AN ALKALINE AMYLOPULLULANASE FROM ALKALOPHILIC BACILLUS SP KSM-1378 - KINETIC EVIDENCE FOR 2 INDEPENDENT ACTIVE-SITES FOR THE ALPHA-1,4 AND ALPHA-1,6 HYDROLYTIC REACTIONS

Alkalophilic Bacillus sp, KSM-1378 produces an alkaline amylopullulanase that hydrolyzes both alpha-1,4 linkages in amylose, amylopectin, and glycogen and alpha-1,6 linkages in pullulan, The hydrolytic activities against amylose and pullulan were specifically inhibited by maltotriose (K-i=0.5mM), isomaltitol (K-i=5.2mM), and methyl alpha-D-galactoside (K-i=40mM) and by beta-cyclodextrin (K-i=0.9mM), alpha-cyclodextrin (K-i=11mM), and raffinose (K-i=31mM), respectively, in a competitive manner in each case, Inhibition by N-bromosuccinimide of the alpha-amylase activity was prevented by amylose but not by pullulan, while inhibition by N-bromosuccinimide of the pullulanase activity was prevented by pullulan but not by amylose, Kinetics of reactions in the simultaneous presence of amylose and pullulan indicated that the observed rates of formation of products closely matched those predicted by a kientic model in which the alpha-1,4 and alpha-1,6 hydrolytic reactions were catalyzed at two independent active sites, Incubation of the enzyme at 40 degrees C and pH 9.0 caused complete inactivation of the amylase activity within 4 days, but the pullulanase activity remained at the original level under the same conditions, This alkaline amylopullulanase can, therefore, be considered to be a ''two-headed'' enzyme molecule.