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

Abstract

Fish processing waste can be used to produce valuable by-products such as chymotrypsin which has applications in the food, leather, chemical and clinical industries. In this study, a reverse micelles system of AOT/isooctane was used to extract chymotrypsin from crude aqueous extract of red perch intestine. The effects of pH and KCl concentration of the backward extraction step on the total volume (TV), volume ratio (VR), total activity (TA), enzyme activity (AE), specific activity (SA), purification fold (PF), protein concentration (Cp) and recovery yield (RY) were studied. Changing the pH from 6.5 to 8.5 and the KCl concentration from 0.5 to 2.0 M during the backward extraction step had no effects on the TV or VR. Increasing the pH from 6.5 to 7.5 increased AE, SA, Cp, PF and RY by up to 47.06%, 30.0%, 27.0%, 26.9% and 18.47%, respectively but they all then declined with further increases in the pH. Similar trends were observed when the KCl concentration was increased from 0.5 to 1.5 M. The decreases in these parameters were due to the denaturation of protein under high pH. The highest AE, Cp and RY were achieved with pH 7.5 and 1.0 M KCl concentration while the highest SA and PF were achieved with pH 7.5 and 1.5 M KCl concentration. Addition of isobutyl alcohol in the backward extraction step increased the TV, AE, TA, Cp, SA, PF and RY by 13.6%, 336.4%, 342.6%, 81.1%, 146.4%, 146.2% and 345.8%, respectively. Alcohol reduced the interfacial resistance for the reverse micelles and, thus, destroyed the reverse micelles structure. The values of AE, TA, SA, PF and RY obtained with reverse micelles methods were much higher (2.3 fold) than those obtained with the ammonium sulphate method.