Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-06-24
2003-05-20
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S183000, C435S200000, C435S201000, C435S202000, C435S203000, C435S204000, C435S205000, C435S206000, C435S207000, C435S208000, C435S209000, C435S210000, C510S114000
Reexamination Certificate
active
06566114
ABSTRACT:
The present invention relates to microbial mannanases, more specifically to microbial enzymes exhibiting mannanase 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 paper and pulp, textile, oil drilling, cleaning, laundering, detergent and cellulose fiber processing industries.
BACKGROUND OF THE INVENTION
Mannan containing polysaccharides are a major component of the hemicellulose fraction in woods and endosperm in many leguminous seeds and in some mature seeds of non-leguminous plants. Essentially unsubstituted linear beta-1,4-mannan is found in some non-leguminous plants. Unsubstituted beta-1,4-mannan which is present e.g. in ivory nuts resembles cellulose in the conformation of the individual polysaccharide chains, and is water-insoluble. In leguminous seeds, water-soluble galactomannan is the main storage carbohydrate comprising up to 20% of the total dry weight. Galactomannans have a linear beta-1,4-mannan backbone substituted with single alpha-1,6-galactose, optionally substituted with acetyl groups. Mannans are also found in several monocotyledonous plants and are the most abundant polysaccharides in the cell wall material in palm kernel meal. Glucomannans are linear polysaccharides with a backbone of beta-1,4-linked mannose and glucose alternating in a more or less regular manner, the backbone optionally being substituted with galactose and/or acetyl groups. Mannans, galactomannans, glucomannans and galactoglucomannans (i.e. glucomannan backbones with branched galactose) contribute to more than 50% of the softwood hemicellulose. Moreover, the cellulose of many red algae contains a significant amount of mannose.
Mannanases have been identified in several Bacillus organisms. For example, Talbot et al., Appl. Environ. Microbiol., Vol.56, No. 11, pp. 3505-3510 (1990) describes a beta-mannanase derived from
Bacillus stearothermophilus
in dimer form having molecular weight of 162 kDa and an optimum pH of 5.5-7.5. Mendoza et al., World J. Microbiol. Biotech., Vol. 10, No. 5, pp. 551-555 (1994) describes a beta-mannanase derived from
Bacillus subtilis
having a molecular weight of 38 kDa, an optimum activity at pH 5.0 and 55° C. and a pI of 4.8. JP-A-03047076 discloses a beta-mannanase derived from Bacillus sp., having a molecular weight of 37±3 kDa measured by gel filtration, an optimum pH of 8-10 and a pI of 5.3-5.4. JP-A-63056289 describes the production of an alkaline, thermostable beta-mannanase which hydrolyses beta-1,4-D-mannopyranoside bonds of e.g. mannans and produces manno-oligosaccharides. JP-A-63036775 relates to the Bacillus microorganism FERM P-8856 which produces beta-mannanase and beta-mannosidase at an alkaline pH. JP-A-08051975 discloses alkaline beta-mannanases from alkalophilic Bacillus sp. AM-001 having molecular weights of 43±3 kDa and 57±3 kDa and optimum pH of 8-10. A purified mannanase from
Bacillus amyloliquefaciens
useful in the bleaching of pulp and paper and a method of preparation thereof is disclosed in WO 97/11164. WO 91/18974 describes a hemicellulase such as a glucanase, xylanase or mannanase active at an extreme pH and temperature. WO 94/25576 discloses an enzyme from
Aspergillus aculeatus,
CBS 101.43, exhibiting mannanase activity which may be useful for degradation or modification of plant or algae cell wall material. WO 93/24622 discloses a mannanase isolated from
Trichoderma reseei
useful for bleaching lignocellulosic pulps.
WO 95/35362 discloses cleaning compositions containing plant cell wall degrading enzymes having pectinase and/or hemicellulase and optionally cellulase activity for the removal of stains of vegetable origin and further discloses an alkaline mannanase from the strain C11SB.G17.
It is an object of the present invention to provide a novel and efficient enzyme exhibiting mannanase activity also in the alkaline pH range, e.g. when applied in cleaning compositions or different industrial processes.
SUMMARY OF THE INVENTION
The inventors have now found novel enzymes having substantial mannanase activity, i.e. enzymes exhibiting mannanase activity which may be obtained from a bacterial strain of the genus Bacillus and have succeeded in identifying DNA sequences encoding such enzymes. The DNA sequences are listed in the sequence listing as SEQ ID No. 1, 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31; and the deduced amino acid sequences are listed in the sequence listing as SEQ ID No. 2, 6, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 and 32, respectively. It is believed that the novel enzymes will be classified according to the Enzyme Nomenclature in the Enzyme Class EC 3.2.1.78.
In a first aspect, the present invention relates to a mannanase which is i) a polypeptide produced by Bacillus sp. I633, ii) a polypeptide comprising an amino acid sequence as shown in positions 32-330 of SEQ ID NO:2, or iii) an analogue of the polypeptide defined in i) or ii) which is at least 65% homologous with said polypeptide, is derived from said polypeptide by substitution, deletion or addition of one or several amino acids, or 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 mannanase activity and comprising a sequence of nucleotides as shown in SEQ ID NO: 1 from nucleotide 94 to nucleotide 990; (b) species homologs of (a); (c) polynucleotide molecules that encode a polypeptide having mannanase activity that is at least 65% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 32 to amino acid residue 330; (d) molecules complementary to (a), (b) or (c); and (e) degenerate nucleotide sequences of (a), (b), (c) or (d).
The plasmid pBXM3 comprising the polynucleotide molecule (the DNA sequence) encoding a mannanase of the present invention 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 May 29, 1998 under the deposition number DSM 12197.
Within another aspect of the invention there is provided 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 mannanase activity and comprising a sequence of nucleotides as shown in SEQ ID NO: 1 from nucleotide 94 to nucleotide 990; (b) species homologs of (a); (c) polynucleotide molecules that encode a polypeptide having mannanase activity that is at least 65% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 32 to amino acid residue 330; and (d) degenerate nucleotide sequences of (a), (b), or (c); and a transcription terminator.
Within yet another aspect of the present invention there is provided a cultured cell into which has been introduced an expression vector as disclosed above, wherein said cell expresses the polypeptide encoded by the DNA segment.
Further aspects of the present invention provide an isolated polypeptide having mannanase activity selected from the group consisting of (a) polypeptide molecules comprising a sequence of amino acid residues as shown in SEQ ID NO:2 from amino acid residue 32 to amino acid residue 330; (b) species homologs of (a); and a fusion protein having mannanase activity comprising a first polypeptide part exhibiting mannanase activity and a second polypeptide part exhibiting cellulose binding function, the second polypeptide preferably being a cellulose binding domain (CBD), such as a fusion protein represented by SEQ ID NO:4.
Within another aspect of the present in
Andersen Lene Nonboe
Bjørnvad Mads Eskelund
Kauppinen Markus Sakari
Schnorr Kirk
Schülein Martin
Lambiris Elias J.
Novozymes A/S
Prouty Rebecca E.
Rao Manjunath N.
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