Cloning and characterization of a thermostable intracellular α-amylase gene from the hyperthermophilic bacterium Thermotoga maritima MSB8

• DOI: 10.1016/j.resmic.2003.09.005
• Publication Date: 2003-10-11T09:41:53Z
• Authors: Woo Jin Lim; Sang Ryeol Park; Chang Long An; Jong Yeoul Lee; Su Young Hong; Eun Chule Shin; Eun Ju Kim; Jong Ok Kim; Hoon Kim; Han Dae Yun
• Keywords: Thermotoga maritima; Thermostability; Alpha-amylase; Molecular mass

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).