Cleaning compositions for solid surfaces – auxiliary compositions – Cleaning compositions or processes of preparing – Enzyme component of specific activity or source
Reexamination Certificate
2000-03-29
2003-08-19
Prouty, Rebecca E. (Department: 1652)
Cleaning compositions for solid surfaces, auxiliary compositions
Cleaning compositions or processes of preparing
Enzyme component of specific activity or source
C435S193000
Reexamination Certificate
active
06608018
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to isolated polypeptides having branching enzyme activity and isolated nucleic acid sequences encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.
2. Description of the Related Art
Branching enzyme (EC 2.4.1.18) hereinafter denoted BE, catalyzes transglycosylation to form the alpha-1,6-glucosidic linkages (branch points) of glycogen and amylopectin in microorganisms, plants and higher organisms. Glycogen and amylopectin are highly branched starch materials used for energy storage in microorganisms, plants and higher organisms. Not only does BE form a branch between different molecules (intermolecular transfer), it also catalyzes the transfer of a multi-branched glucan to another site on the same molecule (intramolecular transfer).
Highly branched starch materials have unique properties like high solubility, low viscosity and less tendency to retrograde compared to unmodified starch, which make them interesting for use in adhesive compositions including surface sizing and coating in the paper industry as described in EP 0 690 170, food and drink additives and anti-starch retrogradation agents as described in U.S. Pat. No. 4,454,161.
Branching enzymes from several different organisms have been isolated and disclosed, e.g.: U.S. Pat. No. 4,454,161 describes a BE from
Bacillus megaterium;
Boyer and Preiss, Biochemistry, vol. 16 (16), pp. 3693-3699, 1977, describe a BE from
Escherichia coli;
Zevenhuizen, Biochem. Biophys. Acta (81), pp. 608-611, 1964, describes a BE from
Arthrobacter globiformis;
Walker and Builder, Eur. J. of Biochem., vol. 20 (1), pp. 14-21, 1971, describe a BE from
Streptococcus mitis;
Kiel et al., Gene, vol. 78 (1), pp. 9-18, 1989, describe a BE from the cyanobacterium Synechococcus sp. PCC7942; Rumbak et al., Journal of Bacteriology, vol. 173 (21), pp. 6732-6741, 1991, describe a BE from Butyrivibrio fibrisolvens; Kiel et al., DNA Sequence, vol. 3 (4), pp. 221-232, 1992, describe a BE from
Bacillus caldolyticus.
A common characteristic of the above mentioned references is that none of the reported temperature optima are higher than 45° C. Only two branching enzymes have been disclosed which are characterized by having a higher temperature optimum: Kiel et al., Molecular & General Genetics, vol. 230 (1-2), pp. 136-144, 1991 (also in EP 0 418 945): a
Bacillus stearothermophilus
1503-4R branching enzyme having a temperature optimum of 53° C.; and Takata et al., Applied and Environmental Microbiology, vol. 60 (9), pp. 3096-3104, 1994: a
Bacillus stearothermophilus
TRBE14 branching enzyme having a temperature optimum of around 50° C.
Because of the increased reaction rates obtained at higher temperatures it is industrially advantageous to use branching enzymes with a high temperature optimum. Thereby a higher capacity is obtainable with the same amount of enzyme, and a better production economy is achieved. Furthermore it is beneficial to run processes at high temperatures due to prevention of infections.
It is an object of the present invention to provide improved polypeptides having branching enzyme activity and nucleic acids encoding the polypeptides.
SUMMARY OF THE INVENTION
The present invention relates to isolated polypeptides having branching enzyme activity, and a temperature optimum of at least 60° C.
The present invention also relates to isolated nucleic acid sequences encoding the polypeptides and to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.
DETAILED DESCRIPTION OF THE INVENTION
Polypeptides Having Branching Enzyme Activity
“branching enzyme activity” is 1,4-alpha-glucan branching activity which catalyzes the formation of 1,6-alpha-glucosidic linkages of glycogen or amylopectin (EC 2.4.1.18). For purposes of the present invention, branching enzyme activity may be determined according to the modified version of the procedure described by Takata et al., Applied and Environmental Microbiology (1994), p. 3097 (assay A), which is described in the Materials and Methods section herein under the heading “Branching enzyme activity”.
In a first aspect, the present invention relates to isolated polypeptides having branching enzyme activity, and having a temperature optimum of at least 60° C.; preferably the temperature optimum is in the range of 60° C. to 120° C.; more preferably the temperature optimum is in the range of 60° C. to 100° C.; even more preferably the temperature optimum is in the range of 60° C. to 80° C.; most preferably the temperature optimum is in the range of 60° C. to 70° C.; and even most preferably the temperature optimum is 65° C.
In a first embodiment, the polypeptides of the present invention retain about 70% relative activity in the range of pH 6 to pH 8; preferably about 80% relative activity in the range of pH 6 to pH 7; more preferably the pH optimum is around pH 7.
The conditions used for determining the temperature and pH optima are those described in the Materials and Methods section herein under the heading “Branching enzyme activity”.
In a second embodiment, the polypeptides of the present invention have an amino acid sequence which has a degree of homology to amino acids 2 to 621 of SEQ ID NO:2 (i.e. the mature polypeptide) or to the amino acid sequence encoded by the nucleic acid sequence contained in plasmid pT7Blue contained in
E. coli
DSM 12607 of at least about 65%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least about 95%, and even most preferably at least about 97%, which have branching enzyme activity (hereinafter “homologous polypeptides”). In a preferred embodiment, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from the amino acid sequence of SEQ ID NO:2.
For purposes of the present invention, alignments of sequences and calculation of homology scores may be done using a full Smith-Waterman alignment, useful for both protein and DNA alignments. The default scoring matrices BLOSUM50 and the identity matrix are used for protein and DNA alignments respectively. The penalty for the first residue in a gap is −12 for proteins and −16 for DNA, while the penalty for additional residues in a gap is −2 for proteins and −4 for DNA. Alignment may be made with the FASTA package version v20u6 (W. R. Pearson and D. J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448, and W. R. Pearson (1990) “Rapid and Sensitive Sequence Comparison with FASTP and FASTA”, Methods in Enzymology, 183:63-98). Multiple alignments of protein sequences may be made using “ClustalW” (Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680). Multiple alignment of DNA sequences may be done using the protein alignment as a template, replacing the amino acids with the corresponding codon from the DNA sequence.
Preferably, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO:2 or an allelic variant thereof; or a fragment thereof that has branching enzyme activity. In a more preferred embodiment, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO:2. In another preferred embodiment, the polypeptide of the present invention consists of the amino acid sequence of SEQ ID NO:2 or an allelic variant thereof; or a fragment thereof that has branching enzyme activity. In another preferred embodiment, the polypeptide of
Garbell Jason
Lambiris Elias
Novozymes A/S
Prouty Rebecca E.
Steadman David J
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