Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Lyase
Reexamination Certificate
2000-09-26
2002-08-06
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Lyase
C435S200000, C435S252300, C435S252330, C435S320100, C536S023200, C536S023700
Reexamination Certificate
active
06429000
ABSTRACT:
The present invention relates to a pectin degrading enzyme preparation; preferably to microbial pectin degrading enzymes, more specifically to microbial enzymes exhibiting pectin degrading activity as their major enzymatic activity in the neutral and alkaline pH ranges, especially to cloned pectin degrading enzymes derived from
Bacillus licheniformis;
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-polygalaturonase (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, Kiebsiella 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.
WO 98/45393 discloses detergent compositions containing protopectinase with remarkable detergency agains 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 and complex multi-component enzyme preparations from such microorganisms may be difficult to optimise for various applications, 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 or different industrial processes.
SUMMARY OF THE INVENTION
The inventors have now found several pectin degrading enzymes, especially alkaline pectin degrading enzymes, which are endogeneous to a bacterial strain of the genus Bacillus, more specifically to the strain
Bacillus licheniformis,
and have succeeded in identifying DNA sequences encoding such enzymes.
Accordingly, in a first aspect the present invention relates to an enzyme preparation consisting essentially of a pectin degrading enzyme derived from or endogeneous to a strain of
Bacillus licheniformis
or highly related Bacillus species, the enzyme preferably being a pectate lyase (EC 4.2.2.2), a pectin lyase (EC 4.2.2.10) or a polygalacturonase (EC 3.2.1.15).
The DNA sequences of two pectate lyases of the invention are listed in the sequence listing as SEQ ID No. 3 and 7, respectively, and the deduced amino acid sequences are listed in the sequence listing as SEQ ID No. 4 and 8, respectively. It is believed that these novel enzyme will be classified according to the Enzyme Nomenclature in the Enzyme Class EC 4.2.2.2. However, it should be noted that the enzyme of the invention also exhibits catalytic activity on pectin (which may be esterified) besides the activity. on pectate and polygalacturonides conventionally attributed to enzymes belonging to EC 4.2.2.2.
The DNA sequence of a pectin lyase of the invention is listed in the sequence listing as SEQ ID No. 1 and the deduced amino acid sequence is listed in the sequence listing as SEQ ID No. 2. It is believed that this novel enzyme will be classified according to the Enzyme Nomenclature in the Enzyme Class EC 4.2.2.10.
The DNA sequence of a polygalacturonase of the invention is listed in the sequence listing as SEQ ID No. 5 and the deduced amino acid sequence is listed in the sequence listing as SEQ ID No. 6. It is believed that this novel enzyme will be classified according to the Enzyme Nomenclature in the Enzyme Class EC 3.2.1.15.
Accordingly, in a second aspect, the present invention relates to a pectate lyase which is i) a polypeptide produced by
Bacillus licheniformis,
ATCC 14580, or ii) a polypeptide comprising an amino acid sequence as shown in positions 28-341. of SEQ ID NO:8, or iii) an analogue of the polypeptide defined in i) or ii) which is at least 45% homologous with said polypeptide, or iv) is derived from said polypeptide by substitution, deletion or addition of one or several amino acids, provided that the arginines in position 233 and 238 are conserved and the derived polypeptide is at least 42% homologous with said polypeptide, or v) is immunologically reactive with a polyclonal antibody raised against said polypeptide in purified form.
Within one aspect, the present invention provides an isolated polynucleotide molecule selected from the group consisting of (a) polynucleotide molecules encoding a polypeptide having pectate lyase activity and comprising a sequence of nucleotides as shown in SEQ ID No: 10: 7 from nucleotide 82 to nucleotide 1026; (b) species homologs of (a); (c) polynucleotide molecules that encode a polypeptide having pectate lyase activity that is at least 70% identical to the amino acid sequence of SEQ ID NO: 8 from amino acid residue 28 to amino acid residue 341; (d) molecules complementary to (a), (b) or (c); and (e) degenerate nucleotide sequences of (a), (b), (c) or (d).
The plasmid pSJ1678 comprising the polynucleotide molecule (the DNA sequence) encoding the pectate lyase of the present invention as represented by the amino acid sequence SEQ ID NO:8 has been transformed into a strain of the
Escherichia coli
which was deposited by the inventors according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Federal Republic of Germany, on Sep. 25, 1997 under the deposition number DSM 11789.
In a third aspect, the present invention relates to a pectate lyase which is i) a polypeptide produced by
Bacillus licheniformis,
ATCC 14580, or ii) a polypeptide comprising an amino acid sequence as shown in positions 28-221 of SEQ ID NO:4, or iii) an analogue of the polypeptide defined in i) or ii) which is at least 60% homologous with said polypeptide, or iv) is derived from said polypeptide by substitution, deletion or addition of one or several amino acids, provided that the lysines in positions 133 and 155 and the arginine in position 158 are conserved and the derived polypeptide is at least 66% ho
Andersen Lene Nonboe
Bjørnvad Mads Eskelund
Lange Niels Erik Krebs
Schnorr Kirk
Schülein Martin
Lambiris Elias
Nashed Nashaat T.
Novozymes A/S
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