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

Lisha Zhao, Suzanne M Budge, Abdel E Ghaly*, Marianne S Brooks and Deepika Dave

Fish processing waste can be used to produce commercially valuable by-products, such as pepsinogen, which has application in food, manufacturing industries, collagen extraction, gelatin extraction, and in regulating digestibility.An important acidic protease, pepsin, is synthesized and secreted in the gastric membrane in an inactive state called pepsinogen (PG). 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 PEG molecular weight (PEG 1000, 1500, 3000 and 4000) and concentration (16, 18, 20, 22 and 24%) on the partitioning of PG were studied, and parameters including total volume (TV), volume ratio (VR), total enzyme activity (AE), protein content (Cp), specific enzyme activity (SA), partition coefficient (Kp), purification fold (PF), and recovery yield (RY) were evaluated. PEG molecular weight and PEG concentration also had significant effects on each parameter. TV and VR decreased with increased salt concentration, since salt formed hydrogen bonds with water molecules and formed a more compact and ordered water structure. PG partitioned predominantly in the PEG-rich top phase due to its negative charge. AE, CP, SA, PF and RY increased with increased salt concentration and then decreased, while KP had an opposite pattern. The PEG 3000 (20%), PEG 1000 (24%), PEG 4000 (16%) and PEG 1000 (18%) concentrations gave the highest TV, VR, CP and KP, respectively. PEG 1500 with 18% concentration gave the highest AE, SA, PF and RY (86.2%). As PEG 1500 at 18% concentration gave the highest RY (86.2%). It was selected as the optimum PEG molecular weight and PEG concentration. (NH4)2SO4 at 15%, which gave the highest RY (71.7%), was selected as the optimum salt type and salt concentration. 15% (NH4)2SO418% PEG 1500 was the optimal ATPS combination, and presented a better partition. The values of SA and PF and RY obtained with ATPS method were much higher (2 fold in case of SA and PF, and 1.2 fold in case of RY), than those obtained with the Ammonium Sulphate Fractionation (ASF) method.