Xiong Ya-Hong, Liu Jian-Zhong, Song Hai-Yan, Ji Liang-Nian
Biotechnology Research Center and Key Laboratory of Gene Engineering of Ministry of Education, Zhongshan University, Guangzhou 510275, China.
J Biotechnol. 2005 Oct 10;119(4):348-56. doi: 10.1016/j.jbiotec.2005.04.008.
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.
通过硫酸铵沉淀、DEAE-纤维素阴离子交换色谱、超滤和Sephacryl HR-200色谱，从黑曲霉SA-13-20中纯化核糖核酸酶至均一水平。该酶的分子量和等电点分别为40.1kDa和5.3。测定了其pH和温度依赖性动力学参数。该核糖核酸酶对以RNA为底物表现出最强的亲和力，在pH 3.5和65℃时对底物水解的催化效率最高。它呈现米氏动力学，催化常数（k(cat)）为118.1s(-1)，米氏常数（K(m)）为57.0μg ml(-1)。还测定了催化和热变性的热力学参数。黑曲霉SA-13-20核糖核酸酶催化的活化能（E(a)）为50.31kJ mol(-1)，在65℃时该酶催化的活化自由能（ΔG(#)）、活化焓（ΔH(#)）和活化熵（ΔS(#)）分别为69.76、47.50和-65.83Jmol(-1)K(-1)。该酶变性的活化能（E(a,d)）为200.53kJ mol(-1)，在45℃时该酶变性的活化自由能（ΔG(d)(#)）、活化焓（ΔH(d)(#)）和活化熵（ΔS(d)(#)）分别为79.18kJ mol(-1)、197.88和373.09Jmol(-1)K(-1)。
