Purification and characterization of deoxyribonuclease from earthworm Eisenia foetida / 北京大学学报(医学版)

ABSTRACT

Objective:To purify a kind of deoxyribonuclease from earthworm Eisenia foetida (named earthworm DNase, EDNase) and study its characteristics. Methods: Acetone precipitation, ion-exchange chromatography, high performance liquid chromatography, SDS-PAGE, CapiUary electrophoresis isoelectric focusing and MALDI-TOP MS were used for the study. Results: This purified protocol improved 137-fold purification and 45.6 % recovery of enzyme activity. The molecular mass of EDNase was estimated to be 63 000. Mg2+ , Mn2+ and Ca2+ were strong inhibitors of EDNase, while Na+ slightly increasd the enzyme activity. The enzyme was completely stable in the pH range from 4. 4 to 5.2 and had a pH optimum of 4.8. The optimum temperature was 37℃ and the enzyme was stable up to 40 ℃. The pI of the enzyme was 6. 20. Km and Vmax for the enzyme were 1.52 g/L and 4. 89 mg/(mL ·min), respectively, with calf thymus DNA as substrate. The enzyme was able to degrade chromosemal DNA, linear λbacteriophage DNA as well as supereoiled plasmid DNA, but didn' t display any RNase activity. Conclusion: This kind of deoxyribonuclease possesses unique characteristics, which is different from the deoxyribonucleases which we have known before.