V. A. Toptikov*, Ye. A. Shesterenko, Yu. A. Shesterenko

Medical Biotechnology and Enzymology Laboratory, Department of Biomedicine,
A.V. Bogatsky Physico-Chemical Institute, National Academy of Sciences of Ukraine, Odesa;
*e-mail: v.a.toptikov@gmail.com

Received: 02 July2025; Revised: 28 August 2025;
Accepted: 28 November 2025; Available on-line:           2025

Collagenases have found practical applications in both medicine and the food industry, but the search for novel collagenase sources remains an active area of studies. Rapana, a predatory mollusk that primarily feeds on bivalves rich in connective tissue, has emerged as a potential source of collagenolytic enzymes. This study aimed to isolate collagenase-active preparation from Rapana venosa salivary glands and characterize its properties. The salivary gland extract was purified by acetone precipitation followed by ammonium sulfate treatment. Electrophoresis was performed by the Laemmli protocol under both reducing and non-reducing conditions. Proteolytic activity was determined spectrophotometrically using collagen or gelatin as a substrate. The preparation consisted of five protein fractions and exhibited enzymatic polymorphism. A 13.8-fold purification of collagenase activity was achieved, at least 22% of total proteins displayed collagenolytic activity, while 88% showed gelatinolytic activity. The optimum of preparation activity was found in acidic (pH 4.5) and alkaline (-9.5) ranges, with thermal optimum at 46°C. At room temperature, about 90% of activity was maintained for 8 h. Serine protease inhibitors did not affect enzyme activity, metal ion chelators completely inhibited it. Reducing agents enhancing SH-groups increased enzyme activity, disulfide bond regeneration or SH-group modification decreased it. The data obtained showed that the collagenase-active enzyme preparation from Rapana venosa salivary glands consists mainly of metalloproteinases and cysteine proteases, exhibiting high stability.

Keywords: collagenase, proteolytic enzymes, purification, Rapana venosa, salivary glands