Site-specific immobilization of cytochrome c on mesoporous silica through metal affinity adsorption to enhance activity and stability
Site-specific immobilization of cytochrome c on mesoporous silica through metal affinity adsorption to enhance activity and stability
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DOI：
10.1039/c1nj20255c
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发表时间：
2011-01-01
影响因子：
3.3
通讯作者：
Lee, Chia-Hung
中科院分区：
文献类型：
--
作者：
Cheng, Shih-Hsun;Kao, Kun-Che;Lee, Chia-Hung
我们报道了一种水热稳定性和高活性的细胞色素c(Cytc)通过金属亲和作用固定在介孔二氧化硅(SBA-15)的纳米孔道中。由于汞-硫键的亲和力很强，我们在SBA-15表面修饰了4-氨基苯基汞(APMA)基团。结果表明，含有半胱氨酸残基(Cys-102)的酵母酶分子(酵母Cytc)具有很强的吸附能力，具有高负载量、高催化活性和对水热过程和有机溶剂高度稳定的特点。为了比较金属亲和作用和传统的共价键(二硫键)对含半胱氨酸的半胱氨酸的固定作用，我们用3-巯丙基三甲氧基硅烷(MPTS)对SBA-15表面进行修饰，以进一步与半胱氨酸残基形成二硫键。半胱氨酸残基可以通过形成二硫键共价连接到硫醇修饰的SBA-15上。此外，SBA-15的硫醇基团与Cytc的血红素Fe(III)的非特异性配位可能破坏催化中心，导致Fe(III)离子的浸出。我们以前对分子模型的研究表明，通过半胱氨酸残基固定化Cytc可以提供正确的催化中心方向，活性中心可以很容易地接近底物分子。因此，我们开发了一种快速高效的方法，通过APMA配体来固定化含有半胱氨酸酶的酶，既可以保护蛋白质的折叠，又可以控制方向来优化稳定性和催化活性。
We report a hydrothermally stable and highly reactive cytochrome c (cyt c) immobilized in the nanochannels of mesoporous silica (SBA-15) through a metal affinity interaction. Due to the strong affinity of mercury-sulfuric bonds, we modified the SBA-15 surfaces with 4-aminophenylmercuric acetate (APMA) groups. As a result, an enzyme molecule (yeast cyt c) with a cysteine residue (cys-102) demonstrated strong adsorption, which provided high cyt c loading amounts, highly catalytic activity, and high stability against hydrothermal processes and organic solvents. To compare the immobilization of cysteine-containing cyt c through metal affinity interactions and a traditional covalent bond (a disulfide bond), we modified the SBA-15 surfaces with 3-mercaptopropyl-trimethoxysilane (MPTS) for further production of a disulfide bond with the cysteine residue of cyt c. The cysteine residue of the cyt c can covalently link to thiol-modified SBA-15 through the formation of a disulfide bond. In addition, a non-specific coordination from the thiol groups of SBA-15 to the heme Fe(III) of cyt c may destroy the catalytic center and cause the leaching of Fe(III) ions. Our previous studies of the molecular model have shown that the immobilization of cyt c through the cysteine residue can provide a correct orientation of the catalytic center, where the active site can easily approach the substrate molecules. Therefore, we have developed rapid and highly efficient approaches to immobilize a cysteine-containing enzyme through APMA ligands, which can both protect the protein folding and control the orientation to optimize the stability and catalytic activity.