Chemistry: molecular biology and microbiology – Process of utilizing an enzyme or micro-organism to destroy... – Resolution of optical isomers or purification of organic...
Reexamination Certificate
1999-07-19
2001-03-20
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Process of utilizing an enzyme or micro-organism to destroy...
Resolution of optical isomers or purification of organic...
C435S107000
Reexamination Certificate
active
06204050
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of biocatalysis. More specifically, this invention pertains to the discovery of novel 4-hydroxyproline epimerases and to their use to convert either trans-4-hydroxy-L-proline or cis-4-hydroxy-D-proline to a mixture of trans-4-hydroxy-L-proline and cis-4-hydroxy-D-proline.
BACKGROUND OF THE INVENTION
The agricultural and pharmaceutical industry seeks production of compounds in high yield, and when a compound contains one or more chiral centers, it is often desirable to produce a single isomer. The products of the present invention are useful as precursors for chemicals of high value in these industries. Specifically, cis-4-hydroxy-D-proline (CHDP) and trans-4-hydroxy-L-proline (THLP) are useful for preparing agrochemicals and pharmaceuticals.
CHDP is prepared commercially by the chemical epimerization of THLP (Greenstein, J. P. and Winitz, M.,
Chemistry of the Amino Acids,
vol.3, chapter 29, John Wiley and Sons: New York (1961)). Required for the synthesis of CHDP, THLP is prepared commercially from hydrolyzed animal gelatin, which contains approximately 13% of the desired amino acid (U.S. Pat. No. 3,860,607). Chemically epimerizing THLP to CHDP uses either basic reaction conditions (e.g., barium hydroxide under pressure at 200° C. for 6 h) or acidic reaction conditions (e.g., refluxing acetic acid/acetic anhydride). Producing mixtures of THLP and CHDP by chemical epimerization typically (1) uses high temperatures, (2) requires separating the base or acid from the desired hydroxyproline diastereomers before separating CHDP from THLP, and (3) produces highly caustic or acidic waste streams. These conditions introduce undesirable cost and handling risks.
Enzymes, as opposed to harsh chemicals, can also be used in epimerization reactions. Reactions carried out with the aid of enzymes or intact microorganisms have been used with increasing frequency and increasing success to catalyze synthetic chemical reactions. Biological processes are commonly perceived as being less harmful to the environment than chemical manufacturing processes. Amino acid racemases catalyze formation of a racemic mixture from either the D or L form of the free amino acid by equilibrating configuration at the &agr;-carbon. Amino acid epimerases also catalyze equilibration of configuration at the &agr;-carbon. However, since epimerases act on compounds possessing an additional asymmetric carbon, a diastereomer of the substrate is formed rather than its antipode.
4-Hydroxyproline epimerase (EC 5.1.1.8, also known as hydroxyproline 2-epimerase) catalyzes the conversion of THLP to CHDP and CHDP to THLP. The enzyme was the first amino acid-racemizing enzyme to be characterized in an essentially homogenous state, permitting direct investigation of cofactor status (Adams et al.,
J. Biol. Chem.
239:1525-1535 (1964)). An approximately equimolar mixture of THLP and CHDP is obtained when starting with either diastereomer. The enzyme also epimerizes cis-4-hydroxy-L-proline (CHLP) or trans-4-hydroxy-D-proline (THDP) to a mixture of CHLP and THDP. 3-Hydroxyprolines have also been reported to be substrates for the enzyme.
4-Hydroxyproline epimerase has been isolated from
Pseudomonas putida
(Finlay et al.,
J. Biol. Chem.
245:5248-5260 (1970)),
Pseudomonas striata
(Adams et al.,
J. Biol. Chem.
239:1525-1535 (1964)),
Pseudomonas fluorescence
(Ito et al.,
Anal. Biochem.
151:510-514 (1985)),
Pseudomonas aeruginosa
(Manoharan et al.,
J. Biosci.
2:107-120 (1980)),
Pseudomonas ovalis
and Alcaligenes sp. (Drawert et al.,
Chem., Mikrobiol., Technol. Lebensm.
5:165-169 (1978)), Achromobacter (Jayaraman et al.,
Indian J Biochem.
2:153 (1965)), and
Aerobacter aerogenes
(Adams et al.,
Methods Enzymol.
17B:266-306 (1971)). In addition, the enzyme has been isolated from a fermentation of Pseudomonas No. 109 which was cultured in the presence of 4-hydroxy-L-proline (JP 60221083).
