Methods for producing sialyloligosaccharides in a dairy source

Details Methods for producing sialyloligosaccharides in a dairy source Methods for producing sialyloligosaccharides in a dairy source

2001-09-18

2004-03-16

Prats, Francisco (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S101000, C435S099000, C435S274000, C536S124000, C536S127000

Reexamination Certificate

active

06706497

ABSTRACT:

1. INTRODUCTION
This invention relates to methods for producing &agr;(2-3) sialyloligosaccharides in a dairy source or cheese processing waste stream by contacting the dairy source or cheese processing waste stream with a catalytic amount of at least one &agr;(2-3) trans-sialidase. In preferred embodiments, the methods of the invention are applied to produce &agr;(2-3) sialyllactose in a dairy source or cheese processing waste stream. Methods for isolating the &agr;(2-3) sialyloligosaccharides produced according to the methods of the invention are also provided. The invention additionally relates to a method for producing &agr;(2-3) sialyllactose in milk using a transgenic mammal containing an &agr;(2-3) trans-sialidase encoding sequence operably linked to a regulatory sequence of a gene expressed in mammary tissue.
2. BACKGROUND OF THE INVENTION
2.1. Sialyloligosaccharides in Cheese Waste Streams
Whey is a major by-product of cheese manufacturing, which, for environmental reasons, presents a difficult waste disposal problem. In the United States alone, fluid whey is being produced at a rate of about 62.6 billion pounds annually. Whey is typically composed of about 5 wt. % lactose, 1 wt. % protein and about 0.5 wt. % salts, where the balance of the mixture is water. A major effort by many cheese making countries is presently underway to develop uses for this commodity, which formerly was considered a cheese processing waste product.
Although the protein concentrate obtained by ultrafiltration of whey has become a valuable commodity in the food industry and has found applications in animal feed, fertilizer, fermentation, and food filler, the majority of the resulting lactose-rich ultrafiltered permeate is still considered a disposable fraction.
Recently, several sialyloligosaccharides have been found to have valuable application as pharmaceutics. See, e.g. U.S. Pat. No. 5,270,462 to Shimatani et al. Sialyllactose has been shown to neutralize enterotoxins of various pathogenic microbes including
Escherichia coli, Vibrio cholerae
and Salmonella. See, e.g. U.S. Pat. No. 5,330,975 to Hiroko et al. It has also been shown that &agr;(2-3) sialyllactose (&agr;-Neu5Ac-(2-3)-Gal-&bgr;-(1-4)-Glc) interferes with colonization of
Helicobacter pylori
and thereby prevents or inhibits gastric and duodenal ulcers. See e.g. U.S. Pat. No. 5,514,660 to Zopf et al. Sialyllactose has additionally been proposed to inhibit immune complex formation by disrupting occupancy of the Fc carbohydrate binding site on IgG and to be useful in treating arthritis. See, e.g. U.S. Pat. No. 5,164,374 to Rademacher et al.
To date, commercially available sialyloligosaccharides have been very expensive due to their low quantity in natural sources. For example, &agr;(2-3) sialyllactose and &agr;(2-6) sialyllactose isolated from bovine colostrum, is sold for $75.60 and $83.30 per milligram, respectively (Sigma Chemical Company, 1997).
A focused effort has been directed toward harvesting sialyloligosaccharides from the vast supply of whey made available as a cheese processing waste product. Processes for isolating sialyloligosaccharides have utilized such techniques as ultrafiltration, ion-exchange resins and phase partition chemistry. U.S. Pat. No. 4,001,198 to Thomas and U.S. Pat. No. 4,202,909 to Pederson; U.S. Pat. No. 4,547,386 to Chambers et al.; U.S. Pat. No. 4,617,861 to Armstrong; U.S. Pat. Nos. 4,971,701 and 4,855,056 to Harju et al.; U.S. Pat. No. 4,968,521 to McInychyn; U.S. Pat. No. 4,543,261 to Harmon et al.; U.S. Pat. Nos. 5,118,516 and 5,270,462 to Shimatani; JP Kokai 01-168,693; JP Kokai 03-143,351; JP Kokai 59-184,197; JP Kokoku 40-1234; JP Kokai 63-284,199 and Japanese Patent Publication No. 21234/1965, each of which is herein incorporated by reference in its entirety. Yields of up to 6 grams of &agr;(2-3) sialyllactose sialyloligosaccharide per kilogram of cheese processing waste stream have been reported. U.S. Pat. No. 5,575,916 to Brian et al. which is herein incorporated by reference in its entirety.
