Production and properties of a raw-starch-degrading amylase from the thermophilic and alkaliphilic Bacillus sp. TS-23

The optimum temperature and initial medium pH for amylase production by Bacillus sp. TS-23 were 55 degrees C and 8.5 respectively. Maximum amylase activity was obtained in a medium containing peptone and soluble starch as nitrogen and carbon sources. Activity staining revealed that two amylases with molecular masses of 150 and 42 kDa were produced when maltose, soluble starch or amylose was used as carbon source for growth, whereas only the 150 kDa protein was detected in the medium containing water-insoluble carbon sources. A raw-starch-degrading amylase was purified from culture supernatant of Bacillus sp, TS-23. The molecular mass of the purified amylase was estimated at 42 kDa by electrophoresis. The enzyme had a pi of 4.2, The optimal pH and temperature for activity were 9.0 and 70 degrees C respectively. The thermoactivity of the purified enzyme was enhanced in the presence of 5 mM Ca2+; under this condition, enzyme activity could be measured at a temperature of 90 degrees C, The enzyme was strongly inhibited by Hg2+, Pb2+, Zn2+, Cu2+ and EDTA, but less affected by Ni2+ and Cd2+. The enzyme preferentially hydrolysed high-molecular-mass substrates with an alpha-1,4-glucosidic bond except glycogen. The raw starches were partly degraded by the purified amylase to yield predominantly oligosaccharides with degrees of polymerization 3, 4 and 5.