Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Patent
1990-06-11
1994-10-04
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
435 691, 435136, 435138, 4351723, 4352523, 4353201, 536 232, 935 14, 935 60, 935 72, C12N 1553, C12N 1563, C12N 1574
Patent
active
053525990
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a novel enzyme, namely L-sorbosone dehydrogenase, a process for producing the same and a process for producing 2-keto-L-gulonic acid utilizing said enzyme. Moreover, the present invention relates to genetic engineering techniques which provide an improved method for the cloning and expression of the gene of said enzyme and transformed microorganisms capable of producing 2-keto-L-gulonic acid with high efficiency.
The compound 2-keto-L-gulonic acid (2-KGA) is an important intermediate in the synthesis of ascorbic acid (Vitamin C). Numerous microorganisms are known to produce 2-KGA from D-sorbitol or L-sorbose, for example, members of the genera Acetobacter and Pseudomonas, though levels of 2-KGA produced are less than 6g/L (Japan Patent Publication No. 40,154/1976). Generally the pathway of D-sorbitol to 2-KGA can be illustrated as follows (Makover et al. 1975, Biotechnol. and Bioeng. 17, 1485-1514):
Reactions to convert L-sorbosone to 2-KGA using microorganisms are known. 2-KGA production from L-sorbosone using cell free extracts of microorganisms was reported in several prior publications.
In U.S. Pat. No. 3,907,639, microorganisms belonging to the genera Acetobacter, Pseudomonas, Escherichia, Serratia, Bacillus, Staphylococcus, Aerobacter, Alcaligenes, Penicillium, Candida and Gluconobacter were reported to be capable of such a conversion.
Furthermore, Kitamura et al. (Europ. J. Appl. Microbiol., (2) 1, 1975) reported that a L-sorbosone oxidizing enzyme found in Gluconobacter melanogenes IFO 3293 required neither coenzyme nor an electron acceptor for the development of its enzyme activity.
However, no disclosure has been made up to now on a purified enzyme having the activity to oxidize L-sorbosone to 2-KGA not depending on coenzymes, e.g. nicotinamide-adenine dinucleotide (NAD) or nicotinamide-adenine dinucleotide phosphate (NADP), etc. It has been found that the purified enzyme isolated from the membrane fraction of cells of specific microorganisms catalyzes the oxidation of L-sorbosone to 2-KGA independently of coenzymes such as NAD and NADP. The present invention has been accomplished based on this finding.
It is an object of the present invention to provide a novel coenzyme independent L-sorbosone dehydrogenase which catalyzes oxidation of L-sorbosone to 2-KGA. It is another object to provide a process for producing said novel L-sorbosone dehydrogenase by a fermentation method. It is also an object to provide an improved process for the production of 2-KGA from L-sorbosone with the aid of said novel L-sorbosone dehydrogenase or a microorganism which produces said enzyme.
Furthermore, another aspect of the object of the present invention is to provide gene engineering techniques which enable the production of said L-sorbosone dehydrogenase by an improved method and also enable the production of 2-KGA from L-sorbosone using said enzyme produced by a recombinant microorganism or using said recombinant microorganism by fermentation. In this respect, DNA comprising the gene encoding said novel L-sorbosone dehydrogenase, vectors and recombinant organisms containing said DNA are also within the scope of the present invention.
It is to be understood that with regard to the partial amino acid sequences given in connection with the DNA, the DNA fragments, the recombinant DNA molecules, the recombinant microorganisms and the processes involved, the functional equivalents of said amino acid sequences, i.e. equivalents which achieve the same goal are also included.
DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the restriction map of the plasmid pVK 102.
FIG. 2 illustrates the restriction maps of the plasmids of the present invention.
FIG. 3 illustrates the restriction map of a DNA containing the structural gene encoding L-sorbosone dehydrogenase.
FIG. 4 illustrates the DNA sequence encoding L-sorbosone dehydrogenase originated from Gluconobacter oxydans IFO 12258.
The underlined portions indicate partial amino acid sequences also determined by amino acid sequence analysis of
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Fujiwara Akiko
Hoshino Tatsuo
Shinjoh Masako
Epstein William H.
Gould George M.
Hoffmann-La Roche Inc.
Moore William W.
Wax Robert A.
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