Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2001-10-09
2004-06-01
Rao, Manjunath (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S004000, C435S006120, C435S069100, C435S183000, C435S200000, C536S023200, C536S023700, C510S114000, C510S392000, C510S515000
Reexamination Certificate
active
06743616
ABSTRACT:
TECHNICAL FIELD
The present invention relates to mutant &agr;-amylases having improved productivity.
BACKGROUND ART
&agr;-Amylases [EC.3.2.1.1.] have been used in a wide range of industrial fields such as starch industry, brewing industry, fiber industry, pharmaceutical industry and food industry. Among them, those capable of degrading starches at high random are suited for detergents. Conventionally known as such are, as well as &agr;-amylases derived from
Bacillus licheniformis
, liquefying alkaline &agr;-amylases derived from the alkaliphilic strain Bacillus sp. KSM-AP1378 (FERM BP-3048) (WO94/26881) and improved enzymes having improved heat resistance and oxidant resistance (WO98/44126).
The present inventors have recently found liquefying alkaline &agr;-amylases derived from the alkaliphilic strain Bacillus sp. KSM-K38 (FERM BP-6946) and having chelating-agent- and oxidation-resistance (Japanese Patent Application No. Hei 10-362487, Japanese Patent Application No. Hei 10-362488); and improved enzymes having improved heat resistance (Japanese Patent Application No. Hei 11-163569).
In addition to such properties, enzymes for detergents are required to have high productivity in consideration of their industrial production. Although various trials have been made to improve the heat resistance or oxidant resistance of &agr;-amylases for detergent by using protein engineering technique, neither improvement of productivity has been considered sufficiently nor an attempt of production increase by mutation of a structural gene has been reported yet.
An object of the present invention is to provide mutant &agr;-amylases having excellent productivity.
DISCLOSURE OF THE INVENTION
The present inventors introduced, in microorganisms, mutant &agr;-amylase structural gene constructed by site-directed mutagenesis and evaluated productivity of &agr;-amylases. As a result, it has been found that since an &agr;-amylase gene has a site taking part in the improvement of productivity, introduction, into a microorganism, of a recombinant gene having this site mutated makes it possible to produce &agr;-amylases having drastically improved productivity.
In one aspect of the present invention, there is thus provided a mutant &agr;-amylase which is derived from an &agr;-amylase having an amino acid sequence represented by SEQ ED No. 2 or showing at least 60% homology thereto by substitution or deletion of at least one amino acid residue corresponding to any one of Pro
18
, Gln
86
, Glu
130
, Asn
154
, Arg
171
, Ala
186
, Glu
212
, Val
222
, Tyr
243
) Pro
260
, Lys
269
, Glu
276
, Asn
277
, Arg
310
, Glu
360
, Gln
391
, Trp
439
, Lys
444
, Asn
471
and Gly
476
of the amino acid sequence.
In another aspect of the present invention, there is also provided a mutant &agr;-amylase derived from an &agr;-amylase having an amino acid sequence represented by SEQ ID No. 4 or showing at least 60% homology thereto by substitution or deletion of at least one amino acid residue corresponding to any one of Asp
128
, Gly
140
, Ser
144
, Arg
168
, Asn
181
, Glu
207
, Phe
272
, Ser
375
, Trp
434
and Glu
466
of the amino acid sequence.
In a further aspect of the present invention, there is also provided a gene encoding this mutant &agr;-amylase, a vector containing the gene, a cell transformed with the vector and a production method of a mutant &agr;-amylase which comprises cultivating the transformed cell.
In a still further aspect of the present invention, there is also provided a detergent composition containing this mutant &agr;-amylase.
REFERENCES:
patent: 5635468 (1997-06-01), Ara et al.
patent: 5989169 (1999-11-01), Svendsen et al.
patent: 6309871 (2001-10-01), Outtrup et al.
patent: 6410295 (2002-06-01), Andersen et al.
patent: 6436888 (2002-08-01), Svendsen et al.
patent: 1 065 277 (2001-01-01), None
patent: 98362487 (1998-12-01), None
patent: 98362488 (1998-12-01), None
patent: 2000-184882 (2000-07-01), None
patent: 2000-184883 (2000-07-01), None
patent: 2001-54392 (2001-02-01), None
patent: 2001054392 (2001-02-01), None
patent: WO 9426881 (1994-11-01), None
patent: WO 96/23873 (1996-08-01), None
patent: WO 98/05748 (1998-02-01), None
patent: WO 98/44126 (1998-10-01), None
patent: WO 00/60058 (2000-10-01), None
Yuuki (a) et al. SwissProt database accession No. P06278, Jan. 1, 1988.*
Yuuki (b) et al. PIR database accession No. A91997, Jun. 30, 1987.*
A. Tsukamoto, et al., Biochemical and Biophysical Research Communications, vol. 151, No. 1, pp. 25-31, XP-000605386, “Nucleotide Sequence of the Maltohexaose-Producing Amylase Gene From an Alkalophilic Bacillus sp. #707 and Structural Similarity to Liquefying Type &agr;-Amylases”, Feb. 29, 1988.
K. Igarashi, et al., Biochemical and Biophysical Research Communications, vol. 248, No. 2, pp. 372-377, XP-002901159, “Improved Thermostability of a Bacillus &agr;-Amylase by Deletion of an Arginine-Glycine Residue is Caused By Enhanced Calcium Binding”, 1998.
Araki Hiroyuki
Endo Keiji
Hagihara Hiroshi
Hayashi Yasuhiro
Igarashi Kazuaki
Kao Corporation
Rao Manjunath
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