Enzyme with rhamnogalacturonase activity

Details Enzyme with rhamnogalacturonase activity Enzyme with rhamnogalacturonase activity

1995-09-25

1998-09-22

Wax, Robert A.

Chemistry: molecular biology and microbiology

Process of utilizing an enzyme or micro-organism to destroy...

Treating animal or plant material or micro-organism

435200, 435208, 435274, C12N 924, C08B 100, C08B 3004

Patent

active

058112910

DESCRIPTION:

BRIEF SUMMARY
CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT/DK94/00097 filed Mar. 4, 1994, which is incorporated herein by reference.


FIELD OF INVENTION

The present invention relates an enzyme exhibiting rhamnogalacturonase activity, a DNA construct encoding the enzyme, an enzyme preparation comprising the enzyme, and the use of a rhamnogalacturonase for degradation or modification of plant cell materials.


BACKGROUND OF THE INVENTION

Plants contain rhamnogalacturonans, i.e. polysaccharides with more or less regularly alternating rhamnose and galacturonic acid residues in the backbone. The rhamnogalacturonans may have mono, oligo, and polysaccharide side-branches. Rhamnogalacturonans are part of the pectin-polymers, a major component of the plant cell walls.
Most pectin-polymers are composed of smooth regions, i.e. linear homogalacturonan, and hairy (ramified) regions. The hairy regions consist of a rhamnogalacturonan backbone with side-branches of varying length. The side-branches includes monosaccharides like xylose, galactose and arabinose, and oligo and polysaccharides like araban, galactan and arabinogalactan. Further the rhamnogalacturonan backbone is methylated and acetylated. The composition of the very complex structure of the hairy regions vary according to the source of the plant cell wall, cf Fry (1988), Schols et al. (1990b), O'Neill et al. (1990), Voragen and Schols (1992) and Carpita and Gibeaut (1993).
The enzymatic liquefaction and degradation of plant materials (e.g. fruits, vegetables, cereals, oil fruits and seeds) by technical processes involves combinations of pectolytic, cellulolytic and proteolytic enzyme preparations. However the hairy regions of pectin cause problems in such processes, because they are resistant to degradation of most technical enzyme preparations. A more extensive degradation of hairy regions is desired in many processes in order to improve the liquefaction and degradation of the plant material. For instance, an extensive degradation is important in processing of clear liquids and in processing of viscous plant cell wall containing material where a viscosity reduction is otherwise difficult to obtain. Furthermore, a more specific enzymatic degradation of the hairy regions is desirable for e.g. production of cloudy liquids, purification of pectin and soluble dietary fibres.
For these processes a degradation of the backbone of pectin hairy regions is of major importance. The degradation of the backbone of the hairy regions is performed by enzymes designated rhamnogalacturonases (RGases). RGases are believed to hydrolyse the bond between rhamnose and galacturonic acid. In order to facilitate the activity of RGases it may be desirable to reduce the degree of acetylation of the backbone, e.g. by use of the enzyme rhamnogalacturonan acetyl esterase (cf Searle-van Leeuwen et al., 1992). Furthermore, a reduced degree of branching of parts of the hairy regions may be desirable. The reduced degree of branching may be obtained by enzymes which attacks the side-branches, like galactanases, arabinanases, beta-galactosidases, alpha-arabinosidases and beta-xylosidases.
The isolation and purification of a RG'ase from Aspergillus aculeatus is described by Schols et al. (1990a). WO 92/19728, the contents of which are incorporated by reference herein, discloses partial amino acid sequences of different RGases isolated from the Aspergillus sp. A. aculeatus and A. japonicus and from Irpex lacteus. EP 570 075 discloses an Aspergillus RGase gene and the construction of recombinant Aspergillus strains which overexpress RGase.
The RGase described by Schols et al., (1990a) has been found to have a similar degradation pattern to the A. aculeatus RGase described in WO 92/19728 and has been found to be immunologically cross-reactive with said RGase. RGase of this type is termed RGase II in the following disclosure.
Furthermore, in an article of Colquhoun (1990), the composition of a mixture of oligosaccharides obtained by enzymatic degradation of the modified hair

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Ngo et al. (1994) Computational complexity, protein structure prediction, and the ILevinthal paradox. In: The Protein Folding Problem and Tertiary Structure Prediction. Eds. Merz et al. Birkhauser et al. Boston, MA, pp. 491-495, Jun. 1994.
Thornton et al. (1995) Protein Engineering: Editoral Overview. Current Opinion in Biotechnology 6(4): 367-369, Aug. 1995.
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Schols et al., Carbohydrate Research, vol. 206, pp. 105-115, 1990.
de Ruiter et al. (1990) Identification by n.m.r. spectroscopy of oligosaccharides obtained by treatment of the hairy regions of apple pectin with rhamnogalacturonase. Carbohydrate Research 206: 131-144, May 1990.
Sakamoto et al. (1993) Studies on protopectinase-C mode of action: Analysis of the chemical structure of the specific substrate in sugar beet protopectin and characterization of the enzyme activity. Biosci. Biotech. Biochem. 57 (11): 1832-1837, Jun. 1993.
Thibault et al. (1993) Studies on apple protopectin VI: extraction of pectins from apple cell walls with rhamnogalacturonase. Cabohydrate Polymers 22: 203-210, Aug. 1993.
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