Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
1998-12-01
2001-06-05
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S191000, C435S136000, C435S252100
Reexamination Certificate
active
06242233
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention concerns a novel enzyme, namely aldehyde dehydrogenase (ADH), a process for producing ADH and a process for producing 2-keto-L-gulonic acid (2-KGA) from L-sorbosone utilizing said enzyme. 2-KGA is an important intermediate for the production of vitamin C.
Some microorganisms are known to convert L-sorbosone to 2-KGA. For example, in U.S. Pat. No. 3,907,639, the microorganisms belonging to the genera Acetobacter, Pseudomonas, Escherichia, Serratia, Bacillus, Staphylococcus, Aerobacter, Alcaligenes, Penicillium, Candida and Gluconobacter are reported to be capable of effecting the conversion. Furthermore, Kitamura et al. (Eur. J. Appl. Microbiol., 2, 1, 1975) report that the enzyme oxidizing L-sorbosone found in
Gluconobacter melanogenus
IFO 3293 requires neither a coenzyme nor an electron acceptor for the development of enzyme activity. Makover et al. (Biotechnol. Bioeng. 17, 1485, 1975) report the presence of L-sorbosone dehydrogenase activity in the particulate fraction of
Pseudomonas putida
ATCC 21812 and of
Gluconobacter melaogenus
IFO 3293. They also indicate that neither nicotinamide adenine dinucleotide (NAD) nor nicotinamide adenine dinucleotide phosphate (NADP) acts as a coenzyme for the enzyme. T. Hoshino et al. (Agric. Biol. Chem., 55, 665, 1991) purified and characterized L-sorbosone dehydrogenase from
Gluconobacter melanogenus UV
10, which requires NAD or NADP as a coenzyme.
In the context of the present invention, microorganisms belonging to the genus Gluconobacter have been studied more closely and, as a result, it has been found that the further novel ADH which catalyzes the oxidation of L-sorbosone to 2-KGA can be obtained from said microorganisms. Furthermore, it has been found that the purified ADH provided by the present invention oxidizes L-sorbosone to 2-KGA in the presence of electron acceptors, such as 2,6-dichlorophenolindophenol (DCIP) and phenazine methosulfate (PMS), ferricyanide or cytochrome c, but that NAD, NADP and oxygen are not suitable as electron acceptors. Thus, the ADH provided by the present invention is clearly distinct from the known L-sorbosone dehydrogenase.
SUMMARY OF THE INVENTION
This invention provides a purified aldehyde dehydrogenase, wherein the dehydrogenase: has a molecular weight of 150,000±6,000 Da and comprises two homologous subunits or has a molecular weight of 230,000±9,000 Da and comprises three homologous subunits, each subunit having a molecular weight of about 75,000±3,000 Da; has L-sorbosone, D-glucosone, D-glucose, D-galactose, D-mannose, L-gulose, D-xylose, D-ribose, and D-arabinose dehydrogenase activity; utilizes as cofactors pyrroloquinoline quinone and heme c; has an optimum pH of from 7.0 to 8.5; and is inhibited by Co
2+
, Cu
2+
, Fe
2+
, Ni
2+
, Zn
2+
, monoiodoacetate and ethylenediamine tetraacetic acid.
This invention provides a process for producing the aldehyde dehydrogenase described above, comprising disrupting one or more cells of a Gluconobacter organism containing the dehydrogenase, and purifying the aldehyde dehydrogenase from the disrupted cells.
This invention provides a process for producing a carboxylic acid from its corresponding aldehyde which comprises contacting the aldehyde with the purified aldehyde dehydrogenase of this invention, in the presence of an electron acceptor.
REFERENCES:
patent: 3907639 (1975-09-01), Makover et al.
patent: 5437989 (1995-08-01), Asakura
patent: 5932463 (1999-08-01), Asakura
patent: 0 278 447 B1 (1988-08-01), None
patent: 0 606 621 A2 (1994-07-01), None
patent: 0 758 679 A1 (1997-02-01), None
patent: 0 790 301 A2 (1997-08-01), None
Kitamura et al., “Metabolism of L-Sorbose by Enzymes fromGluconobacter melanogenusIFO 3293”, Eur. J. Appl. Microbiol. Vol. 2, pp. 1-7 (1975).
Makover et al., “New Mechanisms for the Biosynthesis and Metabolism of 2-Keto-L-Gulonic Acid in Bacteria”, BioTech and BioEng. vol. 7, pp. 1485-1514 (1975).
Hoshino et al., “Isolation and Characterization of NAD(P)-Dependent L-Sorbosone Dehydrogenase fromGluconobacter melanogenusUV10”, Agric. Biol. Chem., vol. 55(3), pp. 665-670 (1991).
Kondo, et al., Characterization of the Genes Encoding the Three-Component Membrane-Bound Alcohol Dehydrogenase fromGluconobacter suboxydansand Their Expression inAcetobacter pasteurianus,Applied and Environmental Microbiology, vol. 63, No. 3, pp. 1131-1138 (1997).
Matsushita, et al., “Function of Multiple Heme c Moieties in Intramolecular Electron Transport and Ubiquinone Reduction in the Quinohemoprotein Alcohol Dehydrogenase-Cytochrom c Complex ofGluconobacter suboxydans,”Journal of Biological Chemistry, vol. 271, No. 9, pp. 4850-4857 (1996).
Thurner, et al., “Biochemical and Genetic Characterization of the Acetaldehyde Dehydrogenase Complex fromAcetobacter europaeus,”Arch. Microbiol., vol. 168, No. 2, pp. 81-91 (1997).
Hoshino Tatsuo
Miyazaki Taro
Sugisawa Teruhide
Bryan Cave LLP
Haracz Stephen M.
Marx Irene
Roche Vitamins Inc.
Waddell Mark E.
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