Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-10-13
2001-06-26
Slobodyansky, Elizabeth (Department: 1602)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
Reexamination Certificate
active
06251650
ABSTRACT:
FIELD OF INVENTION
The present invention relates to the field of molecular biology and methods for the isolation and expression of foreign genes in recombinant microorganisms. More specifically, the invention relates to the isolation, sequencing, and recombinant expression of nucleic acid fragments (genes) encoding a stereospecific, nitrile hydratase (NHase) activity capable of catalyzing the hydrolysis of certain racemic nitrites to the corresponding R- or S- amides. Additionally, the invention relates to the co-expression of the nitrile hydratase nucleic acid fragment with a nucleic acid fragment encoding a stereospecific amidase activity capable of converting a racemic mixture of R- and S- amides to the corresponding enantiomeric R- or S- carboxylic acids.
BACKGROUND
Many agrochemicals and pharmaceuticals of the general formula X—CHR—COOH are currently marketed as racemic or diastereomer mixtures. In many cases the physiological effect derives from only one enantiomer/diastereomer where the other enantiomer/diastereomer is inactive or even harmful. Methods for synthesizing enantiomers are becoming increasingly important tools for the production of chemicals of enantiomer purity. To date, however, no recombinant, stereospecific NHase has been described capable of catalyzing the hydrolysis of certain racemic nitrites to the corresponding R- or S- amides.
Methods for the selective preparation of stereo-specific amides from nitrites are known and incorporate microorganisms possessing nitrile hydratase activity (NHase). These NHases catalyze the addition of one molecule of water to the nitrile, resulting in the formation of the amide free product according to Reaction 1:
R—CN+H
2
O→RCONH
2
Reaction 1
Similarly, methods for the stereospecific production of carboxylic acids are known and incorporate microorganisms possessing an amidase (Am) activity. In general amidases convert the amide product of Reaction 1 to the acid free product plus ammonia according to Reaction 2:
RCONH
2
→RCOOH+NH
3
Reaction 2
A wide variety of bacterial genera are known to possess a diverse spectrum of nitrile hydratase and amidase activities including Rhodococcus, Pseudomonas, Alcaligenes, Arthrobacter, Bacillus, Bacteridium, Brevibacterium, Corynebacterium, and Micrococcus. For example, nitrile hydratase enzymes have been isolated from
Pseudomonas chlororaphis
, B23 [Nishiyama, M. J.,
Bacteriol.,
173:2465-2472 (1991)]
Rhodococcus rhodochrous
J1 [Kobayashi, M.,
Biochem. Biophys. Acta,
1129:23-33 (1991)] Brevibacterium sp. 312(Mayaux et al.,
J. Bacteriol.,
172:6764-6773 (1990)), and Rhodococcus sp. N-774 [Ikehata, O., Nishiyama, M., Horinouchi, S., Beppu, T.,
Eur. J. Biochem.,
181: 563-570 (1989)]. No disclosure of any stereoselective activity is made for any of these enzymes. Only two disclosures have been made for stereoselective nitrile hydratase activity in native bacterial strains. The Applicants have disclosed a stereospecific nitrile hydratase from
P. putida
NRRL-18668 [WO 92/05275 (1990)].
Wildtype microorganisms known to possess nitrile hydratase activity have been used to convert nitriles to amides and carboxylic acids. For example, EPA 326,482 discloses the stereospecific preparation of aryl-2-alkanoic acids such as 2-(4-chlorophenyl)-3-methylbutyric acid by microbial hydrodysis of the corresponding racemic amide using members of Brevibacterium and Corynebacteriumn. Similarly, U.S. Pat. No. 4,366,250 teaches the use of Bacillus, Bacteridium, Micrococcus and Brevibacterium in a method for the preparation of L-amino acids from the corresponding racemic amino nitriles. Finally, WO 92/05275 teaches a biologically-catalyzed method for converting a racemic alkyl nitrile to the corresponding R- or S-alkanoic acid through an intermediate amide using members of the bacterial genera Pseudomonas spp. (e.g., putida, aureofaciens, Moraxella spp.) and Serratia (e.g.,
Serratia liquefaciens
).
