Recovery of biological activity in reversibly inactivated proteins by three phase partitioning

Three phase partitioning (TPP) is carried out by adding ammonium sulphate and t-butanol to an aqueous solution of proteins. Under optimized conditions, the protein appears as an interfacial precipitate between upper t-butanol and lower aqueous phases. It is shown that TPP can be used to recover biological activities of cellulase, cellobiase and β-glucosidase from their urea-denatured forms. The activities were originally present in a commercial preparation and TPP led to simultaneous purification as well. The best results were 94% regain of activity with 73-fold purification for cellulase, 98% regain of activity with 65-fold purification for cellobiase and 90% regain of activity with 101-fold purification for β-glucosidase. Working with more concentrated protein solutions was possible but poor results were obtained beyond 10 mg ml−1 of the starting protein concentration.