Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1992-04-27
1994-10-25
Patterson, Jr., Charles L.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
426 10, 426 20, 426635, 162 87, 435 691, 435200, 4352523, 4353201, 536 221, 536 231, 536 232, 536 234, 536 2374, 536 241, C12N 942, C12N 924, C07H 2104, C12P 2106
Patent
active
053588649
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the field of molecular biology. In particular, the present invention relates to the cloning and overexpression of a fungal DNA sequence encoding a protein having the activity of a xylanase. The present invention also provides methods for the production and use of a single xylanase which is obtainable in a form which is free of other xylanases, and indeed from other enzymes in general.
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.
A cross-sectional diagram of a plant cell is depicted in FIG. 1. 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 crosslinked 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, 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).
For certain applications, however, complete degradation of the entire hemicellulose into monomers is not necessary or is not desirable. In the liquefaction of arabinoxylan, for example, one needs simply to cleave the main xylan backbone into shorter units. This may be achieved by the action of an endo-xylanase, which ultimately results in a mixture of xylose monomer units and oligomers such as xylobiose and xylotriose. These shorter subunits are then sufficiently soluble for the desired use.
Filamentous fungi are widely known for their capacity to secrete large amounts of a variety of hydrolytic enzymes such as .alpha.-amylases, proteases and amyloglucosidases and various plant cell wall degrading enzymes such as cellulases, hemicellulases, and pectinases. Among these, multiple xylan-degrading enzymes have been recognized, which have been shown to possess a variety of biochemical and physical properties. This heterogeneity in xylanase function allows for t
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de Graaff Leendert H.
Harder Abraham
Hille Jan D. R.
van den Broeck Henriette C.
van Ooyen Albert J. J.
Gist-brocades, N.V.
Kim Hyosuk
Patterson Jr. Charles L.
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