In addition to the use of wildtype organisms, recombinant organisms containing heterologous genes for the expression of nitrile hydratase are also known for the conversion of nitriles. For example, Cerebelaud et al., (WO 9504828) teach the isolation and expression in
E. coli
of nitrile hydratase genes isolated from
C. testosteroni
. The transformed hosts effectively convert nitriles to amides where the nitrile substrate consists of one nitrile and one carboxylate group. However, WO 9504828 does not teach a stereospecific conversion of nitriles.
Similarly, Beppu et al., (EP 5024576) disclose plasmids carrying both nitrile hydratase and amidase genes from Rhodococcus capable of transforming
E. coli
where the transformed host is then able to use isobutyronitrile and isobutyroamide as enzymatic substrates. However, EP 5024576 does not teach a stereospecific conversion of nitriles or amides.
As with nitrile hydratases, microorganisms possessing amidase activity have been used to convert amides to carboxylic acids. In U.S. Ser. No. 08/403911, Applicants disclose a method for converting an (S)-amide, or stereospecifically converting a mixture of (R)- and (S)-amides to the corresponding enantiomeric (S)-carboxylic acid by contacting said amide with
Pseudomonas chlororaphis
B23 in a solvent. This method uses a wildtype microorganism and does not anticipate a recombinant catalyst or heterologous gene expression. Blakey et al.,
FEMS Microbiology Letters,
129:57-62 (1995) disclose a Rhodococcus sp. having activity against a broad range of nitriles and dinitriles and able to catalyze regio-specific and stereo-specific nitrile biotransformations.
Genes encoding amidase activity have been cloned, sequenced, and expressed in recombinant organisms. For example, Azza et al., (
FEMS Microbiol
. Lett. 122, 129, (1994)) disclose the cloning and over-expression in
E. coli
of an amidase gene from Brevibacterium sp. R312 under the control of the native promoter. Similarly, Kobayashi et al., (
Eur. J. Biochem.,
217, 327, (1993)) teach the cloning of both a nitrile hydratase and amidase gene from
R. rhodococcus
J1 and their co-expression in
E. coli.
What is needed and inventive over the prior art is a method for the stereospecific conversion of racemic alkyl nitriles to the corresponding R- or S-alkanoic acids using a recombinant organism.
SUMMARY OF THE INVENTION
This invention relates to nucleic acid fragments encoding:
1) the &agr; subunit of a stereospecific nitrile hydratase enzyme, said gene having at least a 64% base homology with the &agr; subunit coding region of the
Rhodococcus rhodochrous
J1 L-NHase gene [Kobayashi, M., Biochem. Biophys. Acta, 1129:23-33 (1991)] and said enzyme capable of catalyzing the hydrolysis of racemic aryl-2-alkane nitrites to the corresponding R- or S- amides; and
2) the &bgr; subunit of a stereospecific nitrile hydratase enzyme, said gene having at least a 52% base homology with the &bgr; subunit coding region of the
Rhodococcus rhodochrous
J1 L-NHase gene and said enzyme capable of catalyzing the hydrolysis of racemic aryl-2-alkane nitrites to the corresponding R- or S- amides.
Another embodiment of the invention is a nucleic acid fragment comprising the nucleic acid fragments encoding both the &agr; and &bgr; subunits of a stereospecific nitrile hydratase enzyme described above, said enzyme capable of catalyzing the hydrolysis of racemic aryl-2-alkane nitrites to the corresponding R- or S- amides.
A further embodiment of the invention is a nucleic acid fragment encoding the &agr; subunit of a stereospecific nitrile hydratase enzyme, said nucleic acid fragment having the nucleotide sequence as represented in SEQ ID NO.:3 and said enzyme capable of catalyzing the hydrolysis of racemic alkyl nitriles to the corresponding R- or S- amides.
A further embodiment of the invention is a nucleic acid fragment encoding the &bgr; subunit of a stereospecific nitrile hydratase enzyme, said nucleic acid fragment having the nucleotide sequence as represented in SEQ ID NO.:4 and said enzyme capable
Fallon Robert Donald
Nelson Mark James
Payne Mark Scott
E. I. Du Pont de Nemours and Company
Slobodyansky Elizabeth
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