Applications of pullulan in aqueous two-phase systems for enzyme production, purification and utilization

Pullulan, a microbial polysaccharide, was employed with polyethylene glycol (PEG) to form an aqueous two-phase system. The phase diagram of the PEG-Pullulan systems as well as the partition of enzymes depended strongly on the molecular weight of PEG. This behavior provided a basis for the separation of cellulase from β-galactosidase. The extractive hydrolysis of lactose, using a commercial preparation of β-galactosidase, could also be performed in a PEG-Pullulan system (16% PEG 1400, 14% Pullulan). The enzyme was effectively contained in the bottom phase while a high percentage of the glucose produced could be removed in the top phase. The enzyme activity was preserved very well after several repeated hydrolyses. Furthermore, it was demonstrated that the PEG-Pullulan system was capable of retaining Trichoderma reesei in the bottom phase, allowing the production of cellulase which could be intermittently removed in the top phase. Such a system permitted a semicontinuous production scheme although the enzyme production was observed to decrease after five cycles. In view of the low cost and low viscosity of pullulan, this polysaccharide could be considered a suitable replacement for dextran, a component widely used in aqueous two-phase formulation.