Enzyme immobilization in porous silicon: Quantitative analysis of the kinetic parameters for glutathione-S-transferases
Enzyme immobilization in porous silicon: Quantitative analysis of the kinetic parameters for glutathione-S-transferases
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DOI：
10.1021/ac0486185
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发表时间：
2005-04-01
影响因子：
7.4
通讯作者：
Miller, BL
中科院分区：
文献类型：
--
作者：
DeLouise, LA;Miller, BL
多孔硅基质是用于构建生物传感器的有吸引力的材料，并且还可用于生产固定化酶生物反应器。在努力获得一个定量的了解固定化对酶活性的影响，我们比较了活性的谷胱甘肽-S-转移酶固定在电化学蚀刻的多孔硅膜（类似于6.5 μ m厚）的酶在溶液中。通过改变谷胱甘肽浓度同时保持1-cbloro 2，4-二硝基苯的固定饱和浓度进行动力学测量。反应动力学遵循稳态平衡行为。游离酶在溶液中的比活性类似于固定化酶的4倍高，我们测得固定化酶的表观K '（GSH）（m）值为1.0 +/-0.3。最大流速V '（max）与固定化酶浓度成线性关系，但其幅度比溶液中低约20倍。结果表明，大约25%的酶以非活性构象或受阻取向与催化位点结合。最后，水化和暴露于变性剂对固定化酶活性的影响。
Porous silicon matrixes are attractive materials for the construction of biosensors and may also have utility for the production of immobilized enzyme bioreactors. In an effort to gain a quantitative understanding of the effects of immobilization on enzyme activity, we compared the activity of glutathione-S-transferase immobilized in electrochemically etched porous silicon films (similar to 6.5 mu m thick) with the enzyme in solution. Kinetic measurements were made by varying the glutathione concentration while maintaining a fixed saturating concentration of 1-cbloro2,4-dinitrobenzene. The reaction kinetics follow steady-state equilibrium behavior. The specific activity of the free enzyme in solution is similar to 4x higher than the immobilized enzyme, for which we measured an apparent K '(GSH)(m) value of 1.0 +/- 0.3. The maximum velocity, V '(max), is linearly proportional to immobilized enzyme concentration, but the magnitude is similar to 20 times lower than that in solution. Results suggest similar to 25% of the enzyme is bound with the catalytic site in an inactive conformation or in a hindered orientation. Finally, the effects of hydration and exposure to denaturants on the immobilized enzyme activity are presented.