Orientation specific positioning of organophosphorus hydrolase on solid interfaces for biosensor applications.
Orientation specific positioning of organophosphorus hydrolase on solid interfaces for biosensor applications.
复制标题
用于生物传感器应用的有机磷水解酶在固体界面上的方向特异性定位。
DOI：
10.1021/la9007526
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Simonian,AleksandrL
中科院分区：
文献类型：
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作者：
Reeves,TonyE;Paliwal,Sheetal;Wales,MelindaE;Wild,JamesR;Simonian,AleksandrL
蛋白质在固体界面上的固定化是其在生物传感、纯化、分离、去污等技术中成功应用的一个关键方面。虽然固定化可以提高蛋白质的长期稳定性和操作稳定性，但这通常是以固定酶的催化活性的重大损失为代价的。共价连接方法利用氨基酸侧链上的活性基团。当蛋白质通过这些侧链键固定在表面或基质中时，溶剂暴露侧链在酶分子表面上的分布通常会导致取向的集合。根据附着机制和产生的取向，进出活性部位可能受到限制。本研究描述了一种方法，用于设计和实现一种定向的附着在表面等离子体共振传感器表面的酶。该酶，有机磷水解酶，进行结构分析，以确定表面驻留作为修饰的候选，以优化活性位点的可及性，从而提高检测的灵敏度。选择酶二聚体活性位点表面的单一表面赖氨酸进行消除，从而允许催化剂在首选方向上固定。酶的动力学评价表明，表面赖氨酸-丙氨酸变体对神经毒素底物，对氧磷和demeton-S保留了80%的野生型活性。固定化后，尽管传感器表面的酶覆盖率降低了17%，但携带该变体的表面被确定为更活跃。
Protein immobilization on solid interfaces is a crucial aspect of their successful application in technologies such as biosensing, purification, separation, decontamination, etc. Although immobilization can improve the long-term and operational stability of proteins, this is often at the cost of significant losses in the catalytic activity of the tethered enzyme. Covalent attachment methods take advantage of reactive groups on the amino acid side chains. The distribution of the solvent exposed side chains on an enzyme’s molecular surface often results in an ensemble of orientations when the protein is immobilized on a surface or in a matrix through these side chain linkages. Depending on the attachment mechanism and resulting orientation, access to and from the active site could be restricted. This study describes a methodology for the design and implementation of an orientation specific attachment of an enzyme to a surface plasmon resonance sensor surface. The enzyme, organophosphorus hydrolase, was structurally analyzed to identify surface resides as candidates for modification to optimize active site accessibility and, thus, sensitivity of detection. A single surface lysine on the active site face of the enzyme dimer was selected for elimination, thus allowing for the immobilization of the catalyst in the preferred orientation. Kinetic evaluation of the enzymes determined that the surface lysine-to-alanine variant retained 80% of the wild-type activity with the neurotoxin substrates, paraoxon and demeton-S. After immobilization, surfaces bearing the variant were determined to be more active even though the enzyme coverage on the sensor surface was reduced by 17%.