Sunday, August 12, 2012
Columbia Hall, Terrace Level (Washington Hilton)
The raw starch digesting type of amylase (RSDA) presents greater opportunities for process efficiency at cheaper cost and shorter time. Biocatalysts are utilized in environments alien to their natural ones; however chemical modification is lauded as a simple and rapid method towards enzyme stabilization. RSDA from Aspergillus carbonarius was acylated using the monocarboxylic phthalic anhydride or glycosylated with the oxidized cationic polysaccharide, chitosan. Circular dichroism and fluorescent intensity spectra were used to probe structural changes. Activity retention was 87.3% for acylated and 80.9% for glycosylated RSDA. Optimum pH shifted from 5 to 7 after acylation but was not changed following glycosylation. At pH 3 and 10 activity retention was 60.3% and 93.5% for acylated, 72.4% and 98.8% for glycosylated whereas native RSDA had 57.4% and 83.3%. Optimum temperature changed from 30 oC (native) to 30-40 oC and 60 oC for acylated and glycosylated, respectively. After exposure for 20 min at 80 oC, acylated lost 7%, glycosylated, 5.8% while native RSDA lost 29% activity. Rate of inactivation of native RSDA was higher as temperature increased while modified RSDA was optimally stable at higher temperatures. The specificity constants (Vmax/Km) were 73.2 for acylated, 63.1 for glycosylated and 77.1 for native RSDA. Fluorescence spectra proved that the environment of tryptophan was modified; centre of spectral mass decreased for the acylated but increased for glycosylated RSDA. Stabilization observed using the negatively charged anhydride and positively charged chitosan depicts favorable side chain reorientations, and interactions whether electrostatic or hydrophobic leading to enhanced enzyme structure.