Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1994-10-28
1997-03-11
Patterson, Jr., Charles L.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
43525411, 4353201, 4352523, 536 232, C12N 924, C12N 119, C12N 1563, C07H 2104
Patent
active
056100469
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 DNA sequence encoding a protein having the activity of the Aspergillus tubigensis xylanase B (XYL B) protein. 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) aging 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-.crclbar.(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).
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. Furthermore, it has been demonstrated that the actions of specific xylanase enzymes differ from one another, as seen by the varying patterns of xylose monomer and oligomer units resulting from the action of these specific enzymes on an arabinoxylan substrate (Kormelink, F., 1992).
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 degradin
REFERENCES:
Chesson, "Supplementary Enzymes To Improve the Utilization of Pig and Poultry Diets", Recent acvances in Animal Food Nutrition, Haresign, W. and Cole, D. J. A., eds., Butterworth, London, 71-89 (1987).
Carre et al., "Yield and Composition of Cell Wall Residues Isolated from Various feedstuffs Used for Non-Ruminant Farm Animals", J. Sci. Food Agric. 37:341-351 (1986).
McCleary et al., "Enzymic Analysis of Polysaccharide Structure", Adv. Carb. Chem. and Biochem. 44:147-276 (1987).
Wong et al., "Multiplicity of .beta.-1, 4-Xylanase in Microorganisms: Functions and Applications", Microbiol. Rev. 52:305-317 (1988).
Woodward, "Xylanases: Functions, Properties and Applications", Topics in Enzyme Ferment. Biotechnol. 8:9-30 (1984).
Dekker et al., "Hemicellulases: Their Occurrence, Purification, Properties, and Mode of Action", Adv. Carb. Chem. and Biochem. 32:278-353 (1976).
DeGraaff Leendert H.
van den Broeck Henriette C.
van Ooyen Albert J. J.
Visser Jacob
Gist-brocades, N.V.
Lau Kawai
Patterson Jr. Charles L.
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