Xiong Ya-Hong, Liu Jian-Zhong, Song Hai-Yan
Biotechnology Research Center and The Key Laboratory of Gene Engineering of Ministry of Education, Zhongshan University, Guangzhou 510275, P.R.China.
Appl Biochem Biotechnol. 2005 Jun;125(3):201-10. doi: 10.1385/abab:125:3:201.
A new extracellular ribonuclease (RNase) from a mutant of Aspergillus niger, named A. niger SA-13-20 RNase, was purified to homogeneity by (NH4)2SO4 fractionation (50-85%), DEAE-cellulose anion-exchange chromatography, ultrafiltration and Sephacryl HR-200 chromatography. The enzyme was purified up to 54.4-fold with a final yield of 24.5%. There were differences in the molecular weight, pI value and some physico-chemical properties between A. niger SA-13-20 RNase and that from the parent strain. The enzyme is monomeric and its molecular weight and isoelectric point were 40.1 kDa and 5.3, respectively. The N-terminal amino acid sequence of A. niger SA-13-20 RNase was TIDTYSSDSP. The optimum pH, temperature and buffer concentration for the enzymatic reaction were 3.5, 65 degrees C, and 0.175 M, respectively. Metal ions, such as K+, NH4+, Mg2+, and Ca2+ at the concentration of 1.0 mM had a slight activation effect on the enzyme activity and (NH4)2SO4 activated the enzyme significantly. The enzyme was stable at pH lower than 8.5 and was easy to inactivate in strong alkali solution.
从黑曲霉突变体中分离得到一种新的细胞外核糖核酸酶（RNase），命名为黑曲霉SA-13-20 RNase，通过硫酸铵分级沉淀（50%-85%）、DEAE-纤维素阴离子交换色谱、超滤和Sephacryl HR-200色谱法将其纯化至同质。该酶纯化了54.4倍，最终产率为24.5%。黑曲霉SA-13-20 RNase与其亲本菌株的RNase在分子量、pI值和一些理化性质上存在差异。该酶为单体，分子量和等电点分别为40.1 kDa和5.3。黑曲霉SA-13-20 RNase的N端氨基酸序列为TIDTYSSDSP。酶促反应的最适pH、温度和缓冲液浓度分别为3.5、65℃和0.175 M。浓度为1.0 mM的金属离子如K+、NH4+、Mg2+和Ca2+对酶活性有轻微的激活作用，硫酸铵对该酶有显著的激活作用。该酶在pH低于8.5时稳定，在强碱溶液中容易失活。
