Gene analysis, expression, and characterization of an intracellular α-amylase from the extremely halophilic archaeon Haloarcula japonica.

Masahiko Onodera, Rie Yatsunami, Wataru Tsukimura, Toshiaki Fukui, Kaoru Nakasone, Tomonori Takashina, Satoshi Nakamura

Index: Biosci. Biotechnol. Biochem. 77(2) , 281-8, (2013)

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Abstract

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 α-amylase, malA, was identified. The deduced amino acid sequence of MalA, consisting of 663 amino acids, showed homology to α-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.