Characterization of semi-purified collagenase fraction from lobster (Homarus americanus)

A collagenolytic enzyme fraction was isolated from the hepatopancreas of the lobster (Homarus americanus) and semi-purified by the successive steps of acetone precipitation, ammonium sulfate fractionation, ion exchange chromatography on Mono Q column, followed by gel filtration on a Superdex 75 column or by preparative isoelectric focusing using a Rotofor cell.Semi-purified collagenase fractions from the lobster hepatopancreas was electrophoresed in polyacrylamide gels both in the presence or absence of SDS, and shown to have molecular weights ranging from 15,000-66,000. The enzymatically active peak 1 fraction from the isoelectric focusing step in the Rotofor cell migrated as a single band in 12% polyacrylamide gel with few light protein bands.The pH-activity data indicated that the collagenase fraction had two pH optima for the hydrolysis of native collagen, one at pH 4 and the other between pH 7-8.The temperature-activity data for the hydrolysis of native collagen indicated the lobster enzyme exhibited two temperature optima--a minor one at 25$ sp circ$C and a more pronounced one between 40$ sp circ$C and 50$ sp circ$C