Partition of Pepsinogen from the Stomach of Red Perch (Sebastes marinus) by Aqueous Two Phase Systems: Effects of the Salt Type and Concentration

摘要

An important acidic protease, pepsin is synthesized and secreted in the gastric membrane in an inactive state called pepsinogen (PG) and has applications in the food and manufacturing industries, collagen extraction, gelatin extraction and in regulating digestibility. Fish processing waste can be used to produce commercially valuable byproducts such as pepsinogen. In the present study, the purification of pepsinogen from the stomach of red perch using aqueous two phase systems (ATPS) formed by polyethylene glycol (PEG) and salt at 4°C was optimized. The effects of salt type (MgSO4, (NH4)2SO4, Na3C6H5O7 and K2HPO4) and concentration (6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 19%) on the partitioning of PG were studied and parameters including total volume (TV), volume ratio (VR), enzyme activity (AE), protein content (Cp), specific activity (SA), partition coefficient (Kp), purification fold (PF) and recovery yield (RY) were evaluated. Salt type and salt concentration had significant effects on each parameter. MgSO4, (NH4)2SO4, Na3C6H5O7 and K2HPO4 required different critical salt concentrations (9, 12, 12 and 10%, respectively) to form biphasic systems. TV and VR decreased with increased salt concentration since salt formed hydrogen bonds with water molecules and created a more compact and ordered water structure. AE, CP, SA, PF and RY showed a maximum increase with intermediate salt concentration, while KP had the opposite pattern. The highest TV and AE values were obtained at 12% (NH4)2SO4 while the highest SA and PF values were obtained at 12% MgSO4. The highest TV and Cp values were obtained at 12 and 15% Na3C6H5O7, respectively. (NH4)2SO4 at 15% concentration gave the highest RY (71.7%) and was selected as the optimum salt type and concentration. Thus, 15% (NH4)2SO4 18% PEG 1500 was the optimal ATPS combination and presented the best partition. The values of SA and PF and RY obtained with ATPS method were two fold higher than those obtained with the ammonium sulphate fractionation (ASF) method.