Three-phase partitioning and proteins hydrolysis patterns of alkaline proteases derived from fish viscera

•Three-phase partitioning gave the max. recovery of 220% with 6-fold purification.•The best condition is 50% Na-citrate, 1:0.5 crude extract:t-butanol, pH 8.0 at 25 °C.•Effectiveness in protein hydrolysis of the enzyme is better than commercial trypsin.•Three-phase partitioning is rapid, economical, and scalability technique.

In this study, the recovery alkaline proteases from farmed giant catfish viscera were isolated by using three-phase partitioning (TPP). Factors affecting partitioning efficiency such as salts, solvent types, pH, and incubation temperatures were studied. Furthermore, the application of extracted alkaline proteases on proteins hydrolysis was also determined. The system consisted of crude enzyme extract:t-butanol 1:0.5 (w/v), 50% sodium citrate, pH 8.0 with incubation temperature of 25 °C provided the highest enzyme recovery (220%). The smear protein bands with molecular weight of 20, 24, and 215 kDa of TPP fractions appeared on the protein stained gel. Two major clear zones (24 and 130 kDa) in the interphase were observed on casein-substrate gel electrophoresis. Extracted alkaline proteases showed relatively high effective in protein hydrolysis. As a result, TPP provided high enzyme recovery and could be applied to other enzymes. The obtained alkaline proteases can be further applied in preparation of protein hydrolysates.