PURIFICATION AND CHARACTERIZATION OF A PROTEASE FROM THE PYLORIC CAECA OF MENHADEN (Brevoortia spp) AND MULLET (Mugil spp) FROM THE SOUTHWEST ATLANTIC REGION

Abstract

A protease classified as trypsin was isolated and purified from the pyloric caeca of two species of fishes: Brevoortia spp (menhaden) and Mugil spp. (mullet). The characterization of both enzymes as trypsin was based on their molecular weight (24 kDa) determined by SDS-PAGE, their inhibition by some known trypsin inhibitors (PMSF, SBTI, TLCK and benzamidine), and their ability to hydrolyze the synthetic trypsin substrate N-α-Benzoyl-DL-arginine-p-nitroanilide (BAPA). Menhaden trypsin had maximum activity at pH 9.5 and was stable for 30 min between pH 6.0 and 10.0 at 0, 10 and 25C. On the other hand, mullet trypsin showed maximum activity at pH's from 7.8 to 9.0, and was stable over a wider pH range (7.0–10.0). The optimum temperature for menhaden and mullet trypsin was 63C and 60C respectively. Thermostability for menhaden trypsin was up to 50C, whereas mullet trypsin was stable up to 60C. The Km(BAPA) values for menhaden trypsin were conserved in the temperature range from 10 to 30C, while for mullet trypsin the Km(BAPA) was conserved between 10 and 25C.