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;
(1) the middle lamella forms the exterior cell wall. It also serves as the 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; PA1 (2) The primary wall is situated just inside the middle lamella. It is a well-organized structure of cellulose microfibrils embedded in an amorphous matrix of pectin, hemicellulose, phenolic esters and proteins; PA1 (3) The secondary wall is formed as the plant matures. During the plant's growth and ageing phase, cellulose microfibrils, hemicellulose and lignin are deposited. PA1 (a) a DNA fragment encoding a polypeptide having the amino acid sequence represented by amino acids 1 to 306, or a polypeptide precursor of said polypeptide represented by amino acids -27 to 306 in SEQIDNO: 5; PA1 (b) a DNA fragment encoding a polypeptide having the amino acid sequence represented by amino acids 1 to 306, or a precursor of said polypeptide represented by amino acids -27 to 306 in SEQIDNO: 7; PA1 (c) A DNA fragment encoding a variant or portion of the polypeptides represented by amino acid residues 1 to 306 depicted in SEQIDNO: 5 or 7, still having arabinoxylan degrading activity, or a polypeptide precursor thereof; PA1 (d) A DNA fragment coding for a polypeptide having arabinoxylan degrading activity and having the nucleotide sequence represented by nucleotides 784 to 1779 in SEQIDNO: 5 or nucleotides 823 to 1818 in SEQIDNO: 7; PA1 (e) A DNA fragment encoding a polypeptide having arabinoxylan degrading activity, or a part of such polypeptide, which DNA fragment is capable of hybridising to a DNA fragment as represented by nucleotides 784 to 1779 in SEQIDNO: 5 or nucleotides 823 to 1818 in SEQIDNO: 7. The recombinant DNA according to the invention is preferably obtainable from a filamentous fungus, more in particular from an Aspergillus species. Especially preferred recombinant DNA sequences comprise DNA fragments encoding AXDA from Aspergillus niger or tubigensis.
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 substitutents on the xylan backbone. The choice of the specific enzymes is the 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 encoded 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 niger produces an enzyme named arafur B which is active on oligosaccharide, as well as high molecular weight arabinoxylan structures. The enzymatic action of ArafurB is confined to releasing arabinose residues from terminal single substituted xylose residues. No DNA fragments are known sofar.
An activity capable of releasing arabinose residues from non-terminal single substituted xylose residues in both oligosaccharide as well as high molecular weight arabinoxylan structures has been isolated from Aspergillus awamori. This enzyme is named arabinoxylan arabinofuranose hydrolase (AXH). Sofar no DNA fragments and/or sequence data are available. It is clear that not all enzymes involved in arabinoxylan degradation have been detected yet (Kormelink 1990). For instance, no enzyme attacking xylose molecules double substituted with arabinose, has been found yet. The reason for this is that these enzymes are often secreted in low quantities. Molecular cloning and overproduction in a suitable host of these enzymes, although not easy, its a way to obtain sufficient quantities of pure enzymes, which in turn allows to assess their significance in various applications.