Purification and characterization of a serine proteinase from the tuna pyloric caeca

A 15.0 kDa serine proteinase with collagenase activity from pyloric caeca of tuna, Thunnus thynnus, was purified in four steps; acetone precipitation, gel filtration chromatography on a Sephadex G-100, ion-exchange chromatography on a DEAE-Sephadex A-50 and gel filtration chromatography on a Sephadex G-75 column. The purification and yield were 30.5-fold and 0.023%, respectively, as compared with those in the starting crude extract. The optimum pH and temperature for the purified collagenolytic enzyme were around pH7.5 and 55C, respectively. The purified proteinase was strongly inhibited by metal ions (Hg2+ and Zn2+) and serine proteinase inhibitors (PMSF, TLCK and soybean trypsin inhibitor) suggesting it is a serine protease. The K-m and V-max of the purified enzyme for collagen type I were approximately 3.82 mM and 851.5 U, respectively.