Purification of hyperthermophilic archaeal amylolytic enzyme (MJA1) using thermoseparating aqueous two-phase systems

Purification of a recombinant, thermostable α-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii, was investigated in the ethylene oxide–propylene oxide random copolymer (PEO–PPO)/(NH4)2SO4, and poly(ethylene glycol) (PEG)/(NH4)2SO4 aqueous two-phase systems. MJA1 partitioned in the top polymer-rich phase, while the remainder of proteins partitioned in the bottom salt-rich phase. It was found that enzyme recovery of up to 90% with a purification factor of 3.31 was achieved using a single aqueous two-phase extraction step. In addition, the partition behavior of pure amyloglucosidase in polymer/salt aqueous two-phase systems was also evaluated. All of the studied enzymes partitioned unevenly in these polymer/salt systems. This work is the first reported application of thermoseparating polymer aqueous two-phase systems for the purification of extremophile enzymes.