Separation of enzymes by sequential macroaffinity ligand-facilitated three-phase partitioning

Pectinase and cellulase were separated from a commercial enzyme preparation called Pectinex Ultra SP-L. This was carried out using a process called macroaffinity ligand-facilitated three-phase partitioning (MLFTPP). In this method, a water-soluble polymer is floated as an interfacial precipitate by adding ammonium sulfate and tert.-butanol. The polymer (appropriately chosen) in the presence of an enzyme for which it shows affinity, selectively binds to the enzyme and floats as a polymer–enzyme complex. In the first step, pectinase was purified (with alginate as the polymer) 13-fold with 96% activity recovery. In the second MLFTPP step, using chitosan, cellulase was purified 16-fold with 92% activity recovery. Both preparations showed a single band on sodium dodecylsulfate–polyacrylamide gel electrophoresis. This illustrative example shows that the strategy of sequential MLFTPP can be used to separate important biological activities from a crude broth.