Abstract
Lipase recovery from Pacific white shrimp hepatopancreas using a three-phase partitioning (TPP) system in combination with an aqueous two-phase system (ATPS) was studied. TPP system was formed with a simultaneous addition of salt directly to crude extract (CE) followed by an organic solvent addition. The various process parameters required for efficient purification of lipase were optimized. The best lipase yield (87.41%) and purification fold (PF) (3.49-fold) were obtained in the interphase of TPP system, which consisted of the CE to t-butanol ratio of 1:1 (v/v) in the presence of 50% (w/v) (NH4)2SO4. Subsequently, TPP fraction was subjected to ATPS. Effects of phase compositions including PEG molecular weight and concentration, types and concentration of salts, NaCl addition and system pH on lipase partitioning were investigated. With the application of 25% (w/w) PEG1000 and 15% (w/w) MgSO4, at pH 5.0 was found most appropriate since high lipase PF (5.19-fold) and yield (78.46%) in top phase were obtained. The partitioned enzyme exhibited optimal activity at pH 8.0 and 55°C and was stable at a temperature range of 0-40°C and a pH range of 7-10. The partitioned lipase showed high tolerance in the presence of ethanol and methanol. Hence, the combined partitioning systems, TPP-ATPS, were found to be an attractive technique for the recovery and partial purification of lipase from Pacific white shrimp hepatopancreas.
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