Partitioning of beta-mannanase and alpha-galactosidase from Aspergillus niger in Ucon/Reppal aqueous two-phase systems and using temperature-induced phase separation

Enzyme partitioning and recovery with a new aqueous two-phase system based on commercially available hydroxypropyl starch Reppal PES 200 and the thermo-separating polymer Ucon 50-HB-5100 was studied. Ucon is an ethylene oxide-propylene oxide random copolymer. A culture supernatant of Aspergillus niger containing extracellular beta-mannanase and alpha-galactosidase was partitioned in two steps. The primary aqueous two-phase system contained Ucon and Reppal as phase forming polymers. The effect on enzyme partitioning of salt composition, salt concentration, pH and polymer concentration was studied with the aim of obtaining optimal partitioning of target enzymes to the phase containing the thermoseparating Ucon polymer. The partitioning of the enzymes could be strongly influenced by addition of the hydrophobic triethyl ammonium ion and the chaotropic perchlorate ion. Also the effect on cationic surfactant, cetyl trimethyl ammonium bromide, on enzyme partitioning was studied. In the second step, temperature induced phase separation was carried out on the isolated Ucon phase. A water phase and a concentrated aqueous Ucon phase were formed. The enzymes were obtained in the water phase almost free of polymer.