Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-07-27
2004-04-27
Afremova, Vera (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S252100, C435S822000, C424S093400, C514S054000
Reexamination Certificate
active
06727081
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel microorganism isolated from Chinese elm(Ulmus sp.) and a process for preparing immunostimulating exopolysaccharides with anticancer activity by employing the microorganism, more specifically, to a novel Enterobacter sp. isolated from the root bark of Chinese elm(Ulmus sp.) alive, which produces immunostimulating exopolysaccharides with anticancer activity, a process for preparing the exopolysaccharides by culturing the microorganism, the exopolysaccharides thus prepared and novel practical uses thereofs.
2. Description of the Related Art
Polysaccharides have been produced from natural products such as plants and seaweeds, or microorganisms and widely used for the materials for foodstuffs, medicine, etc. In light of the various uses of polysaccharides, intensive research has been actively undertaken to produce polysaccharides with novel structure and sugar composition, and to explore their potential uses.
Recently, it has been reported that polysaccharides existing in exterior of cell wall play an important role as a messenger for signal transduction between cells, which arouse great interests in polysaccharides. Examples of commercialized exopolysaccharides produced by microorganisms include xanthan gum, pullulan, gellan gum, curdlan, hyaluronic acid, etc.
Meanwhile, in Korea and China, Chinese elm (elm tree as common name) had been used for a long time as a therapeutic agent, as well as for relieving famine. The root bark of Chinese elm has been used in a dried form for oriental medicine or folk remedy, and its pharmacological properties are described in ancient literatures of oriental medicine such as Bonchogangmok, Euhakibmoon, Hyangyakjibsungbang, and Dongeubogam. According to such medical literatures, the root bark of Chinese elm is known to be a powerful remedy for a rash and an abscess, effective on gastric ptosis, gastric ulcer, and duodenal ulcer, and also working on a mild stomachache, a digestive disorder, urinating disorder, empyema, otitis media, an abscess, a festered wound such as a swelling and a boil, skin conditioning, and uterine- and breast-associated diseases.
In this connection, the present inventors also found that water-soluble polysaccharides(proteoglycans) extracted from the root bark of Chinese elm have an anticancer activity by immunostimulation.
However, it has been revealed that water-soluble polysaccharides(proteoglycans) extracted from the root bark of Chinese elm may bring about destruction of the plant species and their qualities cannot be controlled in an efficient manner owing to different compositions depending on the area and timing of taking the plants.
Under the circumstances, there are strong reasons for exploring and developing alternative process for preparing the exopolysaccharides of uniform quality in a massive manner.
SUMMARY OF THE INVENTION
The present inventors have made an effort to solve the problems mentioned above, and isolated a novel microorganism of Enterobacter sp. which produces exopolysaccharides with immunostimulating activity, from the root bark of Chinese elm. Furthermore, they found that: the exopolysaccharides produced by fermentation of the said microorganism possess an anticancer activity by immunostimulation which is similar to physiological activity of the water-soluble polysaccharides(proteoglycans) extracted from the root bark of Chinese elm; and, their mass production can be accomplished with a uniform quality by controlling medium and culture conditions, without destruction of the plant species.
One aspect of the present invention is to provide an isolated microorganism identified by accession number KCTC 0687BP.
Another aspect of the present invention provides a method of producing an exopolysaccharide. The method comprises the steps of providing an isolated microorganism identified by accession number KCTC 0687BP, and culturing the microorganism in a medium so as to allow production of an exoploysaccharide. The method further comprises isolating the exopolysaccharide from a mixture comprising the culture medium, the microorganism and the exopolysaccharide. The culture medium comprises a carbon source selected from the group consisting of glucose, sucrose, fructose, rhamnose, galactose, arabinose, mannitol, lactose, gluconate, xylose and mixtures thereof. The culturing is performed at a temperature ranged from about 25° C. to about 38° C. under aeration at a flow rate ranged from about 0.1 vvm to about 1.5 vvm and under agitation at an agitation speed ranged from about 150 to about 500 rpm. The isolation of the exopolysaccharide comprises: removing cells from the culture mixture; and dialyzing a resulting mixture so as to isolate the exopolysaccharide. The isolation further comprises lyophilizing the separated exopolysaccharide. The removal of cells comprises: centrifuging the culture mixture to obtain a supernatant; precipitating a mixture comprising the exopolysaccharide; dissolving the precipitate in a liquid; and removing remaining cells. The present invention further provides a composition obtainable by the method.
Another aspect of the present invention provides a composition comprising an isolated exopolysaccharide from an Enterobacter species, wherein the species is obtained from root bark of Chinese elm, Ulmus species and the exopolysaccharide has a molecular weight ranged from about 100,000 to about 1,000,000. The isolated exopolysaccharide comprises sugar in an amount ranged from about 40 wt. % to about 75 wt. %. The isolated exopolysaccharide comprises acidic sugar in an amount ranged from about 5 wt. % to about 15 wt. %. The isolated exopolysaccharide comprises protein in an amount ranged from about 10 wt. % to about 25 wt. %. The isolated exopolysaccharide comprises glucose, fructose, galactose, fucose and glucuronic acid. The isolated exopolysaccharide comprises 10-30 wt. % glucose, less than 1 wt. % fructose, 10-15 wt. % galactose, 8-12 wt. % fucose and 40-70 wt. % glucuronic acid.
Still another aspect of the present invention provides a method of inducing immune cell proliferation, which comprises providing cells and contacting the exopolysaccharide with the cells, thereby stimulating proliferation of immune cells. The method further comprises identifying immune cells in need of an induction of proliferation and/or measuring immune cell proliferation.
Still another aspect of the present invention provides a method of inhibiting proliferation of cancer cells. The method comprises providing a cancer cell and contacting the exopolysaccharide with the cancer cell. The cancer cells comprising melanoma cells.
Still further aspect of the present invention provides a method of inhibiting cancer cell proliferation in a mammal. The method comprises identifying a mammal in need of an agent that inhibit cancer cell proliferation and providing the mammal with the expolysaccaharide.
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Kim Young Joo
Yang Young Lyeol
Afremova Vera
Knobbe Martens Olson & Bear LLP
Samsung Electronics Co,. Ltd.
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