Pectate lyases

Details Pectate lyases Pectate lyases

2000-03-15

2002-06-04

Prouty, Rebecca E. (Department: 1657)

Chemistry: molecular biology and microbiology

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

Lyase

C435S200000, C435S262000, C435S263000, C435S264000, C435S267000, C435S277000, C435S278000, C530S350000, C510S320000, C510S392000, C426S599000, C426S592000, C426S656000

Reexamination Certificate

active

06399351

ABSTRACT:

The present invention relates to microbial pectate lyases, more specifically to a novel subclass of pectate lyases (EC 4.2.2.2), especially to a novel family of polysaccharide lyases exhibiting pectate lyase activity, ie enzymes which are capable of eliminative cleavage of pectate to give oligosaccharides with 4-deoxy-alpha-D-gluc-4-enuronosyl groups at their non-reducing ends; to a method of producing such enzymes; and to methods for using such enzymes in the textile, detergent and cellulose fiber processing industries.
BACKGROUND OF THE INVENTION
Pectin polymers are important constituents of plant cell walls. Pectin is a hetero-polysaccharide with a backbone composed of alternating homogalacturonan (smooth regions) and rhamnogalacturonan (hairy regions). The smooth regions are linear polymers of 1,4-linked alpha-D-galacturonic acid. The galacturonic acid residues can be methyl-esterified on the carboxyl group to a varying degree, usually in a non-random fashion with blocks of polygalacturonic acid being completely methyl-esterified.
Pectinases can be classified according to their preferential substrate, highly methyl-esterified pectin or low methyl-esterified pectin and polygalacturonic acid (pectate), and their reaction mechanism, beta-elimination or hydrolysis. Pectinases can be mainly endo-acting, cutting the polymer at random sites within the chain to give a mixture of oligomers, or they may be exo-acting, attacking from one end of the polymer and producing monomers or dimers. Several pectinase activities acting on the smooth regions of pectin are included in the classification of enzymes provided by the Enzyme Nomenclature (1992) such as pectate lyase (EC 4.2.2.2), pectin lyase (EC 4.2.2.10), polygalacturonase (EC 3.2.1.15), exo-polygalacturonase (EC 3.2.1.67), exo-polygalacturonate lyase (EC 4.2.2.9) and exo-poly-alpha-galacturonosidase (EC 3.2.1.82).
Pectate lyases have been cloned from different bacterial genera such as Erwinia, Pseudomonas, Klebsiella and Xanthomonas. Also from
Bacillus subtilis
(Nasser et al. (1993) FEBS 335:319-326) and Bacillus sp. YA-14 (Kim et al. (1994) Biosci. Biotech. Biochem. 58:947-949) cloning of a pectate lyase has been described. Purification of pectate lyases with maximum activity in the pH range of 8-10 produced by
Bacillus pumilus
(Dave and Vaughn (1971) J. Bacteriol. 108:166-174),
B. polymyxa
(Nagel and Vaughn (1961) Arch. Biochem. Biophys. 93:344-352),
B. stearothermophilus
(Karbassi and Vaughn (1980) Can. J. Microbiol. 26:377-384), Bacillus sp. (Hasegawa and Nagel (1966) J. Food Sci. 31:838-845) and Bacillus sp. RK9 (Kelly and Fogarty (1978) Can. J. Microbiol. 24:1164-1172) has been reported, however, no publication was found on cloning of pectate lyase encoding genes from these organisms. All the pectate lyases described require divalent cations for maximum activity, calcium ions being the most stimulatory.
Polysaccharide lyases are classified into families according to their three-dimensional structure or folding; conventionally the Clustal W method is used the for family determination. Based on amino acid sequence alignment and the Clustal W method, a polypeptide or protein can be classified into a specific polysaccharide lyase family, ie either a known family or a novel and hitherto unknown family (The Sanger Centre: Protein Families Database of alignments and HMMs; www. sanger.ac.uk). At present known pectate lyases belong to polysaccharide lyase family 1, family 2 and family 9 (ExPASy-molecular biology WWW server of the Swiss Institute of Bioinformatics (SIB)).
WO 98/45393 discloses detergent compositions containing protopectinase with remarkable detergency against muddy soilings.
Generally, pectinase producing microorganisms exhibit a broad range of pectin degrading or modifying enzymes. Often the microorganisms also produce cellulases and/or hemicellulases. Complex multi-component enzyme preparations from such microorganisms may be difficult to optimise for use in various applications, a.o. since they even may contain enzymes with detrimental effect. Thus, it is an object of the present invention to provide a pectin degrading enzyme exhibiting only the desired effects e.g. in detergents, in textile processing or different industrial processes.
SUMMARY OF THE INVENTION
The inventors have now found and identified a novel enzyme having substantial pectate lyase activity which enzyme has excellent performance in various industrial processes. Further, the inventors have succeeded in identifying a DNA sequence encoding the enzyme. It was found that the novel pectate lyase enzyme is a member of a hitherto unknown class of pectate lyases, ie the present enzyme belongs to a hitherto unknown family of polysaccharide lyases. Based on the present disclosure, especially the materials, methods and sequence listings provided herein, it is contemplated that the skilled person can find and identify other members of this novel polysaccharide lyase family, preferably pectate lyases of microbial origin, especially bacterial or fungal pectate lyases.
It is believed that the novel pectate lyase enzymes will be classified according to the Enzyme Nomenclature in the Enzyme Class EC 4.2.2.2.
Accordingly, in a first aspect this invention relates to a pectate lyase enzyme belonging to a polysaccharide family other that family 1,2 and 9, which enzyme is selected from one of a) polypeptide encoded by the DNA sequence of positions 88-1033 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 pectate lyase enzyme comprising an amino acid sequence of at least 35% identity to positions 30-344 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 pectate lyase encoding part of the DNA sequence obtainable from the plasmid in
Escherichia coli
DSM 12712.
In a second aspect, the present invention relates to isolated pectate lyase enzyme, in which the enzyme is (i) free from homologous impurities, and (ii) produced by culturing a cell comprising the DNA sequence of positions 88-1033 of SEQ ID NO:1, wherein the enzyme is produced and isolated.
In third aspect, the invention relates to an isolated polynucleotide molecule encoding a polypeptide having pectate lyase activity selected from the group consisting of (a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ ID NO:1 from nucleotide 88 to nucleotide 1033; (b) species homologs of (a); (c) polynucleotide molecules encoding a polypeptide being at least 35% identical to the amino acid sequence of SEQ ID NO:2 from amino acid residue 30 to amino acid residue 344; (d) molecules complementary to (a), (b), or (c); (e) degenerate nucleotide sequences of (a) or (b); and (f) polynucleotide molecules encoding a polypeptide having pectate lyase activity which polynucleotide molecule hybridises 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 88-1033 of SEQ ID NO:1 and DNA probes comprising a subsequence of positions 88-1033 of SEQ ID NO:1, the subsequence having a length of at least about 100 base pairs.
In a further aspect, the present invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment selected from the group consisting of a) polynucleotide molecules encoding a polypeptide having pectate lyase activity comprising a nucleotide sequence as shown in SEQ ID NO:1 from nucleotide 88 to nucleotide 1033, b) polynucleotide molecules encoding a polypeptide having pectate lyase activity that is at least 35% identical to the amino acid sequence of SEQ ID NO:2 from amino acid residue 30 to amino acid residue 344, and (c) degenerate nucleotide sequences of (a) or (b); and a transcription terminator.
Within yet another aspect of the present inve

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