaryl 7-ACA amidase of the invention. The invention also relates to a process for producing desacetyl 7-ACA, from the desacetyl derivatives of 7-&bgr;-(4-carboxybutanamido)cephalosporanic acid (glutaryl 7-ACA) and other
7-&bgr;-(acylamido)cephalosporanic acids, by contacting such substrates with a glutaryl
7-ACA amidase of the invention.


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Ishiye M, Niwa M. Biochim Biophys Acta Oct. 20, 1992;1132(3):233-9 Nucleotide sequence an

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