title: Purification, kinetic and thermodynamic studies of a new ribonuclease from a mutant of Aspergillus niger.
abstract: Ribonuclease was purified from Aspergillus niger SA-13-20 to homogeneity level by using (NH(4))(2)SO(4) precipitation, DEAE-cellulose anion-exchange chromatography, ultrafiltration and Sephacryl HR-200 chromatography. The molecular weight and isoelectric point of the enzyme was 40.1kDa and 5.3, respectively. The pH- and temperature-dependent kinetic parameters were determined. The RNase showed the strongest affinity with RNA as the substrate, and the highest catalytic efficiency for hydrolysis of the substrate at pH 3.5 and 65 degrees C. It exhibited Michaelis-Menten Kinetics with k(cat) of 118.1s(-1) and K(m) of 57.0 microg ml(-1), respectively. Thermodynamic parameters for catalysis and thermal denaturation were also determined. Activation energy (E(a)) for catalysis of A. niger SA-13-20 RNase was 50.31 kJ mol(-1) and free energy (DeltaG(#)), enthalpy (DeltaH(#)) and entropy (DeltaS(#)) of activation for catalysis of the enzyme at 65 degrees C were 69.76, 47.50 and -65.83 Jmol(-1)K(-1), respectively. Activation energy (E(a,d)) for denaturation of the enzyme was 200.53 kJ mol(-1) and free energy (DeltaG(d)(#)), enthalpy (DeltaH(d)(#)) and entropy (DeltaS(d)(#)) of activation for denaturation of the enzyme at 45 degrees C were 79.18 kJ mol(-1), 197.88 and 373.09 Jmol(-1)K(-1), respectively.
authors.affiliation: Biotechnology Research Center and Key Laboratory of Gene Engineering of Ministry of Education, Zhongshan University, Guangzhou 510275, China.
authors.forename: Ya-Hong
authors.initials: YH
authors.lastname: Xiong
authors.forename: Jian-Zhong
authors.initials: JZ
authors.lastname: Liu
authors.forename: Hai-Yan
authors.initials: HY
authors.lastname: Song
authors.forename: Liang-Nian
authors.initials: LN
authors.lastname: Ji
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics*; D002384:Catalysis; D004789:Enzyme Activation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009154:Mutation; D012260:Ribonucleases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D013816:Thermodynamics
chemical_list: D012260:Ribonucleases