PURIFICATION AND CHARACTERIZATION OF THE ALPHA-AMYLASE OF BACILLUS-FLAVOTHERMUS

Bacillus flavothermus alpha-amylase was purified to homogeneity using a combination of ammonium sulfate precipitation, ion-exchange chromato graphy, and gel filtration. The enzyme displayed maximal activity on s tarch at pH 5.5-6.0 and 60 degrees C and had an isoelectric point of 8 .4 and a K-m of 2.2 mg ml(-1). Diethyl pyrocarbonate inactivated the a mylase at pH 5.5 and 20 degrees C in a monomolecular reaction with a s econd-order rate constant of 250 M(-1) min(-1). The influence of pH on the rate of inactivation suggested the participation of a residue wit h a pK(a) of 6.7. Spectrophotometric studies and reactivation in the p resence of hydroxylamine suggested the modification of histidine(s). A single histidine residue appeared to be essential and the substrate a fforded complete protection indicating its location at the active site of the enzyme. (C) 1997 by Elsevier Science Inc.