Recombinant cephalosporin C amidohydrolase in cephalosporin...

Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound

Reexamination Certificate

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C435S069100, C435S252300, C435S320100, C435S874000, C435S047000, C536S063000, C530S350000

Reexamination Certificate

active

06297032

ABSTRACT:

The present invention relates to an enzyme process for the one step conversion of cephalosporin C or a derivative thereof into 7-aminocephalosporanic acid or a corresponding derivative thereof. The one step conversion is effected using a cephalosporin C amidohydrolase enzyme derived from
Pseudomonas vesicularis
B965, or from any cephalosporin C amidohydrolase producing or potentially producing descendants thereof, or from any expression of DNA, particularly a recombinant DNA molecule, derived from
Pseudomonas vesicularis
B965 as described herein, or any cephalosporin C amidohydrolase producing or potentially producing descendants thereof.
Cephalosporin C is the fermentation product of the cephalosporin biosynthesis pathway and although it has been shown to have some activity against gram-negative microorganisms as an antibiotic itself, the major commercial use of cephalosporin C is as a building block for other cephalosporin-like antibiotics. In particular, the D-&agr;-aminoadipoyl side chain may be removed to give the highly useful intermediate 7-aminocephalosporanic acid (7-ACA) which is a precursor to a wide range of semi-synthetic cephalosporin antibiotics including caphalothin, cephaloridine and cefuroxime.
The current industrial process for producing 7-ACA from cephalosporin C is a chemical cleavage of the D-&agr;-aminoadipoyl side chain. There are several different methods in use (see for instance A Smith in “Comprehensive Biotechnology: the Principles, Applications and Regulations of Biotechnology in Industry, Agriculture and Medicine”, Volume 3 (“The Practice of Biotechnology: Current Commodity Products”), Eds. H. W. Blanch et. al., esp. pp. 163-185, Pergamon Press, Oxford, UK, 1985), but all are essentially imino-halide processes with appropriate protection of amino and carboxyl groups. See, for example, the nitrosyl chloride cleavage developed by Morin et al (J.Am.Chem.Soc., 84, 3400 (1962)) now superseded by the imino ether method developed by Ciba Geigy (Fechtig et al, Helv. Chim. Acta., 51, 1108 (1968)).
These chemical processes have several disadvantages which include: the cost of the chemical reagents for protection and cleavage; the expense of providing the required low operating temperatures (e.g. −20° C.); the cost of the complex, often multi-step plant; the cost of the measures to contain the toxic chemical reagents (e.g. trimethyl silyl chloride, phosphorous pentachloride and chloroacetyl chloride); and the need to purify the highly impure cephalosporin C (or a derivative) starting material.
There is therefore a need to provide a means of converting cephalosporin C (or a derivative thereof) to 7-ACA (or a corresponding derivative thereof) which is cheap, involves simple technology, is environmentally friendly and is safe. Such criteria are me therein using an enzyme process.
The search for efficient microbiological or enzyme processes for converting cephalosporin C (or a derivative thereof) into 7-ACA (or a corresponding derivative thereof) has been largely unsuccessful. There have been reports in the literature of two-stage enzymatic processes for converting cephalosporin C to 7-ACA. These require the initial conversion of cephalosporin C into glutaryl-7-ACA using a D-amino acid oxidase (see for instance U.S. Pat. Nos. 3,658,649 or 3,801,458) followed by cleavage of the glutary side chain to give 7-ACA. Two-stage enzymatic processes have, for example, been described by Shibuya et al, Agric.Biol.Chem., 45, 1561-1567 (1981), but all suffer from the disadvantage of reduced efficiency and increased complexity compared with the one step conversion.
There have also been reports of low activity single-step enzyme reactions. For example, EP0283218, EP0322032, and EP0405846, describe one step conversion of cephalosporin C to 7-ACA using enzymes derived from
Arthrobacter viscosus, Bacillus Megaterium
, and another Bacillus species respectively. EP0474652 describes an enzyme capable of a one step conversion of cephalosporin C to 7-ACA with is derived from
Pseudomonas diminuta
. Despite extensive work in this area it has not been demonstrated that an enzyme isolate as described above is able to be used for production of commercially relevant amounts of 7-ACA.
It is particularly unexpected therefore, that we have isolated a strain of
Pseudomonas vesicularis
which produces an amidohydrolase (also known as amidase or acylase) enzyme that can convert caphalosporin C (or a derivative thereof) into 7-ACA (or a corresponding derivative thereof) by way of a one step conversion. The enzyme activity is only weakly observed in the wild type strain, however, useful activity is achieved following partial purification of the enzyme. Furthermore, the gene coding for the amidohydrolase enzyme activity has also been isolated and sequenced (SEQ ID NO:1) and can therefore be expressed in a recombinant host such as
E.coli
to produce increased amounts of the enzyme. In addition is it a further feature of this invention that the amidohydrolase enzyme isolated is sufficiently robust that it may be immobilised whilst maintaining significant enzymatic activity. Immobilisation of the enzyme is a particularly important step in the adaption of an enzyme for use within large scale fermentation synthesis of 7-ACA. The use of an immobilised enzyme as compared to free enzyme in fermentation has obvious benefits in significantly reducing the requirement for enzyme and subsequent reduction in cost of 7-ACA manufacture.


REFERENCES:
patent: 4774179 (1988-09-01), Shigeaki et al.
patent: 5320948 (1994-06-01), Iwami et al.
patent: 0 475 652 (1992-03-01), None
patent: 0 558 241 (1993-09-01), None
Database WPI, Section Ch, Week 8634, Derwent Publications Ltd., London, GB & JP-A-61 152 286, Jul. 10, 1986, see Abstract.

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