Mutated penicillin expandases

Details Mutated penicillin expandases Mutated penicillin expandases

2002-03-26

2004-03-02

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Transferase other than ribonuclease

C435S047000

Reexamination Certificate

active

06699699

ABSTRACT:

FILED OF THE INVENTION
The present invention relates to a mutated penicillin expandase having high substrate specificity to penicillin G, recombinant cells expressing the mutated expandase, and a process of preparing 7-aminodeacetoxycephalosporanic acid (7-ADCA) using the mutated expandase.
BACKGROUND OF THE INVENTION
7-aminodeacetoxycephalosporanic acid (7-ADCA) is one of the important intermediates for the production of cephalosporins cefalexin, cefradine, and cefadroxil which are antibiotic compounds commonly and long used in humans and animals. The industrial process to synthesize 7-ADCA mainly includes two steps: a chemical ring expansion of penicillin G to phenylacetyl-7-ADCA and an enzymatic side chain cleavage of phenylacetyl-7-ADCA. However, the chemical reaction of the ring expansion is complex and expensive, and the by-products and the organic solvents (such as pyridine and HBr) are toxic to the environment. Therefore, an enzymatic reaction is greatly desirable to replace such chemical reaction.
It has been reported that a natural enzyme, deacetoxycephalosporain C synthase (DAOCS, or expandase), may be responsible for the catalysis of the expansion reaction. Streptomyces sp. (such as
Streptomyces clavuligerus, Streptomyces ambofaciens
and
Streptomyces chartreusis
) can produce the expandase. As illustrated in EP-A-034 1892, the expandase can be obtained from
Streptomyces clavuligerus
, and has been cloned. The expandase has been well studied for its chemical and functional properties, see EP-A-0366354. Unfortunately, the native expandase has less substrate specificity to penicillin G than the normal substrate penicillin N (Rollins, M. J. et al.,
Can. J Microbiol
. 34: 1196-1202 (1988) and Crawford, L. et al.,
Bio/Technology
, 13: 58-62 (1995)) Penicillin G is commercially available at a low cost. In contrast, penicillin N is expensive and not easily available. Furthermore, even though penicillin N is expanded, its side chain cannot be easily removed. Accordingly, the chemical synthesis of 7-ADCA, rather than an enzymatical synthesis, is still used in industrial production.
There are a number of applications directed to producing 7-ADCA by expandase. U.S. Pat. No. 5,731,165 describes a process for the preparation and recovery of 7-ADCA via enzymatic ring expansion activity on penicillin G, using a
Penicillium chrysogenum
transformant strain expressing expandase. U.S. Pat. No. 5,559,005 discloses a bioprocess for preparing 7-amino-cephalosporanic acid (7-ACA) or 7-ADCA using a transformed stain of
Penicillium chrysogenum
having expandase activity capable of accepting adipoyl-6-amino-penicillanic acid (adipoyl-6-APA) as a substrate. Nevertheless, since both adipoyl-6-APA and penicillin G are poor substrates for native expandase in vitro (U.S. Pat. No. 5,559,005), it is not optimistic to have a high expansion efficiency while applied in vivo.
Recently, Chih H. S. et al. (
Biochemical and Biophysical Research Communications
, 287: 507-513 (2001)) discloses a mutated DAOCS comprising an amino acid substitution of N304L. U.S. Pat. No. 5,919,680 describes a mutated expandase which has an altered amino acid sequence from a native expandase, resulting in an altered substrate specificity. Several amino acid positions have been mentioned in that patent, and the mutated expandase created by changing one or more of the mentioned amino acids shows a higher activity ratio of penicillin G to penicillin N in a mixture of these two substrates, but has a lower activity on penicillin G and penicillin N individually than wild-type expandase. Therefore, there is still a need to develop a mutated penicillin expandase having more substrate specificity and enzymatic activity on penicillin G.
SUMMARY OF THE INVENTION
The present invention provides mutated expandase having expansion activities 2 to 32 folds higher on penicillin G than wild-type expandase.
One object of the invention is to provide a mutated penicillin expandase which comprises an amino acid substitution at one or more residue positions corresponding to those of a wild-type expandase selected from the group consisting of methionine 73, glycine 79, valine 275, leucine 277, cysteine 281, glycine 300, asparagine 304 and isoleucine 305, provided that the amino acid substitution at the residue position of asparagine 304 is not N304L. In particular, the invention provides a mutated penicillin expandase which comprises one or more specific amino acid substitutions selected from the group consisting of M73T, G79E, V275I, L277K, C281Y, G300V, N304K, I305L and I305M, wherein the residue positions of the amino acid substitution correspond to those of a wild-type expandase.
Another object of the invention is to provide an isolated nucleic acid molecule encoding the mutated penicillin expandase.
Another object of the invention is to provide a recombinant vector comprising the nucleic acid molecule of the invention and a regulatory sequence operatively linked thereto.
Still another object of the invention is to provide recombinant cells comprising the nucleic acid molecule of the invention.
In another aspect, the invention provides a method to produce a mutated penicillin expandase. In one embodiment of the invention, the method comprises expressing the nucleic acid molecule of the invention and recovering the mutated penicillin expandase. In another embodiment of the invention, the method comprises culturing the recombinant cells of the invention to express the mutated penicillin expandase and recovering the mutated penicillin expandase form the cell culture.
In another aspect, the invention provides a process for producing 7-ADCA comprising treating penicillin G with the mutated penicillin expandase of the invention to produce phenylacetyl-7-ADCA, followed by deacylating the phenylacetyl-7-ADCA to produce the 7-ADCA.
In still another aspect, the invention provides a process for producing 7-ADCA, which process comprises the steps of (a) cultivating penicillin G producing cells which is transformed with the nucleic acid molecule of the invention under conditions suitable for production of penicillin G and expression of the mutated penicillin expandase such that the penicillin G is expanded by the mutated expandase and phenylacetyl-7-ADCA is produced; (b) deacylating the phenylacetyl-7-ADCA to produce the 7-ADCA.
The present invention will be fully understood from the detailed description and figures as given below.


REFERENCES:
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patent: 5731165 (1998-03-01), Bovenberg et al.
patent: 5919680 (1999-07-01), Sutherland et al.
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patent: WO 01/85951 (2001-11-01), None
Chin et al.,Biochemical and Biophysical Research Communications287: 507-513 (2001).
Coque J. et al.,Mol Gen Genet. 1993;326(2-3):453-8.
Wei C.L. et al.,Appl Environ Microbiol. 2003;69(4):2306-12.
Wong E. et al.,Biochem Biophys Res Commun. 2001;283(3):621-6.

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