Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
1999-04-22
2001-03-20
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S072000, C435S320100, C435S252300, C435S254110, C435S419000, C435S254200, C536S023200
Reexamination Certificate
active
06204040
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to the fields of molecular biology, bacteriology and industrial fermentation. More specifically, the invention relates to the identification and isolation of nucleic acid sequences and proteins for subunits of a novel, membrane bound sorbitol dehydrogenase in
Gluconobacter suboxydans
. The invention further relates to the fermentative production of L-sorbose from D-sorbitol and the subsequent production of 2-keto-L-gulonic acid.
2. Related Art
Sorbitol dehydrogenase (SDH) is an enzyme responsible for the efficient conversion of D-sorbitol into L-sorbose during sorbose fermentation in the process of the manufacturing of 2-keto-L-gulonic acid (2-KLG), an important precursor for vitamin C synthesis. Gluconobacter possesses several SDHs, which may be categorized according to their cofactor requirement: (1) NAD-dependent, (2) NADP-dependent and (3) NAD(P)-independent types. Among them, NAD(P)-independent enzyme is believed to be directly involved in efficient production of sorbose during industrial sorbose fermentation (Cummins. J. T. et al.,
J Biol. Chem
., 224, 323; 226, 3 01 (1957)).
The process of manufacturing L-sorbose from D-sorbitol is typically performed by fermentation with an acetic acid bacterium such as
Gluconobacter suboxydans
and
Acetobacter xylinum
. At room temperature, 96-99% of conversion is made in less than 24 hours (Liebster, J. et al.,
Chem. List
., 50:395 (1956)).
L-sorbose produced by the action of SDH is a substrate in the production of 2-keto-L-gulonic acid (2-KLG). A variety of processes for the production of 2KLG are known. For example, the fermentative production of 2-KLG via oxidation of L-sorbose to 2-KLG via a sorbosone intermediate is described for processes utilizing a wide range of bacteria:
Gluconobacter oxydans
(U.S. Pat. Nos. 4,935,359; 4,960,695; 5,312,741; and 5,541,108);
Pseudogluconobacter saccharoketogenes
(U.S. Pat. No. 4,877,735; European Pat. No. 221 707);
Pseudomonas sorbosoxidans
(U.S. Pat. Nos. 4,933,289 and 4,892,823); mixtures of microorganisms from these and other genera, such as Acetobacter, Bacillus, Serratia, Mycobacterium, and Streptomyces (U.S. Pat. Nos. 3,912,592; 3,907,639; and 3,234,105); and novel bacterial strains (U.S. Pat. No. 5,834,231).
A number of enzymes involved in the fermentative oxidation of sorbitol, sorbose and sorbosone are identified in the literature. U.S. Pat. Nos. 5,888,786; 5,861,292; 5,834,263 and 5,753,481 disclose nucleic acid molecules encoding and/or isolated proteins for L-sorbose dehydrogenase and L-sorbosone dehydrogenase; and U.S. Pat. No. 5,747,301 discloses an enzyme with specificity for sorbitol dehydrogenase.
In addition to distinguishing Gluconobacter SDH's on the basis of cofactor requirements, other physical characteristics may be found in the literature that distinguish these different enzymes. For example, the sorbitol dehydrogenase identified in U.S. Pat. No. 5,747,301 is distinguished on the basis of subcellular location (membrane-bound) and a haloenzyme molecular weight of 800±50 kDa (10 homologous subunits of 79±5 kDa). The membrane-bound D-sorbitol dehydrogenase isolated by Shinagawa et al.(E. Shinagawa, K. Matsushita, 0. Adachi and M. Ameyama (Agric. Biol. Chem., 46, 135-141, 1982)) consisted of three kinds of subunits with molecular weights of 63,000, 51,000 and 17,000.
In an effort to improve the productivity of commercial fermentation in the production of 2 KLG, the inventors have identified a novel, membrane-bound sorbital dehydrogenase in a strain of G. suboxydans that is distinct from others described in the literature (Choi, E. S. et al.,
FEMS Microbiol
. Lett., 125:45 (1995)).
SUMMARY OF THE INVENTION
This invention pertains to a novel, membrane-bound sorbitol dehydrogenase of
Gluconobacter suboxydans
. The isolated sorbitol dehydrogenase enzyme comprises three subunits: a first subunit of 75 kDa containing pyrroloquinoline quinone (PQQ) as cofactor; a second subunit of 50 kDa being a cytochrome c; and a third subunit of 29 kDa playing a very important role in the stability and the catalytic activity of the enzyme.
