Sequential precipitation with reversibly soluble insoluble polymers as a bioseparation strategy: purification of beta-glucosidase from Trichoderma longibrachiatum.

Precipitation of proteins/enzymes with reversibly soluble-insoluble polymers is a simple approach which can be easily scaled up. It is shown here that considerable purification may be achieved if more than one polymer (chosen judiciously) is used in sequence for purification. The principle is illustrated with purification of beta-glucosidase from a commercial preparation of Trichoderma longibrachiatum. Precipitation with chitosan removed cellulase activity from the preparation with 4-fold purification and 99% recovery of beta-glucosidase activity in the supernatant. The treatment of the supernatant with Eudragit S-100 precipitated 98% of the enzyme activity. The two different elution procedures gave 82% recovery (with 14-fold purification) and 86% recovery (with 12-fold purification) of the enzyme activity from the precipitate. A low molecular weight protein still present in these preparations could be removed by gel filtration. This finally yielded an electrophoretically homogeneous enzyme with 74% recovery and 29-fold purification.