Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2001-11-09
2003-10-28
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S252300, C435S252330, C435S320100, C536S023200
Reexamination Certificate
active
06638748
ABSTRACT:
TECHNICAL FIELD
The present invention relates to the gene encoding alkaline liquefying &agr;-amylase and fragments thereof, and to recombinant DNA and a transformant bearing the gene or fragments of the gene.
BACKGROUND ART
Alpha-amylase has long been used in a variety of fields. For example, it has been used for the saccharification of grains and potatoes in the fermentation industry, as starch paste removers in the textile industry, as digestives in the pharmaceutical industry, and for the manufacture of thick malt syrups in the food industry. Alpha-amylase is an enzyme which acts on a starch-related polysaccharides such as amylose and amylopectin, hydrolyzing solely the &agr;-1,4-glucoside bond of the polysaccharide molecule. Since 1833, when Payen and Persoz first discovered the enzyme, crystalline samples or electrophoretically homogeneous samples of &agr;-amylase have been obtained from a number of different sources including bacteria, fungi, plant seeds, and animal digestive glands.
The present inventors have recently discovered that the efficacy of dish-washing detergents and laundry detergents for clothes can be greatly improved, particularly on starch dirts, when &agr;-amylase and a debranching enzyme are both incorporated into these detergents (Japanese Patent Application Laid-open (kokai) No. 2-132192). However, most of the &agr;-amylases previously found in the natural world exhibit maximal and stable enzymatic activities in the neutral to acidic pH ranges, but scarcely work in an alkaline solution of pH 9-10. There exist only a small number of amylase enzymes that are known to exhibit maximal activities in the alkaline pH range (so-called alkaline &agr;-amylases and alkali-resistant &agr;-amylases). These alkaline &agr;-amylases and alkali-resistant &agr;-amylase include, an enzyme produced by Bacillus sp. A-40-2 [Horikoshi, K. et al.,
Agric. Biol. Chem.,
35, 1783 (1971)], an enzyme produced by Bacillus sp. NRRL B-3881 [Boyer, E.,
J. Baciteriol.,
110, 992 (1972)], an enzyme produced by Streptomyces sp. KSM-9 (Japanese Patent Application Laid-Open (kokai) No. 61-209528, an enzyme produced by Bacillus sp. H-167 (Japanese Patent Application Laid-Open (kokai) No. 62-208278, an enzyme produced by
Bacillus alkalothermophilus
A3-8 (Japanese Patent Application Laid-Open (kokai) No. 2-49584, and an enzyme produced by Natronococcus sp. Ah-36 (Japanese Patent Application Laid-Open (kokai) No. 4-211369.
As used herein, the term “alkaline &agr;-amylase” refers to &agr;-amylases whose optimum pHs fall within the alkaline pH range, whereas the term “alkali-resistant &agr;-amylase” refers to &agr;-amylases which have optimum pHs within the neutral to acidic range but whose activities in the alkaline range are comparable with those obtained at an optimum pH, and in addition, which retain their stabilities in the alkaline range. By the term “neutral range” is meant the range of pH not less than 6 and less than 8, and the term “alkaline” denotes a pH which is higher than the “neutral range”.
Most of these alkaline &agr;-amylases and alkali-resistant amylases are so-called saccharifying &agr;-amylases which decompose starch or starch-related polysaccharides to glucose, maltose, or maltotriose. As such, these enzymes cause problems if they are used as enzymes for detergents, though they are advantageously used in the manufacture of sugar. Thus, there remains a need for so-called alkaline liquefying &agr;-amylases which exhibit resistance against surfactants used in detergents, and which decompose starch or starch-related polysaccharides in a highly random manner. The present inventors continued an extensive search for microorganisms producing an alkaline liquefying &agr;-amylase suitable as a detergent component, and they discovered that an alkalophilic Bacillus sp. KSM-AP1378 strain, having its optimum pH for growth in the alkaline range, produces an enzyme exhibiting the activity of an alkaline liquefying &agr;-amylase. They elucidated that this enzyme is useful as an additive in detergent compositions for washing dishes and kitchen utensils and for detergent compositions for clothes (WO94/26881).
Amounts of the enzyme produced may be effectively increased by improving a method for culturing an alkaline liquefying &agr;-amylase-producing microorganism, Bacillus sp. KSM-AP1378, or by exploiting mutation. However, in order to produce the enzyme advantageously on an industrial scale, another approach must be taken.
Amounts of an enzyme produced can be enhanced using a genetic engineering approach, and in addition, the catalytic properties of the enzyme can be improved, using a protein engineering approach, by altering the gene encoding the enzyme. However, the gene encoding an alkaline liquefying &agr;-amylase has not yet been obtained.
Accordingly, an object of the present invention is to provide the gene encoding alkaline liquefying &agr;-amylase and fragments thereof, a transformant harboring recombinant DNA comprising the gene, and a method for producing an alkaline liquefying &agr;-amylase using the transformant.
The DNA encoding the alkaline liquefying &agr;-amylase gene may be further used to produce probes to be used in the isolation of additional, homologous alkaline liquefying &agr;-amylase genes from other microorganisms. Thus, an additional object of the present invention is to provide a means of screening for and isolating additional alkaline liquefying &agr;-amylase enzymes.
DISCLOSURE OF THE INVENTION
The present inventors attempted to isolate, from the chromosomal DNA of an alkalophilic Bacillus strain, a DNA fragment containing the gene encoding an alkaline liquefying &agr;-amylase, and as a result, they were successful in isolating an approximately 1.8 kb DNA fragment encoding an alkaline liquefying &agr;-amylase. When they transformed a host microorganism using this DNA fragment ligated to a suitable vector, it was confirmed that the resultant recombinant microorganism produced an alkaline liquefying &agr;-amylase. Moreover, it was found that the amino acid sequence of the alkaline liquefying &agr;-amylase to be encoded is different from that of previously known amylases. The present invention was accomplished based on this finding.
Accordingly, the present invention provides a DNA fragment encoding an alkaline liquefying &agr;-amylase.
The present invention also provides a recombinant DNA comprising the above-described DNA fragment encoding an alkaline liquefying &agr;-amylase.
The present invention also provides a transformed microorganism harboring the above-described recombinant DNA comprising a DNA fragment encoding an alkaline liquefying &agr;-amylase.
The present invention further provides a method for producing an alkaline liquefying &agr;-amylase, by culturing the above-described transformed microorganism and collecting the enzyme.
REFERENCES:
patent: 0410498 (1991-01-01), None
patent: 0670367 (1995-09-01), None
patent: WO 9426881 (1994-11-01), None
patent: WO 9526397 (1995-10-01), None
Tsukamoto et al., “Nucleotide Sequence of the Maltohexaose-Producing Amylase Gene From An AlkalophilicBacillus sp.#707 and Structural Similarity to Liquefying Type&agr;-Amylases,”Biochemical and Biophysical Research Communications, vo. 151, No. 1, pp. 25-31 (1988).
Yukki et al., “Complete Nucleotide Sequence of a Gene Coding for Heat- and pH-Stable &agr;-Amylase ofBacillus licheniformis: Comparison of the Amino Acid Sequences of Three Bacterial Liquefying &agr;-Amylases Deduced from the DNA Sequences,”Journal of Biochemistry, vol. 98, No. 5, pp. 1147-1156 (1985).
Ara Katsutoshi
Hatada Yuji
Ito Susumu
Kawai Shuji
Ozaki Katsuya
Birch & Stewart Kolasch & Birch, LLP
Kao Corporation
Slobodyansky Elizabeth
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