Department: 
Food Science & Technology
Project manager(s): 
Year: 
2013
Financial supporter: 
IROST

In this research, cross-linked enzyme aggregates of thermostable -amylase (from Bacillus licheniformis, Liquozyme), glucoamylase (from Aspergillus niger,Dextrozyme G A), and pullulanase from Bacillus subtilis were prepared. At first, kinetics parameters of free enzymes such as reaction velocity and enzyme specific activity, Km, Vmax, Kd, Kcat were determined. Then, acetone, acetonitril, isopropanol, saturated ammonium sulfate, ethanol, and tert-butanol as aggregators were used. Among them, tert-butanol has the highest remaining activity with 98.16% while, saturated ammonium sulfate with 77.80% has the lowest. The optimum conditions of the immobilization process for these 3 enzymes and CLEAs formation were: glutaraldehyde concentration: 5mM, enzyme mixture ratios ( -amylase: Glucoamylase: Pullulanase) was 1: 2.96: 0.17, Enzyme/ BSA ratio 1:2, and crosslinking time: overnight (20 h), at 2-3 C. The optimum temperature and pH for free -amylase were 95 C, and pH, 5.5, for glucoamylase and pullulanse were 60-62 C, pH 5.5. CLEAs that was formed by tert-butanol and 1: 2 enzyme/ BSA ratio has a remaining activity equal to 98.86%. The optimum temperature of resulted CLEAs was 60-62 C , and optimum pH was 5.5. Moreover, resulted multi-CLEAs has a remaining catalytic activity about 90% (uncontinuously) for 16 cycles and the enzyme half life of immobilized enzymes was increased about 3 times than free enzymes. This enhancing of activity was due to addition of 3: 1 ratio of Ca2+ / Na+ ions. This ions addition improves thermostability and enzyme half lives. The results implied that, addition of Ca2+ and Na+ ions in CLEAs formation environment (with a 3: 1 ratio) improves the immobilized -amylase, Glucoamylase, Pullulanase structural and functional stability. All experiments were conducted with three replicates and reported as mean.