Trypsin from zebra blenny (Salaria basilisca) viscera: Purification, characterisation and potential application as a detergent additive

An alkaline trypsin was purified from the viscera of zebra blenny (Salaria basilisca) by ammonium sulphate (40-80% saturation) precipitation, Sephadex G-100, Mono Q-Sepharose and ultrafiltration. A yield of 12% with a purification-fold of 4.2 was obtained. The trypsin had an apparent molecular weight of 27 kDa. Soybean trypsin inhibitor and phenylmethylsulfonyl fluoride showed a strong inhibitory effect on the purified trypsin. Trypsin had maximal activity at pH 9.5 and 60 degrees C for the hydrolysis of N alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA). It was stable at low temperatures and in the pH range of 7.0-12.0. The N-terminal amino acid sequence of the first 12 amino acids of the purified protease was IVGGRECTEPSQ. S. basilisca trypsin, which showed high homology with other fish trypsins, had a charged Arg residue at position 5, where Tyr is common in marine vertebrates and mammalian trypsins. The trypsin kinetic constants. K-m and k(car), for BAPNA, were 0.6 mM and 1.38 s(-1), respectively. (C) 2011 Elsevier Ltd. All rights reserved.