Co-immobilization of α-amylase and Maltogenic Amylase by Nanomagnetic Combi-Cross-linked Enzyme Aggregates for High Maltose syrup Production From Corn Starch

In this study Co-immobilization of α-amylase and maltogenic amylase by using of lysine functionalized magnetic nanoparticles cross-linked enzyme aggregates method was carried out for high maltose syrup production from corn starch. Nanomanetite (Fe3O4) was prepared by co-precipitation method and then, surface functionalization by lysine amino acid was achieved. Enzyme aggregation was performed by acetonitrile, acetone, tert-butanol, isopropanol, ethanol, and saturated ammonium sulfate that tert-butanol implied high enzyme activity for immobilization. Enzyme ratio of 1:9 (α-amylase: maltogenic amylase) optimum pH 6 and optimum temperature 65º C, 2 mM glutaraldehyde concentration, and enzyme to lysine ratio of 1:0.75 were the best conditions for enzyme immobilization. DLS results indicated that, mean particle size of nanomagnetites was in the range of 81.9-88.9 nm with PDI= 0.242 and zeta potential of -21 and mean particle size of immobilized enzyme was in the range of 99.60-110.5 nm with PDI= 0.088 and zeta potential of -32 respectively. The appearance of new NH2 and NH spectra in FTIR spectroscopy confirmed the enzyme''''s immobilization on magnetic nanoparticles. Moreover, immobilized enzyme preserved 80.36% of its activity after 10 consecutive cycles. Comparative study on kinetic parameters of immobilized enzyme versus free one revealed that, Vmax had not any significant change while, Km was reduced 1.5 time. The enzyme half-life of immobilized enzyme increased 2.5 times at 95º C compared to free one. Enzyme loading of immobilized enzyme on magnetic nanoparticles were determined 82%.