title: Purification and characterization of an extracellular ribonuclease from a Bacillus sp. RNS3 (KX966412).
abstract: Ribonucleases (RNases) catalyze the degradation of ribonucleic acid (RNA) into smaller nucleotides. RNases display angiogenic, neurotoxic, antitumor and immunosuppressive properties. In the present study, an extracellular RNase was successfully purified to homogeneity from a Bacillus sp. RNS3 (KX966412) by salting out at 0-50% ammonium sulphate saturation followed by the gel permeation (Sephadex G-100) chromatography. The multistep purification resulted in 10.4 fold purification of RNase with a yield of 3.12%. The activity of the purified RNase was found to be 2.02U/mg protein. The purified RNase was monomeric with a molecular weight of 66kDa. It exhibited Michalis-Menten kinetics parameters Kcat7.92min-1and Km0.12mg/mL. The antiproliferative activity of the purified RNase was tested against an established Hep-2C (HeLa derived) cancer cell line in vitro. The purified RNase reduced the viability of the Hep-2C cells significantly with an IC50value of 3.53μg/mL. The haemolytic activity of purified RNase was also evaluated and unfortunately, it showed a strong haemolytic activity towards human RBCs.
authors.affiliation: Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla, 171005, India.
authors.forename: Gupta
authors.initials: G
authors.lastname: Shruti
authors.affiliation: Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla, 171005, India.
authors.forename: Singh
authors.initials: S
authors.lastname: Sukhdev
authors.affiliation: Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla, 171005, India. Electronic address: kanwarss2000@yahoo.com.
authors.forename: Kanwar Shamsher
authors.initials: KS
authors.lastname: Singh
mesh_terms: D000818:Animals; D001407:Bacillus / Q000166:cytology* / Q000201:enzymology*; D002417:Cattle; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D005110:Extracellular Space / Q000201:enzymology*; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D012260:Ribonucleases / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology* / Q000633:toxicity
chemical_list: D012260:Ribonucleases
keywords: Antiproliferative activity; Bacillus sp.; Haemolytic activity; Michalis-Menten kinetics; Purification; RNase