Purification and characterization of a novel earthworm DNase

Abstract

A new deoxyribonuclease (DNase), referred to as EWDNase, was isolated from earthworm tissues. The purification protocol included acetone precipitation, chromatography on CM-Sepharose, and gel electrophoresis. The overall purification was 73-fold with a recovery rate of 2.3% and a final specific activity of 2039 U/mg. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis suggested a molecular mass of 30 kD for EWDNase, with an isoelectric point of approximately 7.0. Maximum activity was detected at a pH of 5.6 and a temperature of 40°C. Addition of Mg(2+) and Ca(2+) ions promoted enzyme activity strongly, while Zn(2+) and ethylenediamine tetraacetic acid (EDTA) acted as inhibitors. Liquid chromatography-tandem mass spectroscopy (LC-MS/MS) analysis indicated that there was no known matching sequence. The properties of EWDNase were sufficiently different from previously reported enzymes to suggest that it is a new enzyme requiring further confirmation and characterization.

Keywords: activity assay; deoxyribonuclease; earthworm; isolation and purification.