Site- directed mutagenesis and characteristics analysis of new staphylococcal nuclease( Nuc2)
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摘要: 本研究选取了F60、A62、T102和Y135这4个可能的关键位点,采用定点突变的方法成功构建了5个Nuc2的单点和多点突变体。体外表达并纯化这些核酸酶,对其活性、耐热性和二级结构进行测定。结果表明:经方差分析（p<0.05）,T102M和F60A/A62L/T102M/Y135V的核酸酶活性与野生型相比较显著下降,并且F60A/A62L/T102M/Y135V的酶活性基本丧失;T102M和F60A/A62L的耐热性明显弱于野生型;T102M和F60V/A62L的二级结构中α-螺旋结构的含量明显低于野生型,F60A/A62L/T102M/Y135V的二级结构中包含有无规卷曲。因此,推断T102是维持Nuc2核酸酶活性、耐热性和二级结构稳定的一个关键位点;F60、A62、T102和Y135及其与邻近氨基酸之间的相互作用对维持Nuc2的性质稳定起着重要作用。Abstract: Four amino acid residues( F60,A62,T102 and Y135) were selected and five single and multiple mutants of Nuc2 were constructed. After in vitro expression and purification,enzyme activity,thermostability and secondary structure of these nucleases were determined. The results indicated that the enzyme activity of T102 M and F60 A /A62 L / T102 M / Y135 V had significant differences with wild- type( WT) after variance analysis( p < 0.05),and the nuclease activity of F60 A / A62 L / T102 M / Y135 V was totally disabled. The thermostability of T102 M and F60 A / A62 L was much weaker than that of WT. Besides,the α- helical content of T102 M and F60 V / A62 L in their secondary structure was much lower than that of WT,and it was showed from the CD spectra that the secondary structure of F60 A / A62 L / T102 M / Y135 V was in a state of random coil. Therefore,T102 is a pivotal site to maintain the nuclease activity,thermostability and secondary structure of Nuc2.And the interaction among F60,A62,T102 and Y135 is also significant to maintain its stability of secondary structure.
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