By Nikola Lončar, Nataša Božić, Marinela Šokarda Slavić, Zoran Vujčić, and Payal Khandelwal
Raw starch digesting a-amylase (RSDA) is an industrially important enzyme. The kinetics and mode of action of RSDA are not well understood. Further elucidation of this enzyme's mechanism of action, however, requires highly pure RSDA. Here we demonstrate a single-step purification protocol, using the mixed-mode resin Nuvia cPrime, that yields ~96% pure recombinant enzyme from E. coli. This level of purity is sufficient for further mechanistic studies. Additionally, the protocol developed here is suitable for large-scale purification of RSDA and similar enzymes.