Abstract
An alkalophilic bacterium, Bacillus sp. strain GM8901, grown at pH 10.5 and 50(deg)C, produced five alkaline amylases in culture broth. At an early stage of the bacterial growth, amylase I (Amyl I) was produced initially and then, as cultivation progressed, four alkaline amylases, Amyl II, Amyl III, Amyl IV, and Amyl V, were produced from proteolytic degradation of Amyl I. A serine protease present in the culture medium was believed to be involved in Amyl I degradation. We purified Amyl I from the culture supernatant by ammonium sulfate precipitation, heparin-Sepharose CL-6B column chromatography, phenyl-Toyopearl column chromatography, and Mono Q HR5/5 high-performance liquid chromatography. The molecular weight of Amyl I was estimated to be about 97,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amyl I had an extremely high optimal pH of 11.0 to 12.0 and was stable in a broad pH range of 6.0 to 13.0. Amyl I had an optimal temperature of 60(deg)C and was stable up to 50(deg)C. Thermostability was increased in the presence of Ca(sup2+) and soluble starch. The enzyme required metal ions such as Ca(sup2+), Mg(sup2+), Cu(sup2+), Co(sup2+), Ag(sup+), Zn(sup2+), and Fe(sup2+) for its enzyme activity and was inhibited by 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride. According to the mode of action of Amyl I on starch, Amyl I was classified as an (alpha)- and exo-amylase. Amyl I produced maltotetraose predominantly from starch via intermediates such as maltohexaose and maltopentaose.
Full Text
The Full Text of this article is available as a PDF (635.7 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Boyer E. W., Ingle M. B. Extracellular alkaline amylase from a Bacillus species. J Bacteriol. 1972 Jun;110(3):992–1000. doi: 10.1128/jb.110.3.992-1000.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
- Buonocore V., Caporale C., De Rosa M., Gambacorta A. Stable, inducible thermoacidophilic alpha-amylase from Bacillus acidocaldarius. J Bacteriol. 1976 Nov;128(2):515–521. doi: 10.1128/jb.128.2.515-521.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Pfueller S. L., Elliott W. H. The extracellular alpha-amylase of bacillus stearothermophilus. J Biol Chem. 1969 Jan 10;244(1):48–54. [PubMed] [Google Scholar]
- Robyt J. F., Ackerman R. J. Isolation, purification, and characterization of a maltotetraose-producing amylase from Pseudomonas stutzeri. Arch Biochem Biophys. 1971 Jul;145(1):105–114. doi: 10.1016/0003-9861(71)90015-4. [DOI] [PubMed] [Google Scholar]
- Saito N. A thermophilic extracellular -amylase from Bacillus licheniformis. Arch Biochem Biophys. 1973 Apr;155(2):290–298. doi: 10.1016/0003-9861(73)90117-3. [DOI] [PubMed] [Google Scholar]
- Tao B. Y., Reilly P. J., Robyt J. F. Detection of a covalent intermediate in the mechanism of action of porcine pancreatic alpha-amylase by using 13C nuclear magnetic resonance. Biochim Biophys Acta. 1989 May 1;995(3):214–220. doi: 10.1016/0167-4838(89)90038-1. [DOI] [PubMed] [Google Scholar]
- Toda H., Narita K. Correlation of the sulfhydryl group with the essential calcium in Bacillus subtilis saccharifying alpha-amylase. J Biochem. 1968 Mar;63(3):302–307. [PubMed] [Google Scholar]
- Tsukamoto A., Kimura K., Ishii Y., Takano T., Yamane K. Nucleotide sequence of the maltohexaose-producing amylase gene from an alkalophilic Bacillus sp. #707 and structural similarity to liquefying type alpha-amylases. Biochem Biophys Res Commun. 1988 Feb 29;151(1):25–31. doi: 10.1016/0006-291x(88)90554-2. [DOI] [PubMed] [Google Scholar]