PURIFICATION, KINETIC AND MOLECULAR CHARACTERIZATIONS OF A SERINE COLLAGENOLYTIC PROTEASE FROM GREENSHORE CRAB (CARCINUS-MAENAS) DIGESTIVE GLAND

A serine collagenolytic protease was purified from a water soluble fra ction of greenshore crab digestive gland by acidic precipitation, gel filtration on a Sephadex G-50 column, ion-exchange chromatography on a Fractogel TSK DEAE column, immobilized metal ion affinity chromatogra phy (IMAC) on IDA (Cu2+) Sepharose 6B and ion-exchange chromatography on Hyper D column. The molecular mass of the monomeric Carcinus serine collagenase (CSC) was estimated to be 23,000 by SDS PAGE and its isoe lectric point was found to be 4.0. The CSC is optimally active at pH 7 and 30 degrees C and is stable over a month at room temperature. The CSC activity is strongly inhibited by PMSF, 3,4-DCI, soybean trypsin i nhibitor, alpha 1 proteinase inhibitor and elastatinal. The CSC hydrol yzes native collagen (Type I and III). CSC N-terminal sequence is simi lar to shrimp chymotrypsin-like protease and crab collagenolytic prote ase sequences. Kinetic parameters of the CSC were determined using som e peptidyl-p-nitroanilides. The catalytic efficiency (k(cat)/(K-m) is Leu > Phe > Ala.