ABSTRACT
A novel gene (ANK58566) encoding a cold-active α-amylase was cloned from marine bacterium Bacillus sp. dsh19-1 (CCTCC AB 2015426), and the protein was expressed in Escherichia coli. The gene had a length of 1302 bp and encoded an α-amylase of 433 amino acids with an estimated molecular mass of 50.1 kDa. The recombinant α-amylase (AmyD-1) showed maximum activity at 20 °C and pH 6.0, and retained about 35.7% of activity at 4 °C. The AmyD-1 activity was stimulated by Ca2+ and Na+. However, the chelating agent, EDTA, inactivated the enzyme. Moreover, AmyD-1 displayed extreme salt tolerance, with the highest activity in the presence of 2.0 M NaCl and 60.5% of activity in 5.0 M NaCl. The Km, Vmax and kcat of AmyD-1 in 2.0 M NaCl were 2.8 mg ml-1, 21.8 mg ml-1 min-1 and 933.5 s-1, respectively, at 20 °C and pH 6.0 with soluble starch as substrate. MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) revealed that the end products of starch hydrolysis by AmyD-1 were glucose, maltose, maltotriose, maltotetraose, and malt oligosaccharides. Thus, AmyD-1 is one of the very few α-amylases that can tolerate low temperatures and high salt concentrations, which makes it to be a potential candidate for research in basic and applied microbiology.
