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

Abstract

ObjectivesThe aim of this study was to optimize the backward extraction step of the reverse micelles method while purifying chymotrypsin from fish processing waste. The specific objectives were: (a) to study the effect of pH (6.5, 7.0, 7.5, 8.0 and 8.5) and salt concentration (0.5, 1.0, 1.5 and 2.0 M) and alcohol addition in the backward extraction step of the reverse micelles method on the enzyme activity (A E ), protein concentration (Cp), specific activity (SA), purification fold (PF) and recov- AbstractFish 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 (A E ), 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 A E , 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 A E , 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, A E , 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 A E , TA, SA, PF and RY obtained with reverse micelles methods were much higher (2.3 fold) than those obtained with the ammonium sulphate method.

Mathematical Modelling of Backward Extraction Mixed Reverse Micelle of Amoxicillin by Surface Response Methodology (Rsm)

“…As reported by Ono et al (1996), that when the value of pH was increased during the extraction of haemoglobin using dioleyl phosphoric acid (DOLPA) (in their case), the backward extraction rate dramatically increased and almost reached 90% at a pH of 8.0 and then declined with further increase in the pH. This result also was found similar with the results obtained by Zhou et al (2012) and Goto et al (1998). The effect of aqueous stripping pH and backward extraction time on the final mass of amoxicillin extracted at 10 minutes is shown in Figure 4.…”

The solubilization and extraction parameters of erythromycin from aqueous phase into mixed AOT/SB3-18 reverse micelle phase

“…According to Zhao et al, 19 KCl salt has waterstructure-forming property and causes lesser "screening" of solutes, therefore is favorable for forward extraction in a reverse micelle. This factor has been the reason KCl was chosen for this study.…”