Bougatef A.,Laboratoire Of Genie Enzymatique Et Of Microbiologie |
Balti R.,Laboratoire Of Genie Enzymatique Et Of Microbiologie |
Nasri R.,Laboratoire Of Genie Enzymatique Et Of Microbiologie |
Jellouli K.,Laboratoire Of Genie Enzymatique Et Of Microbiologie |
And 2 more authors.
Journal of Agricultural and Food Chemistry | Year: 2010
Trypsin from the intestine of smooth hound (Mustelus mustelus) was purified by fractionation with ammonium sulfate, Sephadex G-75 gel filtration, and DEAE-cellulose ion exchange chromatography, with a 65-fold increase in specific activity and 15% recovery. The molecular weight of the purified trypsin was estimated to be 24 kDa using size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme showed esterase-specific activity on Nα-p-tosyl-l-arginine methyl ester hydrochloride (TAME) that was four times greater than its amidase-specific activity on Nα-benzoyl-dl-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for the trypsin activity were pH 8.5 and 50 °C, respectively, using TAME as a substrate. The enzyme was extremely stable in the pH range of 7.0-9.0 and highly stable up to 40 °C after 1 h of incubation. The purified enzyme was strongly inhibited by soybean trypsin inhibitor (SBTI) and N-p-tosyl-1-lysine chloromethyl ketone (TLCK), specific inhibitors for trypsin. In addition, smooth hound trypsin showed higher proteolytic activity at high NaCl concentration, demonstrating its potential for protein hydrolysis at high salt content. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECKPHSQ. This sequence showed high homology with trypsins from marine vertebrates and invertebrates. Purified trypsin had a Michaelis-Menten constant (Km) and catalytic constant (Kcat) of 0.387 ± 0.02 mM and 2.62 ± 0.11 s-1, respectively, when BAPNA was used as a substrate. For the hydrolysis of TAME, Km and Kcat were 0.156 ± 0.01 mM and 59.15 ± 2.2 s-1, respectively. © 2010 American Chemical Society.