Integration of affinity precipitation with three phase partitioning methods for bioseparation of laccase from Trametes versicolor

Separation of Trametes versicolor laccase from a commercial preparation was carried out using Three-Phase-Partitioning (TPP) technique. The conditions were optimized and the most favorable ammonium sulphate concentration (70% w/v), crude to t-butanol ratio (1.0:2.0) and pH (4.5) resulted in 56.22% yield with 1.65-fold purity of laccase. For further improvement in purification, an improved version of TPP called Macroaffinity-Ligand-Facilitated Three-Phase Partitioning (MLFTPP) of Trametes versicolor laccase was performed in the presence of the polymer chitosan. The chitosan shows a selective binding to the laccase and is precipitated in the interfacial layer as chitosan-laccase complex. The MFLTPP conditions were then optimized for chitosan and the best conditions were found to be 70% w/v ammonium sulphate concentration, 1.0:1.0 ratio of polymer solution to t-butanol and pH 6.0 that gave 92.3% recovery and 4.97-fold purity. The study was further widened by comparing the efficiency of TPP and MLFTPP in purifying laccases from 3 different sources Trametes veriscolor, Pleurotusostreatus, Pseudomonas desmolyticum. This study, thus, demonstrates the potential of MLFTPP as an industrially exploitable, bioseparation technique for the separation of laccase from a less-established fungal source Trametes veriscolor.