A tri-enzyme magnetic nanobiocatalyst with one pot starch hydrolytic activity

•Tri-enzyme magnetic nanobiocatalyst of α-amylase, glucoamylase, pullulanase was prepared.•Tri-enzyme magnetic nanobiocatalyst was characterized by FT-IR, FE-SEM and XRD.•Tri-enzyme magnetic nanobiocatalyst improved enzyme stability and catalytic efficiency.•Tri-enzyme magnetic nanobiocatalyst gained 100% starch conversion in one pot hydrolysis.•Tri-enzyme magnetic nanobiocatalyst showed excellent reusability in starch hydrolysis.

An efficient tri-enzyme magnetic nanobiocatalyst for one pot starch hydrolysis was constructed by co-immobilization of commercially available alpha amylase, glucoamylase and pullulanase onto amino functionalized iron oxide magnetic nanoparticles using glutarldehyde as linker. Co-immobilization using 208 mM glutaraldehyde concentration with 4 h linking time and 12 h immobilization time achieved maximum activity recovery of alpha amylase (98%), glucoamylase (92%) and pullulanase (95%) in tri-enzyme magnetic nanobiocatalyst. The prepared nanobiocatalyst was characterized by using X-ray diffraction (XRD), field-emission scanning electron microscopy (FE-SEM) and Fourier transform infrared spectrophotometry (FT-IR). For one pot starch hydrolytic activity, shift in optimum pH from 6 to 7 and temperature from 70 to 80 °C with a high tolerance to alkaline pH and high temperature were observed after co-immobilization of enzymes. The thermal stability in terms of half-life of enzymes in the range of 70-90 °C was three times more for tri-enzyme magnetic nanobiocatalyst than free enzymes. Moreover, the catalytic efficiency of enzymes was found to be higher in tri-enzyme nanobiocatalyst than in free form. The tri-enzyme nanobiocatalyst displayed 100% starch conversion whereas the free enzyme mixture achieved 70% starch conversion after one pot starch hydrolysis in batch mode. At the end, the tri-enzyme nanobiocatalyst remained fully active even after 8 times recycling for one pot starch hydrolysis with cumulative reaction time of 720 min.

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