Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters fat – fatty oil – ester-type wax – or...
Patent
1994-11-04
1998-06-16
Benzion, Gary
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters fat, fatty oil, ester-type wax, or...
4351723, 4352404, 4353201, 435 691, 47 58, 47DIG1, 536 245, 536 232, 536 236, 536 241, 424 932, 424 9321, A01H 104, C12N 514, C12N 1500, C07H 1700
Patent
active
057673640
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to nucleotide sequences encoding a plant enzyme, vectors containing said nucleotide sequences, transgenic plants containing said nucleotide sequences, recombinant DNA methods of producing the enzyme, and methods of altering the properties of a plant.
BACKGROUND OF THE INVENTION
Fruit and vegetable cell walls are largely polysaccharide, the major components being pectin, cellulose and xyloglucan (reference 1). Numerous cell wall models have been proposed which attempt to incorporate the essential properties of strength and flexibility (references 2,3,4).
Xyloglucans are 1,4-beta-glucans that are extensively substituted with alpha-1,6-xylosyl side chains, some of which are 1,2 beta-galactosylated. They are found in large amounts in the primary cell walls of dicots but also in certain seeds, where they serve different roles.
Primary cell wall xyloglucan is fucosylated. It is tightly hydrogen bonded to cellulose microfibrils and requires concentrated alkali or strong swelling agents to release it. Xyloglucan is thought to form cross-bridges between cellulose microfibrils, the cellulose/xyloglucan network forming the major load-bearing/elastic network of the wall. DCB mutated suspension culture cells (cell walls lacking cellulose) release xyloglucan into their media, suggesting that xyloglucan is normally tightly bound to cellulose.
Hydrolysis of primary cell wall xyloglucan has been demonstrated in segments of dark grown squash hypocotyls, during IAA induced growth (reference 15). Endohydrolysis of wall xyloglucan is thought to contribute to wall loosening which accompanies cell expansion (reference 16). The average molecular weight of xyloglucan has also been shown to decrease during tomato fruit ripening and this may contribute to the tissue softening which accompanies the ripening process (reference 17).
Certain seeds, e.g. nasturtium, contain up to 30% by weight of xyloglucan, stored in thickened cotyledonary cell walls, which serves as a reserve polysaccharide and is rapidly depolymerised during germination.
An endo 1,4 beta-D glucanase which specifically acts on xyloglucan (i.e. a xyloglucanase) has been isolated and purified to apparent homogeneity from germinating nasturtium (Tropaeolum majus L.) seeds (reference 11).
The purified xyloglucanase gives a single polypeptide band on SDS polyacrylamide gel electrophoresis, (apparent molecular weight, 29-31 kDa) and isoelectric focusing (isoelectric point, 5.0). The enzyme displays an absolute specificity for xyloglucan and an endo mode of action, as determined by end product analysis following hydrolysis of tamarind seed xyloglucan (reference 15). Although the natural substrate of the enzyme is nasturtium cotyledonary reserve xyloglucan, it has also been shown to hydrolyse fucose containing primary cell wall xyloglucans in vitro (reference 11). At high substrate concentrations, xyloglucan endo-transglycosylase (XET) activity has been demonstrated (reference 18).
Similar enzyme activity has been detected in other plant tissue and shown to be positively correlated with growth rate in different zones of the pea stem (reference 19). It has been proposed that XET is responsible for cutting and rejoining intermicrofibrillar xyloglucan chains and that this causes the wall-loosening required for plant cell expansion. XET activity has also been demonstrated in tomato fruit (xyloglucanase activatable by xyloglucan oligosaccharides) where it is reportedly highest at the "breaker" stage of ripening (reference 20) and may be involved in the softening process.
This Application describes the isolation of a xyloglucan specific endo- (1-4)-Beta-D-glucanase (xyloglucanase/ XET) gene from nasturtium. The enzyme encoded by this novel nucleotide sequence is highly specific for xyloglucan (reference 11).
SUMMARY OF THE INVENTION
In one aspect the invention provides a nucleotide sequence, encoding an enzyme having a xyloglucan-specific endo-(1-4)-Beta-D-glucanase activity, comprising nucleotides 35-919 of the sequence NXG1 show
REFERENCES:
patent: 5328999 (1994-07-01), Bennett et al.
patent: 5516694 (1996-05-01), Nishitani et al.
patent: 5569830 (1996-10-01), Bennett et al.
Tezuka, et al: "Construction of a beta-glucanase hyperproducing Bacillus subtilis using the cloned beta-glucanase gene and a multi-copy plasmid", Agricultural and Biological Chemistry, vol. 53, No. 9, 1989, pp. 2335-2339.
Edwards et al: Purification and properties of a novel xyloglucan-specific endo-(1,4)-beta-D-glucanase from germinated naturtium seeds(Tropaeolum majus L.),The Journal of Biological Chemistry, vol. 261, No. 20, Jul. 1986, pp. 9489-9494. JP-A-3180180, Jun. 8, 1991.
Fanutti et al: A xyloglucan-oligosaccharide-specific.alpha.-D-xylosidase or exo-oligoxyloglucan-.alpha.-xylohydrolase from germinated nasturtium (Tropaeolum majus L.) seeds. Purification, propeties and its interaction with a xyloglucan-specific endo-(1,fwdarw.4)-.beta.-D-glucanase and other hydrolases during storage-xyloglucan mobilization, & plants 1991, Chemical Abstracts Database, abstract No. 115(7)67183y, vol. 184, No. 1, pp. 137-147.
Arrowsmith David A.
de Silva Jacqueline
Edwards Mary E.
Jarman Carl D.
Reid John S. G.
Benzion Gary
Unilever Patent Holdings B.V.
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