Abstract

To stabilize the raw starch digesting amylase from fungus Aspergillus carbonarius(Bainier) Thom IMI 366159, the enzyme was immobilized on an inorganic porous support silica gel using different methods. Immobilization was carried out by spontaneous adsorption and crosslinking (reticulation), initial physical adsorption followed by crosslinking or conjugation on a silica gel activated with glutaraldehyde or polyglutaraldehyde. Concentration of glutaraldehyde, pH and duration of enzyme immobilization greatly influenced immobilization yield. A shift of optimum pH from pH 5 to 6 was observed for reticulated raw starch digesting amylase (RSDA), while other immobilized derivatives remained the same as the soluble enzyme. Immobilized enzyme exhibited increased activity at alkaline pH 8 to 9. Glutaraldehyde and polyglutaraldehyde activated RSDA showed lower activity at acidic pH 3.5 to 4 as compared to the crosslinked enzyme derivatives which had above 75% activity. The temperature optimum for the reticulated derivative was remarkably broadened from 30 to 60°C. All immobilized derivatives were more active and stable at higher temperatures to varying degrees. Soluble amylase lost 30% of its activity after 2 h incubation at 65°C, while 2.9% loss was recorded for reticulated derivative, 6.2% loss was recorded for physically adsorbed and crosslinked derivative, and 1.9 and 10% loss was recorded for polyglutaraldehyde and glutaraldehyde activated derivatives, respectively. Immobilization led to a slight decrease in K_m for all the derivatives. However, spontaneous adsorption and crosslinking (reticulation) of RSDA to silica gel with glutaraldehyde gave the best overall stability results.

Key words: Raw starch digesting amylase, immobilization, crosslinking, starch, stability.