P32: Stabilization of a raw starch digesting amylase via its funtional groups using activated agarose as support matrix

Applications of raw starch digesting amylases (RSDAs) are limited due to instability, product inhibition of enzyme and contamination issues. RSDA from Aspergillus carbonarius was stabilized through immobilization on agarose gel by conjugation, spontaneous crosslinking and adsorption using glycidol, glutaraldehyde or polyglutaraldehyde. Influence of immobilization on kinetics, catalytic, storage and operational stability of immobilized enzyme was evaluated. Optimal condition for glutaraldehyde activation of agarose (GAg-RSDA) was 12% glutaraldehyde for 60 min activation, for crosslinking following enzyme adsorption (AgRSDA-Cross) 3% for 90 min and for spontaneous adsorption and crosslinking (Ag-RSDA-Ret) 2.5% glutaraldehyde for 120 min, with pH optimum of pH7 in all cases. Polyglutaraldehyde activated agarose (PGAg-RSDA) gave the highest immobilization yield (100%) though expressed activity was 86.7% and for glycidol activated RSDA (GlyAg-RSDA) was 80.4%. A pH shift from optimum pH of 5 for the soluble enzyme to pH 6 for AgRSDA-Cross and AgRSDA-Ret, and pH 7 for PGAg-RSDA was seen. PGAg-RSDA and AgRSDA-Cross was most pH stable and retained over 82% activity from pH 3.5 – pH 9 compared to 59% of the soluble enzyme. Thermoinactivation studies showed that RSDA derivatives with the exception of GAg-RSDA retained over 90% activity at 60⁰C while soluble enzyme retained 76% activity. GAg-RSDA, PGAg-RSDA, Gly-RSDA and GAg-RSDA retained approximately 100% activity after 30 days storage at 4⁰C. GlyAg-RSDA retained 100%, PGAg-RSDA 92%, AgRSDA-Cross 90%, GAg-RSDA 87% and Ag-RSDA-Ret 80% activity after 10 batch reuse. Immobilization successfully stabilized RSDA, permits RSDA reuse, energy conservation while providing a two-phase medium limiting inhibition and product contamination.