Extracellular Halophilic Ribonuclease from a Halotolerant Pseudomonas sp. : Purification and Characterization

Abstract

A halotolerant bacterium, Pseudomonas sp. No. 3241, isolated from Thai fish sauce obtained in Suratthani province, produced an extracellular ribonuclease when cultivated aerobically without NaCl in Sehgal and Gibbons complex medium (SGC medium). Ribonuclease was purified by ethanol precipitation, Sephadex G-150 gel filtration and DEAE Toyopearl 650M anion-exchange chromatography. The purity and molecular weight were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The result showed that the molecular weight of ribonuclease from halotolerant Pseudomonas sp. No. 3241 was 61,000 daltons. Purified ribonuclease was optimum at pH 10.0 and at temperature of 40^oC. Purified ribonuclease was stable between pH 6.0 and 10.0 and at temperatures between 30 and 40^oC. This enzyme had marked halophilic enzyme properties that required an optimum NaCl concentration of 3.0 M (18%).