Process Optimization for the Production and Purification of an Extracellular Ribonuclease from a Soil Bacterial Isolate RNS3 Using One-Factor-At-a-Time (OFAT) Approach

Abstract

Ribonuclease (RNase; EC 3.1.2.6) hydrolyzes the phosphodiester linkage(s) of ribonucleic
acid. Bacteria are the most preferred sources of RNase due to their broad biochemical diversity. The present
study involved bacterium isolation, process optimization and purification of RNase from soil sample. Out of 14
bacterial isolates, one isolate (RNS3) was selected based on relatively higher extracellular RNase activity in the
culture broth. The physicochemical parameters attempted to optimize the extracellular RNase production by the
selected bacterial isolate RNS3 included incubation temperature, time of incubation, carbon & nitrogen sources,
pH of the broth and salt (NaCl) concentration in the broth, and supplementation of glucose and peptone,
respectively to the broth showed enhanced RNase production. Use of RNA as a substrate (10 μg/ml) to the
reaction mixture and 45 minutes of incubation enhanced the RNase activity. A partially alkaline pH (8.5) was
suitable for maximum enzyme production by the bacterial isolate RNS3. The extracellular RNase was purified by
successive salting out with ammonium sulfate, dialysis and gel permeation on Sephadex G-50 column. The SDS/
PAGE showed two protein bands having molecular weight of the ~28kDa and ~35kDa. It reflected the dimeric
nature of enzyme having molecular weight of the ~65kDa.