Family 5 xyloglucanases

Details Family 5 xyloglucanases Family 5 xyloglucanases

2001-03-01

2003-10-07

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S069100, C435S183000, C435S195000, C435S200000, C435S262000, C435S263000, C435S264000, C536S023200, C536S023700, C510S114000

Reexamination Certificate

active

06630340

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to xyloglucanases belonging to family 5 of glycosyl hydrolases, preferably to enzymes exhibiting xyloglucanase activity as their major enzymatic activity in the neutral and alkaline pH ranges; to a method of producing such enzymes; and to methods for using such enzymes in the textile, detergent and cellulose fiber processing industries.
2. Description of Related Art
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 cross-linking cellulose-micro fibrils, 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 oligosaccharides 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 (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 family 12 xyloglucanase (EG II) cloned from the fungus
Aspergillus aculeatus
and expressed in the fungus
Aspergillus oryzae.
International Patent Publication WO 99/02663 discloses xyloglucanases cloned from
Bacillus licheniformis
(family 12) and
Bacillus agaradhaerens
(family 5) and expressed in
Bacillus subtilis.
It is an object of the present invention to provide an enzyme with a high xyloglucanase activity, which have an excellent performance in conventional detergent compositions, especially liquid detergents for household laundering.
SUMMARY OF THE INVENTION
The inventors have now found enzymes having substantial xyloglucanase activity, which enzymes belong to family 5 of glycosyl hydrolases and exhibit excellent performance in conventional detergent compositions, especially liquid detergent compositions. All the found xyloglucanases are endogenous to a strain belonging to
Paenibacillus pabuli
or Paenibacillus sp.
Accordingly, the present invention relates to a xyloglucanase enzyme belonging to family 5 of glycosyl hydrolases, which enzyme is endogenous to a strain of Paenibacillus. Preferably, the strain of Paenibacillus belongs to the group consisting of the species
Paenibacillus pabuli
, the strain Paenibacillus sp., DSM 13330, and strains of Paenibacillus sp. having a higher degree of identity with the
Paenibacillus pabuli
type strain ATCC 43899 than the strain Paenibacillus sp., DSM 13330, when subjected to 16S RNA analysis.
The inventors have also succeeded in cloning and expressing a family 5 xyloglucanase from the above species and strains, i.e. the invention relates in further aspects to a family 5 xyloglucanase which is (a) a polypeptide encoded by the DNA sequence of positions 840-1931 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; (c) a xyloglucanase enzyme having a sequence of at least 85% identity to positions 33-395 of SEQ ID NO: 2 when identity is determined by GAP provided in the GCG program package using a GAP creation penalty of 3.0 and GAP extension penalty of 0.1; or (d) a polypeptide encoded by the xyloglucanase encoding part of the DNA sequence obtainable from the plasmid in
Escherichia coli
DSM 13183; and to an isolated polynucleotide molecule encoding a polypeptide having xyloglucanase activity which polynucleotide molecule hybridizes to a denatured double-stranded DNA probe under medium stringency conditions, wherein the probe is selected from the group consisting of DNA probes comprising the sequence shown in positions 840-1931 of SEQ ID NO: 1, positions 693-1896 of SEQ ID NO:3, and DNA probes comprising a subsequence of positions 840-1931 of SEQ ID NO:1 or positions 693-1896 of SEQ ID NO:3, the subsequence having a length of at least about 100 base pairs.
In further aspects, the invention provides an expression vector comprising a DNA segment which is e.g. a polynucleotide molecule of the invention; a cell comprising the

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