A maltotriose-producing alpha-amylase, AmyA, from a newly isolated bacterial strain Microbulbifer thermotolerans DAU221 was purified and characterized in the heterologous host, Escherichia coli, using the pCold I vector. The amyA gene encoded a 761-residue protein composed of a 33 amino acid secretion signal peptide. The purified alpha-amylase with a molecular mass of 80 kDa, approximately, shared a sequence motif characteristic of the glycoside hydrolase family 13. The enzyme was optimally active, at 50 A degrees C in sodium phosphate buffer (pH 6.0), by the traditional one factor-at-a-time method. But the optimal conditions of time, temperature, and pH for production of maltotriose from soluble starch were 1.76 h, 44.95 A degrees C, and pH 6.35 by response surface methodology, respectively. Maltotriose, as the major enzyme reaction product, was analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The enzyme was found to be inhibited by the addition of 10 mM Cu2+, Fe3+, Hg2+, Zn2+, and EDTA, but exhibited extreme stability toward hexane. The K (m) and V (max) values for the hydrolysis of soluble starch were 1.08 mg/mL and 1.736 mmol maltotriose/mg protein/min, respectively.