Epoxide hydrolase

Details Epoxide hydrolase Epoxide hydrolase

2000-07-03

2002-04-30

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

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing oxygen-containing organic compound

C435S195000

Reexamination Certificate

active

06379938

ABSTRACT:

This is the U.S. National Phase under 35 U.S.C. §371 of International Application PCT/BE98/00160, filed Oct. 23, 1998, which claims priority of European Application EP 97870168.8, filed Oct. 24, 1997.
FIELD OF THE INVENTION
The present invention relates to epoxide hydrolase nucleotidic and amino acid sequences and to their use in the enantiomeric hydrolysis of epoxides.
BACKGROUND OF THE PRESENT INVENTION
Epoxides
Epoxides are used as chiral building blocks in the organic synthesis of fine chemicals, especially enantiomerically pure compounds. They are reactive molecules as their ring may be easily open to give a broad range of products. For this reason, they occupy a place of importance among the precursors of pharmaceuticals and speciality chemicals. Some chemical methods exist for preparing them from optically active precursors, but no efficient asymmetric syntheses involving asymmetrisation or resolution methods are known with the exception of the Sharpless-epoxidation method, which is limited to allylic alcohols (Katsuki et al.,
J. Am. Chem. Soc.
102, p. 5974 (1980)). The use of biological reactions to perform epoxides synthesis has also been investigated (see for example the review of de Bont J. A. M.,
Tetrahedron: Asymmetry
4, p. 1331 (1993)).
Other epoxides, like halogenated aliphatic epoxides, are potential pollutants that are released into the environment from various industrial sources or that may be formed during the transformation of other synthetic chemicals. For example, epichlorohydrin (3-chloro-1,2-epoxypropane) is a widely used industrial chemical that is well recognised as mutagenic and carcinogenic.
Epoxide Hydrolases
Epoxide hydrolases (EC3.3.2.3.) are hydrolytic enzymes which catalyse the opening of an epoxide ring converting their substrate to the corresponding diol. One of their most interesting properties is that they are generally highly regio- and enantioselective, allowing the preparation of pure enantiomers.
Epoxide hydrolases have been studied in a variety of organisms. The best studied are those from mammals. They are found mainly in liver, testis, kidney, ovary and lung. They have been intensively characterised because of their involvement in the metabolism of xenobiotics (detoxification of cytotoxic, mutagenic and carcinogenic intermediates) (Seidegard et al.,
Biochemica et Biophysica Acta
695, p. 251 (1983)).
Epoxide hydrolases have been also described in other higher eukaryotes like plants and insects.
Due to their low availability, enzymes from such sources are not of practical value for large-scale processes. The microbial world represents a suitable alternative due to the possibility of cultivating micro-organisms on a large-scale. The use of whole cells to perform the biotransformation of epoxides has been investigated. Microbial epoxide hydrolases have been already described in a variety of micro-organisms. Some examples of such descriptions are summarised hereafter:
Aspergillus niger
LPC521 and
Beauvaria sulfurescens
ATCC7159 possess enantiocomplementary epoxide hydrolases that hydrolyse the two racemic forms of styrene epoxide (Pedragosa-Moreau et al.,
J. Org. Chem.
58, p. 5533 (1993)).
Diplodia gossipina ATCC16391 catalyses the kinetic resolution of racemic indene oxide into 1(S), 2(R) indene oxide (Zhang et al.,
J. Ferment. Bioeng.
80, p. 244 (1995)).
Epichlorhydrin (3-chloro-1,2-epoxypropane) is transformed in (R)-3-chloro-2-propanol by a epoxide hydrolase characterised in Corynebacterium sp strain N-1074 (Nakamura et al.,
J. Bact.
174, p. 7613 (1992)). A similar enzyme has been purified from Pseudomonas sp. strain AD1 (Jacobs et al.,
Eur. J. Biochem.
202, p. 1217 (1991)).
An epoxide hydrolase that catalyses the asymmetric hydrolysis of various racemic epoxides in chiral epoxides and diols has been isolated from Rhodococcus sp. NCIMB 11216 (Mischitz et al.,
Biotechnol. Lett.
17, p. 893 (1995)).
