Stabilization of a raw starch digesting amylase from Aspergillus carbonarius via immobilization on activated and non-activated agarose gel.
Applications of raw starch digesting amylases (RSDAs) are limited due to instability, product inhibition of enzyme and contamination. RSDA from Aspergillus carbonarius was stabilized through immobilization on agarose gel by adsorption, spontaneous crosslinking and conjugation using glycidol, glutaraldehyde or polyglutaraldehyde. Effects of immobilization on kinetics, catalytic, storage and operational stability of immobilized enzyme were evaluated. Polyglutaraldehyde activated agarose RSDA (PGAg-RSDA) gave the highest immobilization yield (100%) with expressed activity of 86.7% while that of glycidol activated RSDA (GlyAg-RSDA) was 80.4%. A shift in pH from optimum of 5 for the soluble enzyme to 6 for RSDA adsorbed on agarose followed by crosslinking with glutaraldehyde (AgRSDA-CROSS) and simultaneous adsorption and crosslinking (AgRSDA-RET), and pH 7 for PGAg-RSDA was seen. PGAg-RSDA and AgRSDA-CROSS were most pH stable and retained over 82% of their activities between pH 3.5 and 9 compared to 59% for the soluble enzyme. Thermoinactivation studies showed that immobilized RSDAs with the exception of GAg-RSDA retained over 90% of their activities at 60°C for 120 min while soluble enzyme retained only 76% activity under the same condition. AgRSDA-CROSS, PGAg-RSDA, Gly-RSDA and GAg-RSDA retained approximately 100% of their activities after 30 days storage at 4°C. GlyAg-RSDA retained 99.6%, PGAg-RSDA 94%, AgRSDA-CROSS 90%, GAg-RSDA 86.5% and Ag-RSDA-RET 80% activity after 10 batch reactions. Immobilization stabilized RSDA and permits processing at higher temperatures to reduce contamination.
Microbiology Department, Faculty of Biological Sciences, University of Nigeria Nsukka, Nsukka, Nigeria. tochukwu.nwagu@unn.edu.ng
Tochukwu N
TN
Nwagu
Bartho N
BN
Okolo
Hideki
H
Aoyagi
D000681:Amylases / Q000302:isolation & purification / Q000378:metabolism*; D001230:Aspergillus / Q000201:enzymology*; D001709:Biotechnology; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism; D005782:Gels; D005976:Glutaral; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012685:Sepharose; D011198:Solanum tuberosum / Q000737:chemistry; D013213:Starch / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D005782:Gels; D013213:Starch; D012685:Sepharose; D000681:Amylases; D005976:Glutaral