Chitosan as a macroaffinity ligand: Purification of chitinases by affinity precipitation and aqueous two-phase extractions

Abstract

(1) Chitosan was found to be a suitable macroaffinity ligand for affinity precipitation of chitinase from Neurospora crassa, cabbage and puffballs. (2) The activity recoveries of 85, 82 and 90% with concomitant fold purifications in terms of specific activities were 27, 15 and 30 with N. crassa, cabbage and puffballs and were obtained with affinity precipitation. These results were obtained with clarified extracts/homogenates as the starting materials. (3) The incorporation of chitosan in poly(ethylene glycol) (PEG)–salt aqueous two-phase system allowed purification of chitinases from these sources directly from unclarified extracts/homogenates. (4) The 96% (w/v) chitosan (of initially introduced into the aqueous two-phase system) partitioned into PEG-phase and this enhanced the partitioning of chitinases into PEG-phase. The chitosan, free as well as bound to chitinases, could be separated from PEG-phase by increasing the pH to 7. (5) By the process of desorption with 2.0 M MgCl 2, 86, 80 and 88% activity recoveries (% expressed in terms of total units of enzyme activities in the crude extract) were obtained in the case of N. crassa, cabbage and puffballs, respectively. The corresponding fold purifications in terms of specific activities were 34, 20 and 38. (6) The purified preparations gave single bands on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and the estimated molecular masses agreed with the reported values in the literature.