Purification and Characterization of an Extracellular T2 Family Ribonuclease (RNase) from Bacillus megaterium NRRL 3712.

BACKGROUND

Ribonucleases of T2 family are ubiquitous cellular components which have played several biological functions in molecular and pharmaceutical fields.

OBJECTIVE

Therefore, a soluble and highly active RNase belonging to T2 family was screened from Bacillus megaterium NRRL 3712, and different cultivation strategies were applied to enhance the production of enzyme.

METHOD

A high-level of an extracellular RNase and cell density was produced using optimal cultivation conditions. A monomeric enzyme with a molecular mass of 45 kDa, was purified to homogeneity using acetone precipitation and ion-exchange chromatography.

RESULTS

Purified enzyme was optimally activity at 45°C and pH 7.0, and it displayed a half-life of 26 min at 64°C. It was quite stable up to 60 min at 40-50°C temperature and over a broad range of pH 4.5-8.0. It showed great substrate specificity with yeast RNA, poly (A), poly (G), poly (C), and poly (U). Kinetic parameters such as Km, Vmax, kcat and kcat Km -1 values against yeast RNA as substrate, were 71.67 µg mL-1, 7866.4 μmol mg-1min-1, 17669.4 sec-1, and 246.53, respectively.

CONCLUSION

The article provides a valuable novel RNase which exhibited great resistance against various organic solvents, detergents and metal ions, whereas its activity was stimulated up to 142% by adding 5 mM EDTA. Hence, dictates its applicability as therapeutic agent and in various other biotechnological fields.