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अमूर्त

Extraction and Purification of Chymotrypsin from the Intestine of Red Perch (Sebastes marinus) Using Reverse Micelles: Optimization of the Forward Extraction Step

Liang Zhou, Suzanne M. Budge, Abdel E. Ghaly, Marianne S. Brooks and Deepika Dave

Fish processing waste can be used to produce valuable by-products, such as chymotrypsin which has application in the food, leather, chemical and clinical industries. Chymotrypsin is an endopeptidase secreted by the pancreatic tissues of vertebrates and invertebrates. Reverse micelles (RM) systems consisting of AOT/isooctane were used for the purification of chymotrypsin from crude aqueous extract of red perch intestine. The effects of pH and AOT concentration in the forward extraction step on the Total Volume (TV), Volume Ratio (VR), protein concentration (Cp), enzyme activity (AE), Total Activity (TA), Specific Activity (SA), Purification Fold (PF) and Recovery Yield (RY) were studied. The TV decreased with increases in the AOT concentration and decreases in pH. The VR slightly decreased with increases in the pH but was not affected by the AOT concentration. The highest AE, Cp, SA, PF and RY were achieved with pH 7.0 and 20 mM AOT concentration. Higher amount of surfactant formed a stable oil-water mixture structure and caused difficulties in the backward extraction step. When the pH was increased from 6.0 to 7.0 and/or the AOT concentration was increased from 1 to 20 mM, the AE, Cp, SA and RY initially increased and then decreased with further increases in the pH and/or the AOT concentration. The increases in these parameters were due to the increased electrostatic interaction between the net charge on the surface of protein molecule and reverse micelles inner layer charge caused by increasing pH and the increased reverse micelles structure caused by increased AOT concentration. The decreases in these parameters were due to the decreased net charge on protein molecules which weakened the electrostatic interaction between chymotrypsin and reverse micelles. The AE, TA, SA, PF and RY obtained with RM method were higher than those obtained with Ammonium Sulphate (AS) method by 2.16-2.82 folds.