Surface-Anchored Poly(2-vinyl-4,4-dimethyl azlactone) Brushes as Templates for Enzyme Immobilization
Surface-Anchored Poly(2-vinyl-4,4-dimethyl azlactone) Brushes as Templates for Enzyme Immobilization
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DOI：
10.1021/la8024952
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发表时间：
2008-12-02
期刊：
影响因子：
3.9
通讯作者：
Gopalan, Padma
中科院分区：
文献类型：
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作者：
Cullen, Sean P.;Mandel, Ian C.;Gopalan, Padma
我们探讨了表面锚定的聚（2-乙烯基-4，4-二甲基吖内酯）（PVDMA）刷作为潜在的模板蛋白质固定。刷生长使用原子转移自由基聚合从表面锚定的引发剂和其特征在于由椭圆偏振，原子力显微镜，和X-射线光电子能谱的组合。核糖核酸酶A作为一种模型酶固定化，通过氮内酯的亲核攻击的胺基赖氨酸位于蛋白质。RNase A的表面密度从5 μ m到50 μ m呈线性增加。对于50 nm厚的聚（2-乙烯基-4，4-二甲基吖内酯）刷，结合7.5 μ g/cm（2）的RNA酶A。RNase A固定化的动力学和热力学，相对于表面密度的活性，以及pH和温度的依赖性进行了研究。结合动力学的类朗缪尔模型表明，动力学受RNA酶A的吸附速率控制，吸附速率常数k（ads）为2.8 × 10（-8）μ g（-1）s（-1）cm（3）。在固定化RNase A为1.2 μ g/cm（2）（类似于3.0个单层）时，最大相对活性接近0.95，接近游离RNase A的活性。固定化RNase A具有与游离RNase A类似的温度和pH依赖性，表明构象没有显著变化。PVDMA模板扩展到其他生物技术相关的酶，如脱氧核糖核酸酶1，葡萄糖氧化酶，葡糖淀粉酶和胰蛋白酶，具有比通过其他手段固定的酶更高或相当的相对活性。PVDMA刷提供了一种有效的途径，通过开环的吖内酯，而不需要激活或预处理，同时保持高的相对活性的结合酶的蛋白质。
We explored surface-anchored poly(2-vinyl-4,4-dimethyl azlactone) (PVDMA) brushes as potential templates for protein immobilization. The brushes were grown using atom transfer radical polymerization from surface-anchored initiators and characterized by a combination of ellipsometry, atomic force microscopy, and X-ray photoelectron spectroscopy. RNase A was immobilized as a model enzyme through the nucleophilic attack of azlactone by the amine groups in the lysines located in the protein. The surface density of RNase A increased linearly from 5 to 50 urn. For 50 nm thick poly(2-vinyl-4,4-dimethyl azlactone) brushes, 7.5 mu g/cm(2) of RNase A was bound. The kinetics and thermodynamics of RNase A immobilization, the activity relative to surface density, and the pH and temperature dependence were examined. A Langmuir-like model for binding kinetics indicates that the kinetics are controlled by the rate of adsorption of RNase A and has an adsorption rate constant, k(ads), of 2.8 x 10(-8) mu g(-1) s(-1) cm(3). A maximum relative activity of similar to 0.95, which is near the activity of free RNase A, was reached at 1.2 mu g/cm(2) (similar to 3.0 monolayers) of immobilized RNase A. The immobilized RNase A had a similar temperature and pH dependence as free RNase A, indicating no significant change in conformation. The PVDMA template was extended to other biotechnologically relevant enzymes, such as deoxyribonuclease 1, glucose oxidase, glucoamylase, and trypsin, with relative activities higher than or comparable to those of enzymes immobilized by other means. PVDMA brushes offer an efficient route to immobilize proteins via the ring opening of azlactone without the need for activation or pretreatment while retaining high relative activities of the bound enzymes.