Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1993-12-15
1996-07-23
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435208, 435209, 536 221, 536 231, 536 232, 536 237, 536 2374, C12N 924, C12N 940, C07H 1900, C07H 2100
Patent
active
055388841
DESCRIPTION:
BRIEF SUMMARY
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of PCT/DK92/00143 filed May 1, 1992, which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention comprises a rhamnogalacturonase (in the following usually abbreviated RGase), a corresponding DNA sequence, an RGase containing enzyme preparation and a use of the enzyme preparation.
Thus, the invention relates to genetic engineering and provides partial amino acid sequences of an RGase. These partial amino acid sequences can be .used for construction of DNA probes which can be used for screening a genomic library from organisms expressing such enzyme, or a cDNA library, thereby obtaining DNA sequences, which can be used either for an overproduction of RGase, if inserted in the microorganism species, from which the parent DNA molecule originated, or for production of RGase without accompanying closely related enzymes, if inserted in a host microorganism, which in its not-transformed condition does not produce any enzymes closely related to RGase.
2. Description of Related Art
Plant cell walls comprising rhamnogalacturonans are of complex nature. Many publications deal with the polysaccharides serving as building blocks, of which these cell walls consist, and their importance with respect to the growing, ripening and processing of fruits and vegetables. Especially pectins have been studied frequently, because they are among the most important components in this respect. Pectins are proposed to consist of highly carboxyl-methylated linear homogalacturonan regions which alternate with "hairy" (ramified) regions that comprise highly branched rhamnogalacturonans mainly. Whereas the linear homogalacturonan regions are very well known and characterized, the structure of the so-called hairy regions is still not fully characterized, and thus is the subject of many investigations. But besides the scientific interest it is very important to be able to degrade these hairy regions for technical reasons. The enzymatic liquefaction of plant material like e.g. fruits, vegetables, cereals, oil fruits and seeds by technical processes involves combinations of pectolytic, cellulolytic and proteolytic enzyme preparations. This enzymatic treatment solubilizes the hairy regions and other pectic fragments, which originate from the insoluble cell wall protopectin. On one hand the solubilization of these polysaccharides is wanted, e.g. for the production of cloudy liquids and soluble dietary fiber containing solutions. On the other hand these polysaccharides cause problems during the processing of the clear liquids, because they are resistant to complete degradation of most technical enzyme preparations. Only one enzyme preparation (from Aspergillus aculeatus) has so far been described, which can degrade the rhamnogalacturonan backbone of the hairy regions. Therefore, it is of great importance for scientific (studies of the structures of these complex polysaccharides) and technical (liquefaction of plant material) reasons to obtain more knowledge about enzymes that can degrade these hairy regions. Especially for the industries dealing with modifications of plant cell walls for e.g. human nutrition and for animal feed (e.g. liquefaction of fruits, vegetables, cereals, oil fruits and seeds), it is of great importance to provide a great variety of different RGases (in respect to mode of action, pH and temperature range) in order to be able to exploit the desirable actions of RGases under widely varying technical process conditions.
RGase is described in the poster "Rhamnogalacturonase; a novel enzyme degrading the highly branched rhamnogalacturonan regions in apple pectic substances" from Wageningen Agricultural University, Department of Food Science, Biotechnion, Bomenweg 2,6703 HD Wageningen, The Netherlands. From this poster is appears that a rhamnogalacturonase, the origin of which is not described, is well suited for degradation of the backbone of a modified "hairy region" (MHR) in plant cell walls. Also,
REFERENCES:
patent: 4478939 (1984-10-01), Alder-Nissen
patent: 4478940 (1984-10-01), Alder-Nissen et al.
H. A. Schols, Carbohydrate Research, vol. 206, pp. 105-115, 1990.
I. J. Colquhoum et al., Carbohydrate Research, vol. 206, pp. 131-144, 1990.
J. M. Brillouet, Rev. Fr. Oenol., vol. 122, pp. 43-54, 1990 (no translation).
Christensen Flemming M.
D orreich Kurt
Dalb.o slashed.ge Henrik
Halkier Torben
Mikkelsen Jan M.
Kim Hyosuk
Lambiris, Esq. Elias J.
Novo Nordisk A S
Wax Robert A.
Zelson Esq. Steve T.
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