title: Enzymatic properties of a novel liquefying alpha-amylase from an alkaliphilic Bacillus isolate and entire nucleotide and amino acid sequences.
abstract: A novel liquefying alpha-amylase (LAMY) was found in cultures of an alkaliphilic Bacillus isolate, KSM-1378. The specific activity of purified LAMY was approximately 5,000 U mg of protein-1, a value two- to fivefold greater between pH 5 and 10 than that of an industrial, thermostable Bacillus licheniformis enzyme. The enzyme had a pH optimum of 8.0 to 8.5 and displayed maximum activity at 55 degreesC. The molecular mass deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 53 kDa, and the apparent isoelectric point was around pH 9. This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltopentaose, maltohexaose, and maltose as major end products after completion of the reaction. Maltooligosaccharides in the maltose-to-maltopentaose range were unhydrolyzable by the enzyme. The structural gene for LAMY contained a single open reading frame 1, 548 bp in length, corresponding to 516 amino acids that included a signal peptide of 31 amino acids. The calculated molecular mass of the extracellular mature enzyme was 55,391 Da. LAMY exhibited relatively low amino acid identity to other liquefying amylases, such as the enzymes from B. licheniformis (68.9%), Bacillus amyloliquefaciens (66.7%), and Bacillus stearothermophilus (68.6%). The four conserved regions, designated I, II, III, and IV, and the putative catalytic triad were found in the deduced amino acid sequence of LAMY. Essentially, the sequence of LAMY was consistent with the tertiary structures of reported amylolytic enzymes, which are composed of domains A, B, and C and which include the well-known (alpha/beta)8 barrel motif in domain A.
authors.affiliation: Tochigi Research Laboratories of Kao Corporation, Ichikai, Haga, Tochigi 321-3497, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Igarashi
authors.forename: Y
authors.initials: Y
authors.lastname: Hatada
authors.forename: H
authors.initials: H
authors.lastname: Hagihara
authors.forename: K
authors.initials: K
authors.lastname: Saeki
authors.forename: M
authors.initials: M
authors.lastname: Takaiwa
authors.forename: T
authors.initials: T
authors.lastname: Uemura
authors.forename: K
authors.initials: K
authors.lastname: Ara
authors.forename: K
authors.initials: K
authors.lastname: Ozaki
authors.forename: S
authors.initials: S
authors.lastname: Kawai
authors.forename: T
authors.initials: T
authors.lastname: Kobayashi
authors.forename: S
authors.initials: S
authors.lastname: Ito
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002412:Cations / Q000494:pharmacology; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D002412:Cations; D004269:DNA, Bacterial; D000516:alpha-Amylases