| Abstract: | The objective of this study was to isolate alkaline proteases from farmed giant catfish viscera by using a thermoseparating aqueous two-phase system (T-ATPS). Different phase partitioning parameters—type of salts, NaCl addition, and temperature—were optimized. The optimum system contained 40% EOPO3900-10% MgSO4 with 17%NaCl, induced phase separation at 55°C, and provided the highest recovery (77.98 %) and purity (PF: 21.50-fold) with a partition coefficient (KE) of 11.90 and a volume ratio (VR) of 0.19. In the recycling step, the mixture of the bottom salt-rich phase and bottom EOPO-rich phase in the ratio of 0.5:1.5 (w/w) provided the highest recovery and purity. Hence, the total recovery of 91.62% was obtained from the separation system. Three major clear zones (24, 36, and 130 kDa) can be distinctively observed on casein-substrate gel electrophoresis for protease activity staining. Major protein components of perilla seed hydrolysates were completely hydrolyzed by the alkaline proteases at 70 units while those of red kidney bean isolate were more resistant to protein hydrolysis. Fifty units of Flavourzyme® hydrolyzed completely both plant proteins into small peptides or amino acids. As a result, T-ATPS could be used as an alternative method for the separation of enzymes from various sources with acceptable recovery. In addition, the obtained alkaline proteases can be further used in preparation of protein hydrolysates. |
