Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-09-01
2002-12-31
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S183000, C435S200000, C435S209000, C435S262000, C435S263000, C435S264000, C510S114000
Reexamination Certificate
active
06500658
ABSTRACT:
The present invention relates to xyloglucanase enzymes which are endogeneous to a strain belonging to Malbranchea, especially enzymes exhibiting xyloglucanase activity in the pH range 4-11; to a method of producing such enzymes; and to use of such enzymes in the textile, detergent and cellulose fiber processing industries.
BACKGROUND OF THE INVENTION
Xyloglucan is a major structural polysaccharide in the primary (growing) cell wall of plants. Structurally, xyloglucans consists of a cellulose-like beta-1,4-linked glucose backbone which is frequently substituted with various side chains. The xyloglucans of most dicotyledonous plants, some monocotyledons and gymnosperms are highly branched polysaccharides in which approx. 75% of the glucose residues in the backbone bear a glycosyl side chain at O-6. The glycosyl residue that is directly attached to the branched glucose residue is invariably alfa-D-xylose. Up to 50% of the side chains in the xyloglucans contain more than one residue due to the presence of beta-D-galactose or alfa-L-fucose-(1-2)-beta-D-galactose moieties at O-2 of the xylose residues (C. Ohsumi and T. Hayashi (1994) Plant and Cell Physiology 35:963-967; G. J. McDougall and S. C. Fry (1994) Journal of Plant Physiology 143:591-595; J. L. Acebes et al. (1993) Phytochemistry 33:1343-1345). On acid hydrolysis, the xyloglucan extracted from cotton fibers yielded glucose, xylose, galactose and fucose in the ratio of 50:29:12:7 (Hayashi et al., 1988).
Xyloglucans produced by solanaceous plants are unusual in that typical only 40% of the beta-1,4-linked glucose residues bear a glycosyl side chain at O-6. Furthermore, up to 60% of the xylose residues are substituted at O-2 with alfa-L-arabinose residues and some solanaceous plants, such as potato, also have xyloglucans with beta-D-galactose substituents at O-2 of some of the xylose residues (York et al (1996)).
Xyloglucan is believed to function in the primary wall of plants by crosslinking cellulose-microfibrils, forming a cellulose-xyloglucan network. This network is considered necessary for the structural integrity of primary cell-walls (Carpita et al., 1993). Another important function of xyloglucan is to act as a repository for xyloglucan subunit oligo-saccharides that are physiologically active regulators of plant cell growth. Xyloglucan subunits may also modulate the action of a xyloglucan endotransglycosylase (XET), a cell-wall associated enzyme that has been hypothesized to play a role in the elongation of plant cell walls. Therefore xyloglucan might play an important role in wall loosening and consequently cell expansion (Fry et al., 1992).
The seeds of many dicotyledonous species contain xyloglucan as the major polysaccharide storage reserve. This type of xyloglucan, which is localized in massive thickenings on the inside of the seed cotyledon cell wall, is composed mainly of glucose, xylose and galactose (Rose et al., 1996).
Seeds of the tamarind tree
Tamarindus indica
became a commercial source of gum in 1943 when the gum was found useful as a paper and textile size. Sizing of jute and cotton with tamarind xyloglucan has been extensively practiced in Asia owing to the low cost of the gum and to its excellent properties. Food applications of tamarind xyloglucan include use in confections, jams and jellies and as a stabilizer in ice cream and mayonnaise (Whistler et al., 1993).
Xyloglucanase activity is not included in the classification of enzymes provided by the Enzyme Nomenclature (1992). Hitherto, this enzymatic activity has simply been classified as glucanase activity and has often been believed to be identical to cellulolytic activity (EC 3.2.1.4), i.e. activity against &bgr;-1,4-glycosidic linkages in cellulose or cellulose derivative substrates, or at least to be a side activity in enzymes having cellulolytic activity. However, a true xyloglucanase is a true xyloglucan specific enzyme capable of catalyzing the solubilisation of xyloglucan to xyloglucan oligosaccharides but which does not exhibit substantial cellulolytic activity, e.g. activity against the conventionally used cellulose-like substrates CMC (carboxymethylcellulose), HE cellulose and Avicel (microcrystalline cellulose). A xyloglucanase cleaves the beta-1,4-glycosidic linkages in the backbone of xyloglucan.
