Abstract

Amylase activity was detected in the culture medium of marine Wangia sp. C52, which was isolated from the Southern Okinawa Trough deep-sea sediment. In the present study, a cold-active amylase was purified to homogeneity from the culture broth by ammonium sulfate precipitation and gel filtration chromatography. The molecular mass of the amyalse was 58 kDa, and its isoelectric point was close to 5.6. The optimal pH and temperature were 30°C and 6.0, respectively. In the presence of Ca²⁺ and Co²⁺, the enzyme activity was stimulated while Cu²⁺, Hg²⁺, Mn²⁺, Zn²⁺, Fe³⁺, Al³⁺, EDTA, EGTA and SDS reduced the activity. K_m and V_max values of the purified enzyme for soluble starch were 2.08 ± 0.3 mg/ml and 1.26 ± 0.02 mg/ml/min, respectively. The final purified enzyme had α-helix of 25%, β-sheet of 26% and random coil of 49%, consistent with the theoretical values. This showed that the purified amylase folded with a reasonable secondary structure.

Key words: Cold-active amylase, Wangia sp., purification, enzyme characterization.