Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-04-13
2001-10-30
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
Reexamination Certificate
active
06309871
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to isolated polypeptides having &agr;-amylase activity and isolated nucleic acid sequences encoding the polypeptides. Further, the invention relates to variants of the &agr;-amylases of the invention. 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
For a number of years &agr;-amylase enzymes have been used for a variety of different purposes, the most important of which are starch liquefaction, textile desizing, starch modification in the paper and pulp industry, and for brewing and baking. A further use of &agr;-amylases, which is becoming increasingly important is the removal of starchy stains during washing with a detergent at alkaline pH.
Examples of commercial &agr;-amylase products are Termamyl®, Duramyl™, BAN® and Fungamyl®, all available from Novo Nordisk A/S, Denmark. These and similar products from other commercial sources have an acidic to a neutral pH optimum, typically in the range of from pH 5 to pH 7.5, and they do not display optimal activity in detergent solutions at alkaline pH.
WO 95/26397 discloses an &agr;-amylase from a Bacillus strain which has optimum activity at pH 8. WO 96/23873 describes variants of Bacillus amylases with improved performance under washing conditions.
U.S. Pat. No. 5,147,796 describes an alkaline pullulanase having alpha-amylase activity. FIG. 2
b
of the document shows optimum amylase activity at pH 8-8.5.
M. Takagi et al., J. Ferment. Bioeng., vol 81, No. 6, 557-559 (1996) describe an alkaliphilic alpha-amylase-pullulanase from Bacillus sp. The enzyme has optimum amylase activity at pH 9, but the activity drops rapidly at higher pH, and the activity at pH 10 is lower than at pH 7.
WO 97/00324 (KAO) disclose a gene encoding an alkaline liquefying &agr;-amylase derived from Bacillus sp. strain KSM-AP1378 with the deposited no. FERM BP-3048 suitable for detergents.
It is an object of the present invention to provide novel &agr;-amylases with improved performance in alkaline solutions, especially in alkaline detergent solutions at pH around 9-11.
SUMMARY OF THE INVENTION
The present invention relates to isolated polypeptides having &agr;-amylase activity selected from the group consisting of:
(a) a polypeptide having an amino acid sequence which has at least 85% identity with amino acids for mature polypeptide amino acids 1 to 485 of SEQ ID NO:2 or SEQ ID NO: 4;
(b) a polypeptide encoded by a nucleic acid sequence which hybridizes under medium stringency conditions with (i) the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO: 3, (ii) the cDNA sequence of SEQ ID NO:1 or SEQ ID NO:3, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii);
(c) an allelic variant of (a) or (b);
(d) a fragment of (a), (b) or (c) that has &agr;-amylase activity;
(e) a polypeptide having improved wash performance in alkaline detergent solutions, especially in alkaline detergent solutions at a pH around 9-11, and more preferably, at a pH around 9-10.5;
(f) a temperature optimum determined using the Phadebas method (pH 9.0) in the range between 55 to 65° C.; and
(g) a pI between 7-8 determined by isoelectric focusing (Pharmacia, Ampholine, pH 35-9.3).
Further the invention relates to variants of the &agr;-amylase of the invention. 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.
REFERENCES:
patent: 3642582 (1972-02-01), McClary
patent: WO 96/23873 (1996-08-01), None
patent: WO 97/32961 (1997-09-01), None
Igarashi et al. Biochemical and Biophysical Research Communications 248, 372-377 (1998).
Tsukamoto et al. Biochemical and Biophysical Research Communications 151, 25-31 (1988).
Borchert Torben Vedel
Hoeck Lisbeth Hedegaard
Nielsen Bjarne Ronfeldt
Nielsen Vibeke Skovgaard
Outtrup Helle
Achutamurthy Ponnathapu
Garbell Jason I.
Kerr Kathleen
Lambiras Elias J.
Novozymes A/S
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