Cell-wall degrading enzyme variants

#The present invention relates to variants of microbial cell-wall degrading enzymes, more specifically to variants of enzymes having a pectinase structure similar to that of Bacillus licheniformis enzymes exhibiting pectate lyase 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. The enzyme variants of the invention may exhibit increased thermostability as compared to the parent enzyme.

#Plant cell walls consist of a complicated network of fibrous materials. The composition of the cell walls varies considerably, depending on the source of the vegetable material. However, in general its composition can be summarized as mainly comprising non-starch polysaccharides. These polysaccharides can be found in various forms: cellulose, hemicellulose and pectins.

#The composition of a plant cell wall is both 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 e.g. 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; and 5. Ageing (Chesson (1987), Recent Advances in Animal Food Nutrition, Haresign on Cole, eds.). Butterworth, London, 71-89).

#FIGS. 1A-1C are a multiple sequence alignment of pectate lyases derived from Bacillus licheniformis (pel_bacli) (SEQ ID NO: 2), Bacillus subtilis (pel_bacsu) (SEQ ID NO: 17), and Erwinia chrysanthemi (pel_erwch) (SEQ ID NO: 18). The sequences were aligned using the align3d method of Modeller 5o (Ali, A.; T. L. Blundell, “Definition of general topological equivalence in protein structures: A procedure involving comparison of properties and relationships through simulated annealing and dynamic programming,” J. Mol. Biol., 212, 403-428 (1990)) module of the Insight 2000 molecular modelling package (Biosym Inc.). Default parameters were employed using simple alignment of the sequences and the public PDB files as well as the one enclosed here. The figure was produced using the program ESPript (Gouet, P., Courcelle, E., Stuart, D. and Metoz, F. Bioinformatics, 15, 305-308 (1999)), employing the Blosum matrix.

#Appendix 1 shows the structural coordinates of the Bacillus licheniformis pectate lyase comprising the amino acid sequence of SEQ ID NO: 2.

#Protein Numbering

#In the context of this invention, a specific numbering of amino acid residue positions in cell-wall degrading enzymes, especially pectate lyase enzymes, is employed. For example, by aligning the amino acid sequences of known pectate lyases it is possible to unambiguously allot an amino acid position number to any amino acid residue in any pectate lyase enzyme, if its amino acid sequence is known.

#In FIG. 1, a number of selected amino acid sequences of pectate lyases of different microbial origin are aligned.