Abstract

HERNANDEZ-SAMANO, A.C. et al. Extraction and characterization of sea cucumber Isostichopus fuscus proteases, collected at the Gulf of California, Mexico. Rev. Mex. Ing. Quím [online]. 2015, vol.14, n.1, pp.35-47. ISSN 1665-2738.

Enzyme extracts obtained from I. fuscus ventral muscle were studied. Peak activity was observed at pH 2, 6, and 8, and 50-60°C, whereas maximum enzymatic stability occurred at pH 2 to 6 (except pH 3) and 0 to 40°C. More than 74% residual activity was retained after heating at 70°C and 80°C for 1 h, confirming the presence of heat-resistant enzymes. The extracts also retained 59% initial activity after 21-day storage at 4-5°C. Hg²⁺ increased the proteolytic activity in 16%, whereas Cu²⁺ and Mn²⁺ caused partial inhibition, suggesting the presence of cysteine- and metallo- proteases. Extracts were inhibited by pepstatin A, β-mercaptoethanol and EDTA, confirming the predominance of aspartyl-, cystein-, and metallo-, proteases. Four proteins were identified by SDS-PAGE (180.6, 114.5, 91.7 and 52.9 kDa). Zymograms on casein confirmed the presence of proteolytic enzymes. After purification by anion exchange chromatography a 49 kDa protease, possible metalloprotease, was detected. Mass spectrometry of the partially purified protein showed homology with trypsin and chain E, leech-derived tryptase inhibitor trypsin complex (LDTI). Due that no homology was found with any previously described metalloproteases, the presence of a novel enzyme is suggested.

Keywords : sea cucumber; Isostichopus fuscus; ventral muscle; proteases; enzyme characterization.