Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1996-08-05
1998-12-15
Patterson, Jr., Charles L.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
4352523, 4352543, 4353201, 435913, 435917, 536 232, 536 241, C12N 942, C12N 114, C12N 100, C07H 2104
Patent
active
058495592
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to the field of molecular biology. In particular, the present invention relates to the cloning and expression of genes encoding polypeptides showing arabinoxylan degrading activity. These enzymes are suitably used in industrial processes such as baking of bread, paper and pulp processing and in the preparation of feed and food (additives).
BACKGROUND OF THE INVENTION
The composition of a plant cell wall is complex and variable. Polysaccharides are mainly found in the form of long chains of cellulose (the main structural component of the plant cell wall), hemicellulose (comprising various .beta.-xylan chains) and pectin. The occurrence, distribution and structural features of plant cell wall polysaccharides are determined by (1) plant species; (2) variety; (3) tissue type, (4) growth conditions; (5) ageing and (6) processing of plant material prior to feeding.
Basic differences exist between monocotyledons (e.g. cereals and grasses) and dicotyledons (e.g. clover, rapeseed and soybean) and between the seed and vegetative parts of the plant (Chesson, 1987; Carre and Brillouet, 1986).
Monocotyledons are characterized by the presence of an arabinoxylan complex as the major hemicellulose backbone. The main structure of hemicellulose in dicotyledons is a xyloglucan complex. Moreover, higher pectin concentrations are found in dicotyledons than in monocotyledons. Seeds are generally very high in pectic substances but relatively low in cellulosic material.
Three more or less interacting polysaccharide structures can be distinguished in the cell wall: point of attachment for the individual cells to one another within the plant tissue matrix. The middle lamella consists primarily of calcium salts of highly esterified pectins; well-organized structure of cellulose microfibrils embedded in an amorphous matrix of pectin, hemicellulose, phenolic esters and proteins; growth and ageing phase, cellulose microfibrils, hemicellulose and lignin are deposited.
The primary cell wall of mature, metabolically active plant cells (e.g. mesophyll and epidermis) is more susceptible to enzymatic hydrolysis than the secondary cell wall, which by this stage, has become highly lignified.
There is a high degree of interaction between cellulose, hemicellulose and pectin in the cell wall. The enzymatic degradation of these rather intensively cross-linked polysaccharide structures is not a simple process. At least five different enzymes are needed to completely break down an arabinoxylan, for example. The endo-cleavage is effected by the use of an endo-.beta.(1.fwdarw.4)-D-xylanase. Exo-(1.fwdarw.4)-D-xylanase liberates xylose units at the non-reducing end of the polysaccharide. Three other enzymes (.alpha.-glucuronidase, .alpha.-L-arabinofuranosidase and acetyl esterase) are used to attack substituents on the xylan backbone. The choice of the specific enzymes is of course dependent on the specific hemicellulose to be degraded (McCleary and Matheson, 1986).
Enzymes that attack side-chains of the xylan backbone can be of interest because they change the characteristics of the polymer, making it more suitable for certain applications. Furthermore these enzymes may act synergistically with main-chain cleaving endo-xylanases (for an extensive review see Kormelink, 1992, PhD thesis, University of Wageningen).
A DNA fragment encoding an arabinoxylan degrading activity is known. In European patent application 463 706, the isolation, characterisation and gene cloning of an endo-xylanase from Aspergillus tubigensis is described. This enzyme is not capable of attacking side chains of the arabinoxylan backbone.
Enzymatic activities capable of attacking side chains are also known from Aspergillus niger (Kormelink, 1992, supra, Chapters 6 and 7). An enzyme called Arabinofuranosidase A (ArafurA) is characterised by the capacity to release arabinose residues from oligosaccharide structures obtained from arabinoxylans. However, Arafur A is not active on high molecular weight substrates. In addition Aspergillus
REFERENCES:
Conrad, D. et al., "Utilization of Hemicelluloses by Enzymatic Degradation," Wissenschaft und Umwelt (1982) 4:242-245 (summary in English).
Gorbacheva, I. V. et al., "Studies on Xylan-Degrading Enzymes--II. Action Pattern of Endo-1,4-.beta.-Xylanase from Aspergillus niger Str. 14 on Xylan and Xylooligosaccharides," Biochimica et Biophysica Acta, 484 (1977) 94-102.
Kellett, L.E. et al., "Xylanase B and an arabinofuranosidase from Pseudomonas fluorescens subsp. cellulosa contain identical cellulose-binding domains and are encoded by adjacent genes," Biochem. J. (1990) 272:369-376.
Kormelink, F.J.M. et al., "Mode of action of the xylan-degrading enzymes from Aspergillus awamori," Xylans and Xyalases, edited by J. Visser et al. 1992 Elsevier Science Publishers B.V., pp. 141-147.
Kormelink, F.J.M. et al., "Purification and characterization of a (1,4)-.beta.-D-arabinoxylan arabinofuranohydrolase from Aspergillus awamori," Appl Microbiol Biotechnol (1991) 35:753-758.
De Graaff Leendert Hendrik
Gielkens Marcus Matheus Catharina
Van Der Wouw Monique Josina Andrea
Van Ooijen Albert Johannes Joseph
Visser Jacob
Gist-Brocades B.V.
Patterson Jr. Charles L.
Slobodyansky Elizabeth
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