, 2006). TLCK is a well-known trypsin-specific inhibitor, inactivating only trypsin-like enzymes by forming a covalent bond with the histidine residue from the catalytic site and then blocking the substrate-binding

portion at Autophagy inhibitor supplier the active centre ( Jeong, Wei, Preston, & Marshall, 2000). The purified enzyme from D. rhombeus was also inhibited by 75% by benzamidine (a synthetic trypsin inhibitor), 36% by 2-mercaptoetanol, 22.8% and 71.36% by 2 mM and 4 mM PMSF, respectively (a serine proteinase inhibitor) and 21.5% by EDTA. TPCK (a typical chymotrypsin inhibitor) had no effect on the activity of the purified enzyme. The pattern of action of these inhibitors was characteristic of those reported for trypsins,

thereby supporting the identity of this purified enzyme as trypsin. Kinetics parameters of BApNA hydrolysis rates were examined in the present study (Table 2). Michaelis constant (K  m) indicate the affinity of the enzyme to the substrate, K  cat indicates molecular catalytic constant and NU7441 in vitro Kcat·Km-1 indicates its catalytic efficiency. K  m, K  cat and Kcat·Km-1 values for the trypsin-like enzyme from D. rhombeus   were 0.266 mM, 0.93 s−1 and 3.48, respectively. This K  m value is lower than that reported for trypsin from Priacanthus macrachanhtus   ( Hau & Benjakul, 2006), O. niloticus   ( Bezerra et al., 2005), Salmo salar   ( Outzen, Berglund, Smalas, & Willassen, 1996), bovine ( Asgeirsson, Fox, & Bjarnason, 1989) and swine ( Outzen et al., 1996) and higher than that reported for S. s. caerulea   ( Castillo-Yáñes et al., 2005) and E. japonica   ( Heu et al., 1995). This result indicates the considerable affinity of the purified enzyme from D. rhombeus   to the BApNA substrate. The catalytic constant (K  cat) of the trypsin purified in the present study was higher than Niclosamide the value reported for G. morhua   ( Asgeirsson et al., 1989) and S. salar   ( Outzen et al., 1996). A higher molecular activity (K  cat) denotes a greater amount of substrate molecules that are converted into product by a single enzyme, thus indicating

that the enzyme purified in the present study is as highly active as the other fish trypsin. The catalytic efficiency ( Kcat·Km-1) results reveal that the trypsin purified in the present study is able to hydrolyse a classic trypsin synthetic substrate more efficiently than the trypsin from bovine ( Asgeirsson et al., 1989), swine ( Outzen et al., 1996), P. macracanthus ( Hau & Benjakul, 2006) and S. salar ( Outzen et al., 1996), but less efficiently than that from E. japonica ( Heu et al., 1995), S. officinalis ( Balti, Barkia, Bougatef, Ktari, & Nasri, 2009) and G. morhua ( Asgeirsson et al., 1989). The 15 NH2-terminal amino acids residues in D. rhombeus trypsin were IVGGYECTMHSEAHE. This NH2-terminal amino acid sequence was compared to that of other vertebrates ( Fig. 3). According to Cao et al.