Purification and characterization of trypsin-like enzyme from shrimp Penaeus indicus.

Abstract

Trypsin-like enzyme in hepatopancreas of shrimp Penaeus indicus was obtained as two fractions (P1 and P2) from shrimp extract by an FPLC system using a Mono Q ion ex-change column. The enzyme in the peak P2 was purified. The enzyme has a single polypeptide chain with a molecular weight of 36, 000, although the enzyme showed a molecular weight of 18, 000 on gel filtration. Isoelectric point was estimated to be below 3.0 by isoelectric focusing. The enzyme showed broad pH optimum (pH 6.5-11.0) and stability (pH 6.0-12.0), and was most active at 45°C. The Km value for the synthetic sub-strate (α-N-benzoyl-1-arginine-p-nitoroanilide) was 294μM. The activity of the enzyme was almost completely inhibited by phenylmethansulfonyl fluoride or trypsin inhibitor (soybean). Propanol was a potent activator for the enzyme, the activity was extremely activated by propanol. The activity was approximately 4 fold at 30°C, and 177 fold at 4°C in the presence of propanol of the concentration of 25%.