A strain of Flavobacterium sp. is able to convert trans-1-epoxysuccinic acid in mesotartaric acid (Martin et al.,
Biochem. J.
70, p. 405 (1955)).
The epoxide hydrolase of Nocardia tartaricans catalyses the hydrolysis of cis-epoxysuccinate to give L(+)tartaric acid (Patentschrift DE 2605921). The same reaction could be performed by some other micro-organisms like Achromobacter, Alcaligenes (U.S. Pat. No. 3,957,579),
Acinetobacter tartarogenes, Agrobacterium aureum, Agrobacteriuzn viscosum, Rhizobium validum,
Pseudomonas sp. (Offenlegungsschrift DT 2619311).
A characteristic common to all these examples is the use of whole cells or whole crude extracts of the cells to perform the reaction. The enzyme can be liberated either by breaking the cells by physical disruption or by permeabilising the cell wall and/or the cell membrane by the use of detergents.
Tartaric Acid
Tartaric acid is used by the food industry for various applications (additive in soft drinks, food preservative, raw material for the synthesis of emulsifiers, . . . ). It is possible to synthesise chemically the tartaric acid starting from maleic acid but this process gives a racemic product composed of L(+)-tartaric acid and D(+)-tartaric acid. In food, only the L(+) form of tartaric acid is authorised as the D(+) is considered as harmful for human health.
L(+)-tartaric acid is produced naturally as a by-product during wine fermentation, but the supply of this compound is variable from year to year as it is very dependant of the climate.
The enzymatic hydrolysis of cis-epoxysuccinate by a cis-epoxysuccinate hydrolase allows the obtention of the only L(+) form of tartaric acid. This biotransformation would thus represent a valuable alternative for the production of L(+) tartaric acid.
State of the Art
Rink et al. (
J. of Biological Chemistry
272 (23) (Jun. 6, 1997)) describe the primary structure and catalytic mechanisms of the epoxide hydrolase from the strain
Agrobacterium radiobacter
AD1.
Murdiyatmo et al. (
Biochemical Journal Vol.
284, pp. 87-93 (May 1992)) describe the molecular biology of the 2-haloacid-halidohydrolase-IVa from
Pseudomonas cepacia
MBA4.
Mischitz et al. (
Biotechnology Letters
No. 17(9), pp. 893-898 (1995)) describe the isolation of a highly enantioselective epoxide hydrolase from a strain of Rhodococcus sp. having a molecular weight of 33-35000 kD and obtained from gel filtration chromatography SDS page. Said document states that the optimum temperature of the epoxide hydrolase is 30° C. However, said document never describes the amino acid sequence of said enzyme and the possible nucleotide sequence encoding said enzyme.
Yamagishi & Cho (
Annals of New York Academy of Sciences
Vol. 799, pp. 784-785 (1996)) describe the enzymatic preparation of tartaric acid from cis-epoxysuccinic acid by the strain
Pseudomonas putida
MCI3037.
A similar preparation of tartaric acid is described in the Japanese patent application JP-08245497 by treating cis-epoxytartaric acid with a culture of a Pseudomonas micro-organism.
SUMMARY OF THE INVENTION
The present invention is related to a isolated and purified nucleotide sequence from
Rhodococcus rhodochrous,
preferably a strain having the deposit number LMGP-18079, encoding an epoxide hydrolase.
According to the invention, said nucleotide sequence (genomic DNA, CDNA, RNA) presents more than 50%, preferably more than 70%, more preferably more than 90% homology with the sequence SEQ ID NO 5 described hereafter.
According to a preferred embodiment of the present invention, said isolated and purified nucleotide sequence corresponds to the nucleotide sequence SEQ ID NO 5 or a portion thereof encoding a peptide having an epoxide hydrolase activity.
It is meant by “a portion of the nucleotide sequence SEQ ID NO 5”, a fragment of said sequence SEQ ID NO 5 having more than 100 nucleotides of said nucleotide sequence and encoding a protein characterised by an epoxide hydrolase enzymatic activity similar to the epoxide hydrolase activity of the complete amino-acid sequence SEQ ID NO 6.
Preferably, said portion has an epoxide hydrolase enzymatic activity of more tha

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