Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1994-10-05
1996-12-17
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435196, 435 691, 4352523, 43525233, 4352543, 435137, 536 232, 935 14, 935 28, 935 68, 935 72, C12N 918, C12N 1555, C12N 1580, C12P 740
Patent
active
055852564
DESCRIPTION:
BRIEF SUMMARY
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of PCT/DK/93/00109 filed Mar. 29, 1993, which is incorporated herein by reference.
The invention comprises a novel enzyme, which is a rhamnogalacturonan acetyl esterease (the abbreviation RGAE will usually be used in the following), a corresponding DNA sequence, a vector, a transformed host, a method for production of an RGAE, an enzyme preparation, and a use of the RGAE.
The invention provides the characterization, the detection and description of a novel RGAE, a partial amino acid sequence of this enzyme, partial DNA sequences, and a total amino acid sequence and a total DNA sequence.
RGAE is a hydrolase with the systematic enzyme name rhamnogalacturonan acetic-ester acetylhydrolase, which belongs to the group of acetyl esterases (EC no. 3.1.1.6), which catalyze the hydrolysis of acetic esters to the corresponding alcohols and acetate.
BACKGROUND OF THE INVENTION
Polysaccharides (e.g. pectins) from plants are frequently substituted with is acetyl groups (Rombouts, F. M., J. F. Thibault, C. Mercier, "Oxidative enzyme-catalyzed crosslinking of beet pectins", U.S. Pat. No. 4,672,034). In the applications of polysaccharides these substitutions influence the gelation properties (Williamson G., C. B. Faulds, J. A. Matthew, D. B. Archer, V. J. Morris, G. J. Brownsey, M. J. Ridout, "Gelation of sugarbeet and citrus pectins using enzymes extracted from orange peel", Carbohydrate Polymers 13, 387-397, 1990). In the processing of plant material, e.g. fruits and vegetables, endogenous enzymes are used as processing aids to improve yield and quality of the end product (Pilnik, W., A. G. J. Voragen., "Effect of enzyme treatment on the quality of processed fruits and vegetables", in: Jen J. J., "Quality factors of fruits and vegetables, chemistry and technology", ACS Symp. Ser. 405, American Chemical Society, Washington D.C., 250-269, 1989). Schols et al. isolated and characterized from apple cell walls an acidic polymeric pectin fragment by the use of a technical enzyme preparation containing pectolytic, hemicellulolytic and cellulolytic enzymes. This enzyme resistant polysaccharide, called "modified hairy region" (MHR) consists of a highly branched rhamnogalacturonan backbone, with acetyl groups on the galacturonic acid residues (Schols, H. A., M. A. Posthumus, A. G. J. Voragen, "Structural features of hairy regions of pectins isolated from apple juice produced by the liquefaction process", Carbohydrate Research, 206, 117-129, 1990). Extensive screening of commercial enzyme preparations have led to an Aspergillus aculeatus preparation, which was able to degrade MHR. A novel enzyme called rhamnogalacturonase (RG) was identified and purified from this preparation. During the purification of RG it became obvious that the enzyme works only on saponified MHR and that therefore esterases, particularly acetyl esterases, must play an important role for the degradation of MHR (Schols, H. A., C. C. J. M. Geraeds, M. J. F. Searle-van Leuwen, F. J. M. Kormelink, A. G. J. Voragen, "Rhamnogalacturonase: a novel enzyme that degrades the hairy regions of pectins", Carbohydrate research 206, 105-115, 1990). Enzymes which can deacetylate branched rhamnogalacturonans, like HMR, are therefore needed, as the high degree of acetylation on branched rhamnogalacturonans hinders the action of enzymes with higher activity on deacetylated rhamnogalacturonans.
Several polysaccharides (xylan, mannan and pectin) are known to be acetylated, and the acetyl esterases are known to be very specific against their specific polysaccharide substrate, but some of them exhibit activity on non-polysaccharide substrates, like triacetin and naphthol acetate. An Aspergillus niger acetylesterease, active towards triacetin and beet pectin, has been described by Mathew et al. (Mathew, J. A., S. J. Howson, M. H. J. Keenan, P. S. Belton, "Improvement of the gelation properties of sugarbeet pectin following treatment with an enzyme preparation derived from Aspergillus niger--Comparison with
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Henk A. Schols et al., Carbohydrate Research, vol. 206, pp. 105-115, 1990.
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Massiot, P., et al., 1990, Food Biotechnology, 4(1):364 (Abstract).
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Christensen Flemming M.
Dalb.o slashed.ge Henrik
Dorreich Kurt
Heldt-Hansen Hans P.
Mischler Marcel
Lambiris, Esq. Elias J.
Moore William W.
Novo Nordisk A S
Wax Robert A.
Zelson Esq. Steve T.
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