Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1991-06-28
1994-03-22
Brown, Johnnie R.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 74, 435 84, 536 55, 536 553, 424 946, 424 9461, C12P 1904
Patent
active
052963608
DESCRIPTION:
BRIEF SUMMARY
DESCRIPTION
1. Technical Field
This invention relates to a process for producing ganglioside G.sub.Ml.
2. Prior Art
"Ganglioside" is a general name of the group of sialic acid-containing sphingoglycolipids and is known to exist in the brain of higher animals, especially in the nervous system. In the living body, the gangliosides not only take part in nervous function and recognition, differentiation, proliferation, canceration, senescence, etc. of cells, but also, from a cell sociological view point, are responsible for receptor function of cytotoxins such as cholera toxin and botulinum toxin, peptide hormones such as thyroid hormone, interferons and so on. The gangliosides are also supposed to contribute to negative charges on the surface of cells. In fact, it is reported that the gangliosides extracted from bovine brain promote regeneration of damaged peripheral nerve and useful for treatment of alcoholic polyneuritis, diabetic neuropathy, etc (Iyaku to Yakugaku, 13(6), pp 1407-1412 (1985); Practice, 4(4), pp 452-456 (1987); Iyaku Journal, 22(7), pp 55-62 (1986)).
As described above, gangliosides are important substances having various functions in the living body. Therefore, it is desired to establish a process for collecting each of various gangliosides in a pure form. In order to prepare a specific ganglioside, conventionally attempted are methods using various kinds of chromatography. However, since the starting material, i.e., ganglioside components as extracted from bovine brain or the like is a mixture containing various gangliosides each in trace amounts, it is difficult to obtain the desired products even by conducting cumbersome procedures, and any proper purification methods have not been found yet. Further, although an attempt is made to synthesize gangliosides by chemical technique, its process is complicated and side reactions are likely to occur, entailing difficulty in removing by-products.
On the other hand, neuraminidase is an enzyme which has been considered not to act directly on the gangliosides on its own. As a matter of fact, it is only reported that asialoganglioside G.sub.Al is formed from ganglioside G.sub.Ml in the presence of detergents such as cholates (Masaki Saito, Taisha, 16, p 761 (1977)). Moreover, it has been not known at all that any isozyme of the neuraminidase exists.
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide a process for collecting a specific ganglioside, i.e., ganglioside G.sub.Ml from gangliosides.
Another object of the present invention is to provide a process for producing ganglioside G.sub.Ml in high purity and in good yield.
Namely, the present invention provides a process for producing ganglioside G.sub.Ml characterized in that neuraminidase isozyme S is allowed to act on gangliosides to obtain ganglioside G.sub.Ml.
The present inventors have conducted intensive research and consequently found that the neuraminidase derived from the bacteria of the genus Arthrobacter has a novel isozyme and that, when said neuraminidase isozyme S is allowed to act on gangliosides, ganglioside G.sub.Ml can be selectively obtained with high purity and in good yield. According to the present invention, by-products are not produced, and therefore ganglioside G.sub.Ml obtained can be readily purified. Further, the present invention has the advantage that ganglioside G.sub.Ml can be prepared using said enzyme in the absence of any detergent.
The neuraminidase isozyme S (hereinafter referred to as "Isozyme S" ) to be used in the present invention has the following physicochemical properties :
(1) Action: Selectively producing ganglioside G.sub.Ml from gangliosides.
(2) Molecular weight: About 52000 dalton
The molecular weight was determined according to gel filtration chromatography and SDS-PAGE electrophoresis as described below.
Gel Filtration Chromatography
Gel filtration method was conducted using Sephadex G150 (product of Pharmacia). As the eluent, 50 mM phosphate buffer was used.
SDS-PAGE Electrophoresis
Electrophoresis was conduct
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Ohta Yasuhiro
Sugimori Tsunetake
Tsukada Yoji
Brown Johnnie R.
Marukin Shoyu Co., Ltd.
Webber Pamela S.
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