Although these sources of 4-hydroxyproline epimerase are known, a novel and enhanced source for this biocatalyst would be useful to industry in the production of high value agrochemicals and pharmaceuticals.
SUMMARY OF THE INVENTION
The claimed invention is a process to produce 4-hydroxyproline comprising (a) contacting, in an aqueous reaction mixture, at least one starting material selected from the group consisting of trans-4-hydroxy-L-proline and cis-4-hydroxy-D-proline with an enzyme catalyst in a form selected from the group consisting of whole cells, cell extracts, partially purified enzyme or purified enzyme, the enzyme catalyst characterized by 4-hydroxyproline epimerase activity and derived from
Acinetobacter baumanni
WT-50/50-4A (ATCC 202144) or
Serratia marcescens
WT-L-4A1 (RED) (ATCC 202145); and (b) isolating the diastereomers trans-4-hydroxy-L-proline and cis-4-hydroxy-D-proline from the reaction mixture.
The invention encompasses biologically pure cultures of (a)
Acinetobacter baumanni
WT-50/50-4A designated as ATCC Accession Number 202144 and (b)
Serratia marcescens
WT-L-4A1 (RED) designated as ATCC Accession Number 202145.
Additionally, the invention includes a process to produce 4-hydroxyproline comprising (a) contacting, in an aqueous reaction mixture, at least one starting material selected from the group consisting of trans-4-hydroxy-D-proline and cis-4-hydroxy-L-proline, with an enzyme catalyst in a form selected from the group consisting of whole cells, cell extracts, partially purified enzyme and purified enzyme, the enzyme catalyst characterized by 4-hydroxyproline epimerase activity and derived from
Acinetobacter baumanni
WT-50/50-4A (ATCC 202144) or
Serratia marcescens
WT-L-4A1 (RED) (ATCC 202145); and (b) isolating trans-4-hydroxy-D-proline and cis-4-hydroxy-L-proline from the reaction mixture.
In both processes of the invention, the starting material can be recycled through steps (a) and (b) until it is ultimately converted to the corresponding diastereomer.
The processes are operated at a preferred pH of from about 6.0 to 8.0.
BRIEF DESCRIPTION OF THE BIOLOGICAL DEPOSITS
Applicants made the following biological deposits under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure:
Depositor Identification
International Depository
Reference
Designation
Date of Deposit
Acinetobacter baumanni
ATCC 202144
24 June 1998
WT-50/50-4A
Serratia marcescens
ATCC 202145
24 June 1998
WT-L-4A1 (RED)
As used herein, “ATCC” refers to the American Type Culture Collection international depository located at 10801 University Boulevard, Manassas, Va., 20110-2209, U.S.A. The “ATCC No.” is the accession number to cultures on deposit with the ATCC.
DETAILED DESCRIPTION OF THE INVENTION
Applicants discovered 4-hydroxyproline epimerase activity in two isolated microorganisms,
Acinetobacter baumanni
WT-50/50-4A (ATCC 202144) and
Serratia marcescens
WT-L-4A1 (RED) (ATCC 202145). This epimerase activity usefully converts either trans-4-hydroxy-L-proline (THLP) or cis-4-hydroxy-D-proline (CHDP) to a mixture of CHDP and THLP.
After conversion of THLP or CHDP to a mixture of THLP and CHDP, the two components of the product mixture are separated and the starting material recycled into the epimerization reaction mixture, whereby all of the starting material (THLP or CHDP) is ultimately converted to the corresponding diastereomer (CHDP or THLP, respectively).
The instant invention also relates to the use of 4-hydroxyproline epimerase activity in the two novel microorganisms,
Acinetobacter baumanni
WT-50/50-4A (ATCC 202144) and
Serratia marcescens
WT-L-4A1 (RED) (ATCC 202145), for the conversion of either trans-4-hydroxy-D-proline (THDP) or cis-4-hydroxy-L-proline (CHLP) to a mixture of CHLP and THDP.
After conversion of THDP or CHLP to a mixture of THDP and CHLP, the two components of the product mixture are separated, and the starting material recycled into the epimerization reaction, whereby all of the starting material (THDP or CHLP) is ultimately converted to the corresponding dia
DiCosimo Robert
Fager Susan K.
Gavagan John E.
E. I. Du Pont de Nemours and Company
Saucier Sandra E.
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