Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-03-01
2001-09-04
Gitomer, Ralph (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S124000, C536S127000
Reexamination Certificate
active
06284885
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a process for preparing (1→3)-&bgr;-D-glucan from a fungal cell efficiently by an easy process, (1→3)-&bgr;-D-glucan prepared by the process, and use thereof. Particularly, the present invention relates to a novel process for easily and efficiently preparing (1→3)-&bgr;-D-glucan from a cell wall of a cell of a fungus belonging to the genus Candida, which activates a factor G activation system of a limulus amebocyte lysate and/or a prophenoloxidase cascade system of a body fluid of an arthropod, the thus obtained (1→3)-&bgr;-D-glucan, and use of the (1→3)-&bgr;-D-glucan for a (1→3)-&bgr;-D-glucan measurement.
BACKGROUND ART
(1→3)-&bgr;-D-Glucan is a sugar chain which is broadly distributed in the natural world as a cell wall component of fungi, such as yeast, molds, mushrooms and the like, plants, and the like.
The glucan containing a (1→3)-&bgr;-D-glycoside bond is classified into (1) a straight chain glucan comprising only (1→3)-&bgr;-D-glycoside bond (curdlan, paramylon, or the like), (2) a glucan containing a (1→6)-&bgr;-D-glycoside bond and a (1→3)-&bgr;-D-glycoside bond, and (3) a glucan containing a (1→4)-&bgr;-D-glycoside bond and a (1→3)-&bgr;-D-glycoside bond (lichenans, barley albumen glucan, or the like) Furthermore, the glucan of (2) can be classified into small groups, such as (2-1) laminaran having a zigzag structure derived from brown algae of the genus Eisenia, (2-2) laminaran comprising a zigzag structure having a branch derived from brown algae of the genus Laminaria, (2-3) glucan having a multi-branched dendriform structure derived from a cell wall of most of fungi or algae, (2-4) sclerotan, schizophyllan, lentinan, and the like, having a short chain branched structure, and (2-5) glucan having a repeated branched chain structure derived from a cell wall of bakery's yeast and the genus Candida. Thus, various structures exist (Aketagawa, J., Tamura, H. and Tanaka, S.,
J. Antibact. Antifung. Agents,
23(7): 413-419 (1995)). In this specification, the glucan containing a (1→3)-&bgr;-D-glycoside bond is called “(1→3)-&bgr;-D-glucan” (hereinafter referred to as “BG”). The BG shows various biological activities, such as activation of a reticuloendothelial system including production induction of various cytokines from a macrophage and the like, activation of a complement system, antitumor activities (for example, lentinan prepared from
Lentinus edodes,
schizophyllan prepared from
Schizophyllum commune
and the like are on the market as medicaments showing antitumor activities), and the like.
Also, since this substance group is cited as one of the causal substances of allergic respiratory diseases and there is a report that incorporation of BG into the living body together with endotoxin increases the action of endotoxin, its inflow into blood as a foreign material is not desirable, so that contamination of drugs, medical devices and the like with BG becomes a medically important subject.
Also, since the above-described BG is one of the Ls constituting components commonly present in fungal cell walls, detection of this substance indicates a possibility of being contaminated with other toxic components derived from fungi, so that examinations are also started on the application of BG measurement to the detection of fungi or test on the absence of fungi. Additionally, with the progress of studies on the production of medicines using microorganisms and cultured cells derived from various animals and on the various using cells in a macrophage system, it is important to examine not only microbial contamination at culturing but also acute check on the BG contamination and influence of their contamination upon the cells.
On the other hand, a limulus reagent prepared from a limulus amebocyte lysate (hereinafter sometimes referred to as “lysate”) which has been generally used in an endotoxin test method, can recognize BG and react with BG, and a BG-specific limulus reagent has been developed using a BG-sensitive reactor (factor G) activation system derived from the lysate and is now insured as a diagnostic drug of deep mycosis. Additionally, it was found that a reagent containing a factor in prophenoloxidase cascasde prepared from a body fluid of an arthropod such as a silk worm or the like, can also recognize BG and react with BG (hereinafter, the limulus reagent and the reagent containing a factor in prophenoloxidase cascade are both referred to as “BG measuring reagent”).
Because of a background that measurement of BG in the clinical field is carried out to inspect the presence or absence of contamination with fungi, it is expected that a standard substance for the measurement of BG is an appropriate substance derived from a fungus and there is a great demand for the development of a preparation process of an appropriate standard substance derived from a fungus. However, since the BG prepared from fungi, such as the genus Candida and the like, is contaminated with a large amount of mannan and its contamination with a pyrogenic substance typified by endotoxin is not avoidable, a BG preparation having sufficient purity as a standard substance of the above-described measurement and having a high reactivity with a measuring reagent, has not been obtained, so that curdlan (derived from a Gram-negative bacterium: a bacterium classified separately from fungi) and the like have been used as a standard substance for the measurement of BG.
An example of the currently used processes for the preparation of BG as a constituting component of a cell wall of a fungal cell comprises (1) a step in which the cell is subjected to a physical treatment with a A French press or the like, and then digested with a protease to collect the cell wall part by removing proteins and mannan, (2) a step in which the cell wall is subjected to a high temperature treatment in an autoclave or the like using a high concentration alkali or acid to extract a crude BG, and (3) a step in which the crude BG is subjected to neutralization and/or desalting by dialysis or the like, the BG is separated by adding an organic solvent, such as ethanol or the like, the thus separated material (precipitate) is collected by centrifugation or the like, and dehydrated with an organic solvent, such as acetone or the like, and then a precipitate is collected by centrifugation or the like, followed by pulverization by drying under a reduced pressure or the like (e.g., JP-A-3-119995, etc.).
As described above, a large number of reagents and measuring processes have been developed particularly for detecting BG with a high sensitivity, so that it is necessary to obtain BG which stably reacts as a standard substance with the limulus reagent and the reagent containing a factor in prophenoloxidase cascade, but such conventional BG preparation processes are considerably complex and accompany dangers, such as necessity of using a considerable number of steps, use of a high concentration alkali and acid under a high temperature, and the like. Additionally, since the BG obtained by the above-described processes has a low purity due to the presence of a large amount of contaminants, such as mannan and the like, and therefore is poor in stability, it shows a low reactivity when used as a standard substance in the measurement of BG and thus hinders the obtainment of stable measured values. Consequently, a process for preparing more stable BG having a higher purity from a fugal cell has been desired.
DISCLOSURE OF THE INVENTION
In order to solve the above-described problems, the present inventors have conducted extensive studies and found a process for easily and efficiently preparing BG having a high purity and a high reactivity with a BG measuring reagent from a fungal cell. Also, they found that, surprisingly, the BG prepared from a cell particularly belonging to the genus Candida by this process hardly contains mannan and endotoxin, while contamination of BG with such substances cannot easily be prevented co
Aizawa Maki
Tamura Hiroshi
Tanaka Shigenori
Gitomer Ralph
Khare D.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Seikagaku Corporation
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