Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
2000-10-13
2003-09-09
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S069200, C435S183000, C435S252300, C435S320100, C435S262000, C435S263000, C536S023200, C536S023700, C510S114000
Reexamination Certificate
active
06617143
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to isolated polypeptides having glucanotransferase 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.
DESCRIPTION OF THE RELATED ART
4-&agr;-glucanotransferases (EC. 2.4.1.25) catalyzes the reaction of transferring an &agr;-glucan chain from one &agr;-glucan molecule to another (or to glucose). Glucanotransferases are widely distributed in microorganisms (e.g.
E. coli
) and plant tissues such as potato tubers, germinating barley seeds, sweet potato and spinach.
Recently, glucanotransferases have been found to be capable of catalyzing an intramolecular reaction of an &agr;-glucan. For example, it has been reported that glucanotransferase is capable of catalyzing an intramolecular transglycosylation reaction (cyclization reaction) of amylose, thereby synthesizing cycloamylose having a degree of polymerization (hereinafter “DP”) of 17 or more.
Glucanotransferases can be applied for various industrial purposes. As an example, a glucanotransferase can be utilized in processing an &agr;-glucan for the production of a cyclic glucan. In the case for using an enzyme for an industrial purpose, the reaction is desirably conducted at a temperature as high as possible (about 60° C. or higher) due to the fact that an &agr;-glucan, i.e. the substrate, is thereby prevented from retrogradation and, at the same time, contamination of the system by microorganism is avoided, or at least reduced.
In general, only glucanotransferases (amylomaltases) having a high activity in a moderate temperature interval (typically from about 30 to 45° C.) have been isolated, see, for example,
Agric. Biol. Chem
. 53, 2653-2659 (1989) and
J. Chem. Soc
., 44-53 (1956). More recently, a novel heat-resistant glucanotransferase (amylomaltase) also capable of generating a cyclic glucan has been described in EP 0 884 384 A2.
SUMMARY OF THE INVENTION
Thus, in a first aspect the present invention relates to an isolated polypeptide having glucanotransferase activity, selected from the group consisting of:
(a) a polypeptide having an amino acid sequence which has at least 65% identity with the amino acid sequence shown as amino acids 1 to 501 of SEQ ID NO:2;
(b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with
(i) a complementary strand of the nucleic acid sequence shown as nucleotides i1 to 1503 of SEQ ID NO:1, or
(ii) a subsequence of (i) of at least 100 nucleotides;
(c) an allelic variant of (a) or (b);
(d) a polypeptide encoded by the glucanotransferase encoding part of the DNA sequence cloned into a plasmid present in
Escherichia coli
DSM 13049, or a variant thereof having at least 65% identity to said polypeptide; and
(e) a fragment of said polypeptide having glucanotransferase activity.
In a second aspect the present invention relates to an isolated nucleic acid sequence comprising a nucleic acid sequence which encodes for the polypeptide of the invention.
In a third aspect, the present invention relates to an isolated nucleic acid sequence encoding a polypeptide having glucanotransferase activity, selected from the group consisting of:
(a) a nucleic acid sequence having at least 70% identity with the nucleic acid sequence shown as nucleotides 1 to 1503 of SEQ ID NO:1;
(b) a nucleic acid sequence which hybridizes under low stringency conditions with
(i) a complementary strand of the nucleic acid sequence shown as nucleotides 1 to 1503 of SEQ ID NO:1, or
(ii) a subsequence of (i) of at least 100 nucleotides;
(c) an allelic variant of (a) or (b);
(d) the glucanotransferase encoding part of the DNA sequence which has been cloned into a plasmid present in
Escherichia coli
DSM 13049, or a variant thereof having at least 70% identity to said DNA sequence; and
(e) a subsequence of (a), (b), (c), or (d), wherein the subsequence encodes a polypeptide fragment which has glucanotransferase activity;
or an isolated nucleic acid sequence which is the complementary strand of (a), (b), (c), (d) or (e).
In a fourth aspect the present invention a nucleic acid construct comprising the nucleic acid sequence of the invention operably linked to one or more control sequences capable of directing the expression of the polypeptide in a suitable expression host.
In a fifth aspect the present invention relates to a recombinant expression vector comprising the nucleic acid construct of the invention, a promoter, and transcriptional and translational stop signals.
In a sixth aspect the present invention relates to a recombinant host cell comprising the nucleic acid construct of the invention.
The present invention also relates to methods for producing the polypeptide of the invention; to methods for producing foods and use of the polypeptide of the invention for producing foods as well as to detergent compositions comprising the polypeptide of the invention.
REFERENCES:
patent: 6015783 (2000-01-01), von der Osten et al.
patent: 0 884 384 (1998-12-01), None
Kaneko et al. PIR Database Accession No. S74648, Apr. 25, 1997.*
Kitahata et al., Agric. Biol. Chem., 53 (10), 2653-2659, 1989.
Peat et al., J. Chem. Soc. 1956, 44-53.
Terada et al., Applied and Environmental Microbiology, vol. 65, No. 3, pp 910-915 (1999).
Takaha et al., The Journal of Biological Chemistry, vol. 208, No. 2, pp. 1881-1895 (1998).
Garbell Jason
Lambiris Elias
Novozymes A/S
Prouty Rebecca E.
Rao Manjunath N.
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