Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-01-12
2001-05-01
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S123120, C536S055100, C536S056000, C536S112000, C536S124000, C435S012000, C435S018000, C435S027000, C435S028000, C252S008620, C252S175000, C252S175000, C252SDIG018, C530S300000, C530S345000, C530S350000
Reexamination Certificate
active
06225462
ABSTRACT:
FIELD OF INVENTION
This invention relates to binding of polysaccharides and concerns a cellulose-binding polysaccharide conjugate, products including the polysaccharide conjugate, and targeting methods using the polysaccharide conjugate. In the context of the invention the term “polysaccharide” is intended to cover polysaccharides and oligosaccharides, and references to “polysaccharide” and “polysaccharide conjugate” should be construed accordingly. The term “conjugate” is used to refer to units bound or secured together (physically and/or chemically), with a “polysaccharide conjugate” comprising a polysaccharide bound or secured to another entity.
BACKGROUND TO THE INVENTION
It is known that various naturally occurring polysaccharides such as pea xyloglucan, tamarind seed xyloglucan, etc. bind to cellulose by a polysaccharide:polysaccharide interaction; indeed this binding ability is important in the functioning of plant cell walls.
U.S. Pat. No. 3,297,604 concerns polymer compositions containing galactose oxidized to form a carbonyl group at the C6 position. The active carbonyl group can react in known manner, e.g. to form cyano hydrins, bisulfite addition compounds, oximes, hydrazones, etc. The compositions can also act to cross-link polymers, including cellulose. The polymer, may be, e.g., guar, locust bean gum, etc. There is no disclosure of a polysaccharide conjugate with attached entity of molecular weight of at least 5,000. While the polymer composition itself may be capable of binding to cellulose, this is not unexpected, and there is no disclosure of a polysaccharide conjugate that is capable of binding to cellulose.
U.S. Pat. No. 2,949,397 concerns use of mineral filler coated, at least partially, with water-dispersed organic colloid, to promote retention of filler in cellulose fibres in paper making. The colloid may be e.g. a galactomannans, or substituted mannan such as locust bean gum and guar gum. The coated filler is attracted to cellulose fibres by electrostatic action. The filler and colloid are mixed together, but separate on standing and hence are in the form of a simple mixture not a polysaccharide conjugate.
The paper by Hayashi et al entitled “Pea Xyloglucan and Cellulose” in Plant Physiol. (1987) 83, 384-389 describes investigations of binding of pea xyloglucan to cellulose, using fluorescein-labelled xyloglucan prepared by treating xyloglucan with CNBr and incubating with fluoresceinamine, and also using radioiodinated xyloglucan prepared by reaction of 125, with the fluorescein moiety on xyloglucan. These labels were used to trace the binding of the polysaccharide and are among the smallest molecular label entities known.
The present invention is based on the surprising discovery that polysaccharides with much larger attached entities than those used by Hayashi et al can still bind rapidly with high efficiency to cellulose by polysaccharide:polysaccharide interaction. This is surprising because binding occurs at multiple sites along the backbones of the polysaccharides, rather than at a single binding site as with antibody-antigen interactions, and it would have been predicted that binding would have been disrupted by the attachment of large entities to cellulose-binding polysaccharides. The invention thus opens up the possibility of using polysaccharides to target attached entities to cellulose, e.g. in fabric, paper, etc.
SUMMARY OF THE INVENTION
In one aspect the present invention provides a polysaccharide conjugate comprising a polysaccharide with an attached entity having a molecular weight of at least 5000, the polysaccharide conjugate being capable of binding to cellulose.
The polysaccharide conjugate is preferably capable of binding to cellulose by polysaccharide:polysaccharide interaction.
The polysaccharide may be one that binds naturally to cellulose or has been derivatised or otherwise modified to bind to cellulose. The polysaccharide may be naturally occurring or synthetic.
