Isolation and purification of superoxide dismutase from garlic using two-stage ultrafiltration

The objective of this work is to develop a membrane-based cost-effective process for the separation and purification of superoxide dismutase (SOD) from garlic. It was found that a two-stage ultrafiltration process using a 100 kDa MWCO regenerated cellulose membrane and a 50 kDa MWCO polyethersulfone membrane could successfully be employed to isolate SOD by removing the bigger macromolecules and smaller molecules from the crude feedstock. The effects of solution pH and ionic strength on the transmission of SOD and the presence of impurity proteins with molecular weights close to SOD were quantified using the pulsed sample injection ultrafiltration technique. The effect of other parameters including system hydrodynamics and mass transfer, in terms of stirring speed, and permeate flux on the separation of SOD was also studied using parameter scanning ultrafiltration. Under optimized conditions, the purity of SOD obtained was greater than 98.0% after two-stage ultrafiltration process and the recovery of SOD from the feedstock was close to 90%. The resulting SOD product was analyzed by isoelectric focusing (IEF), SDS-PAGE and circular dichroism (CD), to confirm its isoelectric point, molecular weight and molecular secondary structure of the garlic SOD. The purity and activity as well as effects of metal ions (copper and zinc ions) on SOD specific activities were experimentally determined. (C) 2010 Elsevier B.V. All rights reserved.