The present invention provides nucleic acid molecules for the 3 protein subunits of the Gluconobacter sorbitol dehydrogenase described herein. In a first specific embodiment, the invention provides an isolated nucleic acid molecule drawn to the first SDH subunit (75 kDA) identified by SEQ ID NO:1. In a second specific embodiment, the invention provides an isolated nucleic acid molecule drawn to the second SDH subunit (50 kDA) identified by SEQ ID NO:2. In a third specific embodiment the invention provides an isolated nucleic acid molecule drawn to the third SDH subunit (29 kDA) identified by SEQ ID NO:3. Other related embodiments are drawn to vectors, processes for producing the same and host cells carrying said vectors.
The invention also provides isolated nucleic acid molecules encoding the three subunits of the SDH of the invention. In one specific embodiment, the invention provides a cloned nucleic acid molecule encoding the 75 kDa and 50 kDa subunits. The structural genes for the first and the second subunit of sorbitol dehydrogenase are 2,265 bp and 1,437 bp, respectively, in size and are clustered in the cloned nucleic acid molecule which is a 5.7 kb Pstl DNA fragment that defines the operon. In another specific embodiment, the invention provides a cloned nucleic acid molecule encoding the third, 29 kDa, SDH subunit protein. The structural gene coding for the third subunit is 921 bp in size and found in a 4.5 kb Clal DNA fragment. Other related embodiments are drawn to vectors, processes to make the same and host cells containing said vectors.
The invention is also drawn to isolated polypeptides for the three subunits of the SDH described herein.
The invention also provides a method for the production of D-sorbose comprising: (a) transforming a host cell with at least one isolated nucleotide sequence selected from the group consisting of a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:1; a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2; and a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 3; and (b) selecting and propagating said transformed host cell.
Another aspect of the invention is drawn to a method for production of 2-KLG comprising: (a) transforming a host cell with at least one isolated nucleotide sequence selected from the group consisting of a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:1; a polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2; and a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 3; and (b) selecting and propagating said transformed host cell.
REFERENCES:
patent: 4935359 (1990-06-01), Yin et al.
patent: 4960695 (1990-10-01), Hoshino et al.
patent: 5312741 (1994-05-01), Hoshino et al.
patent: 5541108 (1996-07-01), Fujiwara et al.
patent: 5747301 (1998-05-01), Hoshino et al.
patent: 5753481 (1998-05-01), Niwa et al.
patent: 5834263 (1998-11-01), Niwa et al.
patent: 5861292 (1999-01-01), Niwa et al.
patent: 5888786 (1999-03-01), Niwa et al.
patent: 0 897 984 A2 (1999-02-01), None
patent: 98-069057 (1998-10-01), None
GenBank Accession No. E03223 (Jun. 1997).*
GenBank Accession No. E04355 (Jun. 1997).*
GenBank Accession No. AC005387 (Aug. 1998).*
GenBank Accession No. D86375 (Apr. 1997).*
Choi, E.-S. et al. “Purification of a membrane-bound sorbitol dehydrogenase fromGluoconobacter suboxydans,”FEMS Microbiol. Letts. 125:45-50 (1995).
Shinagawa, E. et al. “Purification and Characterization of D-Sorbitol Dehydrogenase from Membrane ofGluconobacter suboxydansver &agr;,”Agric. Biol. Chem. 46:135-141 (1982).
Kondo, K. and S. Horinouchi, “Characterization of the Genes Encoding the Three-Component Membrane-Bound Alcohol Dehydrogenase fromGluconobacter suboxydansand Their Expression inAcetobacter pasteurianus,”Appl. Environ. Microbiol. 63:1131-1138 (Mar. 1997).
International Search Report for International A
Choi Eui-Sung
Lee Eun-Hae
Rhee Sang-Ki
Achutamurthy Ponnathapu
Korea Research Institute of Bioscience and Biotechnology
Sterne Kessler Goldstein & Fox P.L.L.C.
LandOfFree
Gluconobacter suboxydans sorbitol dehydrogenase, genes and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Gluconobacter suboxydans sorbitol dehydrogenase, genes and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Gluconobacter suboxydans sorbitol dehydrogenase, genes and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2472794