Biologia, Bratislava, 57/Suppl. 11: 221-228, 2002.
ISSN 0006-3088 (Biologia).
Ali Akbar Saboury*
* corresponding author
Received: October 4, 2001 / Accepted: February 12, 2002
The interaction of a-amylase from Bacillus amyloliquefaciens (BAA) with divalent calcium and cobalt cations was studied by equilibrium dialysis, isothermal titration microcalorimetry, UV spectrophotometry and temperature scanning spectrophotometry methods at 27 °C in Tris buffer solution at pH 7.5. There is a set of 17 sites for calcium binding on the enzyme with weak positive cooperativity in binding. The binding of calcium is exothermic (DH= -16.0 kJ mol-1) with mean dissociation constant for binding of 0.55 mM. The binding of calcium stabilized the enzyme against surfactant and thermal denaturation. Moreover, the binding of calcium prevents the spontaneous decrease in biological activity of a-amylase. There is a set of 25 non-cooperative sites for cobalt binding on the enzyme. The binding of cobalt is exothermic (DH= -18.5 kJ mol-1) with mean dissociation constant for binding of 0.12 mM. The enzyme activity increased significantly with increasing concentration of cobalt; however, the temperature of denaturation of the enzyme decreased. So, divalent calcium and cobalt cations act as stabilizer and activator, respectively, for BAA.
Key words: a-amylase, calcium, cobalt, activator, stabilizer, titration calorimetry.