Body wall that mainly consists of collagen and polysaccharides is the edible part of sea cucumber and is easy to go autolysis, while the proteinase(s) responsible for autolysis remains unclear. In the present study, a gelatinolytic metalloproteinase (GMP) from the body wall of sea cucumber Stichopus japonicus was purified to homogeneity by a combination of ammonium sulfate fractionation and chromatographic steps including DEAE-Sephacel, Sephacryl S-200, Q-Sepharose and Phenyl-Sepharose. The molecular mass of GMP as estimated by SOS-PAGE and gelatin zymography was 45 kDa. The enzyme revealed high activity at a slightly alkaline pH range (8.0-9.0) and the optimal temperature was at 40-45 degrees C. Metalloproteinase inhibitors, EDTA, EGTA, and 1,10-phenanthroline, almost completely suppressed the activity, whereas other proteinase inhibitors did not show any effect. Peptide mass fingerprinting of the enzyme obtained 3 peptide fragments with a total of 58 amino acid residues, which was 91.4% identical to an alkaline metalloprotease from Pseudomonas fluorescens, strongly suggesting it is a metalloproteinase. Divalent metal ion Ca2+ is essential for its activity, indicating it is a calcium-dependent metalloproteinase. Furthermore, GMP hydrolyzed collagen effectively at 37 degrees C and gradually even at 4 degrees C, suggesting its involvement in the autolysis of sea cucumber. (C) 2013 Elsevier Ltd. All rights reserved.