The polysaccharide desirably has a 1-4 linked &bgr;-glycan (generalized sugar) backbone structure, which is stereochemically compatible with cellulose, such as a glucan backbone (consisting of &bgr; 1-4 linked glucose residues), a mannan backbone (consisting of &bgr; 1-4 linked mannose residues) or a xylan backbone (consisting of &bgr; 1-4 linked xylose residues). Suitable polysaccharides include xyloglucans, glucomannans, mannans, galactomannans, &bgr;(1-3), (1-4) glucan and the xylan family incorporating glucurono-, arabino- and glucuronoarabinoxylan. See “Physiology and Biochemistry of Plant Cell Walls” (1990) by C. Brett and K. Waldron for a discussion of these materials.
The minimum chain length requirement for cellulose oligomers to bind to cellulose is 4 glucose units. For xyloglucans, the side chains make the binding less efficient and 12 backbone glucose units (i.e. about 25 total sugar units) are required for binding to cellulose. Structural considerations suggest galactomannans are intermediate in binding efficiency, and about 6 to 8 backbone residues are expected to be required for binding to cellulose. The polysaccharide should thus have at least 4, and preferably at least 10, backbone residues, which are preferably &bgr;1-4 linked.
Naturally occurring polysaccharides that bind rapidly and strongly to cellulose by polysaccharide:polysaccharide interaction include xyloglucans such as pea xyloglucan and tamarind seed xyloglucan (TXG) (which has a &bgr; 1-4 linked glucan backbone with side chains of a-D xylopyranose and—D-galactopyranosyl-(1-2)-&agr;-D-xylo-pyranose, both 1-6 linked to the backbone:see Gidley et al Carbohydrate Research, 214 (1991) 200-314 for a discussion of the structure of tamarind seed polysaccharide); and galactomammans, particularly low galactose galactomannans, such as locust bean gum (LBG) (which has a mannan backbone of &bgr; 1-4 linked mannose residues, with single unit galactose side chains linked 1-6 to the backbone), enzyme modified guar (EMG) (guar gum has the same structural units as LBG but has a much higher level of galactose substitution, to the extent that there is not enough accessible mannan backbone through which to bind cellulose. EMG is produced by enzymic removal from guar gum of a controllable percentage of the galactose residues to produce a range of materials that are capable of binding to cellulose, but are cheaper and more consistently available than LBG. See Bulpin et al. in Carbohydrate Polymers 12 (1990) 155-168 for a discussion of EMG), tara glactomannan and cassia galactomannan. These materials are commercially available and thus provide potentially useful sources of suitable polysaccharides. These materials have the advantages of being relatively cheap, and already being accepted for food use.
The polysaccharide desirably has side chain galactose residues susceptible to oxidation by galactose oxidase, for production of an aldehyde group for coupling of a protein entity, as will be described below. TXG, LBG and EMG have such galactose residues.
The attached entity may be selected from a wide range of entities that generally perform a useful function in proximity to cellulose, e.g. in fabric, paper, etc.
For example, the entity may be a protein, such as an enzyme, antibody or antibody fragment.
The enzyme is conveniently an oxidase, peroxidase, catalase or urease. These enzymes work by their substrate diffusing to them, and generate a flux of active product of molecules that diffuse away. Redox enzymes, e.g. oxidases such as glucose oxidase, generate hydrogen peroxide which can act as a bleach. Peroxidase catalyses the oxidation by hydrogen peroxide of a number of substrates. Urease catalyses hydrolysis of urea, releasing a flux of ammonium ions which raises the local pH. Catalase is an oxidoreductase that catalyses conversion of hydrogen peroxide to water and oxygen.
Antibodies or antibody fragments may, for example, be used in separation or purification techniques, as is described below, or in immunoassays.
The attached entity may alternatively be a particle, e.g. of silica, organic polymer, etc. Such part
Berry Mark John
Davis Paul James
Gidley Michael John
Lever Brothers Company, a Division of Conopco, Inc.
Mitelman Rimma
Wilson James O.
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