Kinetics of immobilized cyclodextrin gluconotransferase produced by Bacillus macerans ATCC 8244

The extracellular enzyme cyclodextrin gluconotransferase (CGTase, EC 2.4.1.19) was produced by Bacillus macerans ATCC 8244 at optimized conditions. The CGTase activity was found to be 495 mu g beta-CD/(ml min). The enzyme was partially purified by 40-60% ammonium sulfate precipitation. The enzyme was immobilized by sodium alginate beads and kinetic studies were performed. The enzymatic activity of both free and immobilized CGTase was highest at temperature 60 degrees C and pH 7.5. The analysis of kinetic parameters K-m and V-max was obtained by the action of CGTase on the starch with respect to beta-CD. The enzyme exhibits substrate inhibition and the values are 10 and 5.5 mu g starch/ml for free and immobilized enzyme, respectively. The effect of salt concentration on enzyme activity was studied and it was found that Cu2+, Fe2+, Zn2+, Co2+. Mg2+ and Hg2+ were inhibitory, however Ca2+ showed a relative increase in enzyme activity. The immobilization of the enzyme showed a high operational stability by retaining almost 75% of the initial activity after seventh use. (c) 2006 Elsevier Inc. All rights reserved.