一种核糖核酸酶构象稳定性的研究

Study on the Conformational Stability of a Ribonuclease

Aspergillus niger SA-13-20核糖核酸酶是从突变株A.niger SA-13-20分泌的胞外酶中分离出的一种新的核糖核酸酶。采用紫外光谱、荧光光谱和红外光谱研究了A.niger SA-13-20核糖核酸酶在不同pH值条件下的构象稳定性。紫外光谱和荧光光谱结果均表明该酶蛋白在酸性和弱碱性pH值下构象较稳定,当pH值高于9.6时构象不稳定;红外光谱结合去卷积和曲线拟合技术对蛋白质酰胺Ⅰ带的测定和处理结果表明,在室温下,pH 5.0时该酶蛋白二级结构中α-螺旋、β-折叠、转角和无规结构所占的成分分别为13.28%、42.30%、26.48%和17.95%。测得该酶的热解链温度Tm、解链熵变ΔSm及解链焓变ΔHm分别为70.1℃、644 J.mol-1.K-1及22.1 kJ.mol-1,表明该酶属于耐热能力较强的核糖核酸酶。研究结果有助于揭示该酶结构与功能的关系,推动其在科研和生产等方面的应用。

Aspergillus niger SA-13-20 is a new ribonuclease（RNase） isolated from the extracellular enzymes secreted by a mutant named A.niger SA-13-20.The conformational stability of A.niger SA-13-20 ribonuclease at different pH was studied by ultraviolet（UV）,fluorescence and Fourier transform infrared（FT-IR） spectroscopic techniques.UV and fluorescence spectra showed that the conformation of this protein was stable at acid and weak alkali pH,but unstable at pH above 9.6.The secondary structure of the RNase was determined based on the analysis of amide Ⅰ band of FT-IR spectra by using deconvolution and curve-fitting technology.The results showed that the percentages of α-helix,β-sheet,turn angle and unordered structure were 13.28%,42.30%,26.48% and 17.95% at pH 5.0 at room temperature,respectively.Melting temperature（Tm）,melting entropy（ΔSm） and melting enthalpy（ΔHm） of this RNase were 70.1 ℃,644 J·mol-1·K-1 and 22.1 kJ·mol-1,respectively,which indicated that the RNase belonged to a ribonuclease with higher heat tolerance.These results are helpful to understand the relationship between the structure and the function of this enzyme,and propel its application in the scientific research and industrial production.