Characterization of a VLK-protease from Symbiodinium sp. strain KB8

A cysteine protease was characterized from Symbiodinium sp. strain KB8 isolated from Cassiopea ornata. The protease was purified 36-fold with a yield of 1.8%, by four chromatographic steps using DE-52, DEAE-Toyopearl, MonoQ, and Superdex 200 HR. Among the six substrates tested, the enzyme showed highest activity toward t-butyloxycarbonyl-Val-Leu-Lys-4-methylcoumaryl-7-amide (Boc-VLK-MCA), and Z-LLEMCA (approximately 60% of Boc-VLK-MCA). Other substrates were hydrolyzed at lower rates (less than 25%), indicating that this enzyme uses substrates specific for Boc-VLK-MCA. This enzyme activity was almost completely inhibited by the cysteine protease inhibitors, leupeptin and E64, as well as moderately inhibited by serine protease inhibitors. These results suggest that this enzyme is not metallo and aspartic protease, but likely belongs to cysteine protease. pH optimum of the enzyme was estimated to be pH 4.5 by the analysis in the pH range from 3 to 6. From the triplicate data at six concentrations of the substrate from 0 to 200 μmol, the Km and Vmax values were calculated to be 140 μM and 79 μM min, respectively, for Boc-VLK-MCA. As far as we know, this is the first report on the isolation and characterization of a protease from Symbiodinium.