2.2. Sialidases and Sialyltransferases
Sialic acids are 9-carbon carboxylated sugars which generally occur as the terminal monosaccharides in oligosaccharide chains. In mammalian cells, sialic acids are most frequently linked to &bgr;-galactose with an &agr;(2-3) linkage, and to N-acetylglucosamine and N-acetylgalactosamine with an &agr;(2-6) linkage. Cross et al., 1993
, Annu. Rev. Microbiol
. 47:385-411.
Sialidases catalyze the removal of sialic acid residues from the oligosaccharide chain. Due to the wide variety of substitutions which may occur at various carbons of the sialic acid molecules, there are at least 39 different species of sialic acids. Colli, W., 1993
, FASEB J
. 7:1257-1264. Generally, sialidases exhibit substrate specificity for specific forms of sialic acid linkages. Viral sialidases cleave &agr;(2-3) glycosidic bonds more efficiently than &agr;(2-6) bonds, but bacterial sialidases are not as specific. Cross et al., 1993
, Annu. Rev. Microbiol
. 47:385-411 (citing Corfield et al. 1982
, Sialic Acids: Chemistry, Metabolism and Function
, Vol. 10, New York: Springer-Verlag, pp. 195-261). At low enzyme concentrations, bacterial sialidases exhibit a preference for cleaving &agr;(2-3) or &agr;(2-6) glycosidic bonds. Cross et al., 1993
, Annu. Rev. Microbiol
. 47:385-411.
CMP-sialyltransferases catalyze the transfer of cytidine monophosphate-sialic acid (CMP-sialic acid) residues to acceptor molecules. Although many sialidases exhibit at least some substrate specificity, CMP-sialyltransferases act on specific substrates. Mammalian CMP-sialyltransferases are generally found in the Golgi, however, there is evidence that there may be cell-surface associated CMP-sialyltransferases as well. Cross et al., 1993
, Annu. Rev. Microbiol
. 47:385-411 (citing Roth et al., 1971
, J. Cell Biol
. 51:536-547; Shur, 1991
, Glycobiology
1:563-575; Yogeeswaran et al., 1974
, Biochem. Biophys. Res. Commun
. 59:591-599).
2.3
. Trypanosoma Cruzi
&agr;(2-3)-Trans-Sialidase
Trypanosoma cruzi
(Order Kinetoplastida) is the intracellular parasite responsible for Chagas disease, throughout Iberoamerican countries. Chagas disease primarily affects nerve and muscle cells. One serious manifestation of Chagas disease is a chronic progressive fibrotic myocarditis. Colli, 1993
, FASEB J
. 7:1257-1264. Approximately 16-18 million people are infected with
T. cruzi
. Colli, 1993
, FASEB J
. 7:1257-1264.
T. cruzi
invades a broad range of host cells, and a considerable amount of research has focused on the surface molecules in order to determine which molecules may be involved in parasite/host interaction. Colli, 1993
, FASEB J
. 7:1257-1264. One surface molecule which has generated a great deal of interest is the &agr;(2-3)-trans-sialidase. This molecule has the capability of catalyzing both the removal of sialic acid from a donor saccharide-containing molecule (sialidase activity) and catalyzing the transfer of the sialic acid to an acceptor saccharide-containing molecule (trans-sialidase activity). Schenkman et al., 1992
, J. Exp. Med
. 175:567-575. The gene encoding
T. cruzi
trans-sialidase has been cloned and characterized at the molecular level.
The
T. cruzi
&agr;(2-3) trans-sialidase catalyzes the transfer of sialic acid from a donor terminal &bgr;-galactosyl sialoglycoconjugate to a terminal &bgr;-galactose on an acceptor molecule. Colli, W., 1993
, FASEB J
. 7:1257-1264
. T. cruzi
&agr;(2-3) trans-sialidase does not use CMP-sialic acid as a substrate and prefers sialyl &agr;(2-3)-linked to &bgr;-galactosyl residues as sialic acid donor molecules over sialyl &agr;(2-6)-, &agr;(2-8)-, and &agr;(2-9)-linked sialic acids. Schenkman et al., 1994
, Annu. Rev. Microbiol
. 48:499-523. Furthermore,
T. cruzi
&agr;(2-3) trans-sialidase cannot use free sialic acid as a substrate. Vandekerckhove et al. 1992
, Glycobiology
2:541-548. The
T. cruzi
&agr;(2-3) trans-sialidase has a broad pH optimum centered at 7.0. Cross et al., 1993
, Annu Rev. Microbiol
. 47:385-411.
More detailed analysis of the &agr;(2-3) trans-

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