Gene Analysis, Expression, and Characterization of an Intracellular α-Amylase from the Extremely Halophilic Archaeon Haloarcula japonica

Haloarcula japonica is an extremely halophilic archaeon that requires high concentrations of NaCl to grow. Recently the draft genome sequence of Ha. japonica was determined, and a gene encoding an alpha-amylase, malA, was identified. The deduced amino acid sequence of MalA, consisting of 663 amino acids, showed homology to alpha-amylase family enzymes. The sequence did not contain a secretion signal sequence, indicating that the protein is a cytoplasmic enzyme. Transcription of the malA gene was confirmed by reverse transcription (RT)-PCR, and the transcription start site was determined by a 5'-RACE experiment. The malA gene was cloned and expressed in Ha. japonica. The recombinant MalA was purified and characterized. MalA required a high concentration of NaCl for starch-hydrolyzing activity. It showed higher activity on soluble starch, amylose, and amylopectin, and lower activity on glycogen.