PURIFICATION AND CHARACTERIZATION OF A THERMOSTABLE ALPHA-AMYLASE FROM BACILLUS-LICHENIFORMIS

The extracellular alpha-amylase produced by the Bacillus licheniformis 44MB82-A strain was isolated. The enzyme was purified by two-phase separation in a PEG-Dextran system, followed by gel-filtration and ion exchange chromatography. Its molecular mass was determined as 58 000 by SDS-PAGE and gel filtration. The enzyme was stable at pH values from 6.5 to 8.0 and its pH optimum was pH 6.0-6.5. The temperature optimum was determined as 90-degrees-C. The Michaelis constant for soluble starch was measured as 0.90 g l-1. Thermostability was Ca2+-dependent. The half-life of the purified enzyme was 10 min at 85-degrees-C in buffer without Ca2+. The half-life at pH 6.5 with 1.0 mM CaCl2 added was 30 min, and over 120 min with 5.0 mM CaCl2. The purified enzyme was strongly inhibited by N-bromosuccinimide (NBS) and by EDTA. Dithiothreitol and iodacetamide had no inhibitory effect on the purified enzyme.