Exploring the complex effects of metal ions on d-hydantoinase purification with an immobilized metal affinity membrane

Abstract

In this study, d-hydantoinase (DHTase) was purified using an immobilized metal ion affinity membrane (IMAM), on which the interactions between amino acid residues placed on the protein surface and the chelated metal ions located on the IMAM surface facilitate DHTase purification. Batch DHTase adsorption experiments showed that the adsorption capacity varied remarkably for IMAM with different metal ions. The maximum adsorption of DHTase (1.513 ± 0.12 mg) was reached when using Cu 2+ as the chelated ion, whereas the Co 2+ showed the highest activity with only small amounts of protein adsorption. The Mn 2+, Co 2+, Ni 2+, Fe 2+ and Fe 3+ additions showed a positive effect on DHTase activity. The addition of Cu 2+ showed a varied effect from the inhibition on original DHTase to the promotion on Ni-purified DHTase. The purification folds using IMAM chelated with Co 2+, Ni 2+, and Zn 2+ were in the range of six to seven. SDS–PAGE analysis showed that all of the IMAM-purified DHTase exhibited the same molecular weight, meaning DHTase adsorbed on IMAM was highly specific. The DHTase purified by different metal ions showed various levels of increased activity when adding the corresponding metal ions. The addition of Mn 2+ or Co 2+ displayed a dramatic increase (9- to 10-fold) in activity of DHTase purified by IMAM chelated with the same ion.