title: Cloning and characterization of a thermostable intracellular alpha-amylase gene from the hyperthermophilic bacterium Thermotoga maritima MSB8.
abstract: The gene encoding an intracellular alpha-amylase, AmyB (TM1650), from Thermotoga maritima MSB8, a hyperthermophilic bacterium, was cloned and expressed in Escherichia coli. The AmyB enzyme hydrolyzed alpha-1,4 starch linkage. The amyB gene is 1269 bp in length, encoding a protein of 422 amino acids (calculated molecular mass of 50187 Da). The molecular weight of the enzyme was estimated to be 50000 Da by SDS-PAGE after starch-nondenaturing-PAGE. The amino acid sequence of AmyB showed less than 12% identity to other amylases, but contained four regions that are highly conserved among alpha-amylases. The AmyB alpha-amylase exhibited maximal enzymatic activity at pH 7.0 and its optimum temperature for activity was 70 degrees C. Like the alpha-amylases of many other organisms, the thermostability of T. maritima MSB8 alpha-amylase, AmyB expressed in E. coli was enhanced in the presence of Ca(2+) (10 mM).
authors.affiliation: Division of Applied Life Science, Gyeongsang National University, 660-701, Chinju, Republic of Korea.
authors.forename: Woo Jin
authors.initials: WJ
authors.lastname: Lim
authors.forename: Sang Ryeol
authors.initials: SR
authors.lastname: Park
authors.forename: Chang Long
authors.initials: CL
authors.lastname: An
authors.forename: Jong Yeoul
authors.initials: JY
authors.lastname: Lee
authors.forename: Su Young
authors.initials: SY
authors.lastname: Hong
authors.forename: Eun Chule
authors.initials: EC
authors.lastname: Shin
authors.forename: Eun Ju
authors.initials: EJ
authors.lastname: Kim
authors.forename: Jong Ok
authors.initials: JO
authors.lastname: Kim
authors.forename: Hoon
authors.initials: H
authors.lastname: Kim
authors.forename: Han Dae
authors.initials: HD
authors.lastname: Yun
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006358:Hot Temperature; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000516:alpha-Amylases