Xyloglucanase activity is described by Vincken et al. (1997) who characterizes three different endoglucanases from
Trichoderma viride
(similar to
T. reesei
) which all have high activity against cellulose or CMC and show that the EndoI (which is indeed belonging to family 5 of glycosyl hydrolases, see Henrissat, B. et al. (1991, 1993)) has essentially no (i.e. very little) activity against xyloglucan, and that EndoV (belonging to the family 7 of glycosyl hydrolases) and EndoIV (belonging to the family 12 of glycosyl hydrolases) both have activity against xyloglucan and CMC, respectively, of the same order of magnitude.
International Patent Publication WO 94/14953 discloses a xyloglucanase (EG II) cloned from the fungus
Aspergillus aculeatus.
International Patent Publication WO 98/38288 discloses amino acid sequences of xyloglucanases from
Tiarosporella phaseolina
and
Dichotomocladium hesseltinei
, respectively.
International Patent Publication WO 98/50513 discloses laundry and cleaning compositions containing xyloglucanase enzymes.
International Patent Publication WO 99/02663 discloses xyloglucanases cloned from
Bacillus licheniformis
and
Bacillus agaradhaerens.
Since many important processes, both industrial processes and domestic processes using industrially produced agents, are operating at a high pH in the alkaline range. Accordingly, there is a need for a true xyloglucanase enzyme with a high xyloglucanase activity at an alkaline pH.
SUMMARY OF THE INVENTION
The inventors have now found enzymes having substantial xyloglucanase activity at high pH in the alkaline range, such enzymes being obtained from or endogenous to a strain belonging to the fungal genus Malbranchea.
Accordingly, the present invention relates to an isolated or purified xyloglucanase enzyme obtained from a strain belonging to the genus Malbranchea, preferably to a strain belonging to a species selected from the group consisting of
Malbranchea cinnamomea
(and the three synonyms
Malbranchea sulfurea, Malbranchea pulchella
var.
sulfurea
and
Thermoidium sulfureum
),
Malbranchea graminicola, Malbranchea pulchella, Malbranchea filamentosa, Malbranchea gypsea, Malbranchea albolutea, Malbranchea arcuata, Malbranchea aurantiaca, Malbranchea bolognesii-chiurcoi, Malbranchea chrysosporioidea, Malbranchea circinata, Malbranchea flava, Malbranchea flavorosea, Malbranchea flocciformis, Malbranchea fulva, Malbranchea kambayashii, Malbranchea multicolor, Malbranchea sclerotica, Malbranchea setosa
and
Malbranchea dendritica
, which enzyme exhibits xyloglucanase activity and no activity on cellulose or cellulose derivative substrates. In a preferred embodiment of the invention, the xyloglucanase has an apparent molecular weight of 25±10 kDa, a pI between 3 and 5 and exhibits xyloglucanase activity the pH range 4-11, measured at 50° C.
In a further aspect, the present invention provides a xyloglucanase cloned from a strain belonging to Malbranchea, i.e. a xyloglucanase selected from one of (a) a polypeptide comprising a fragment encoded by the DNA sequence of positions 141-806 of SEQ ID NO:1; (b) a polypeptide produced by culturing a cell comprising the sequence of SEQ ID NO:1 under conditions wherein the DNA sequence is expressed; and (c) a xyloglucanase enzyme having a sequence of at least 80% identity to positions 1-222 of SEQ ID NO:1 when identity is determined by GAP provided in the GCG program package verion 10.0 using a GAP creation penalty of 8 and GAP extension penalty of 2.
In another aspect, the present invention provides an isolated or purified polypeptide having xyloglucanase activity which is obtained from a strain of the genus Malbranchea and has
i) xyloglucanase activity in the pH range 4-11, measured at 50° C.;
ii) a molecular mass of 25&p
Kauppinen Markus Sakari
Schulein Martin
Stringer Mary Ann
Wu Wenping
Gorbell Jason I.
Lambiris Elias J.
Novozymes A/S
Prouty Rebecca E.
Rao Manjunath N.
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