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

Abstract

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.

Keywords: Bacillus megaterium; cultivation; ion-exchange chromatography; purification; ribonuclease; screening..