Delouise Lisa A, Miller Benjamin L
Department of Dermatology and the Center for Future Health, University of Rochester Medical Center, Rochester, New York 14642, USA.
Anal Chem. 2005 Apr 1;77(7):1950-6. doi: 10.1021/ac0486185.
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 (approximately 6.5 microm thick) with the enzyme in solution. Kinetic measurements were made by varying the glutathione concentration while maintaining a fixed saturating concentration of 1-chloro-2,4-dinitrobenzene. The reaction kinetics follow steady-state equilibrium behavior. The specific activity of the free enzyme in solution is approximately 4x higher than the immobilized enzyme, for which we measured an apparent K'(m)(GSH) value of 1.0 +/- 0.3. The maximum velocity, V'(max), is linearly proportional to immobilized enzyme concentration, but the magnitude is approximately 20 times lower than that in solution. Results suggest approximately 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.
多孔硅基质是构建生物传感器的有吸引力的材料，也可能对固定化酶生物反应器的生产有用。为了定量了解固定化对酶活性的影响，我们将固定在电化学蚀刻多孔硅膜（约6.5微米厚）中的谷胱甘肽 - S - 转移酶的活性与溶液中的酶活性进行了比较。通过改变谷胱甘肽浓度同时保持1 - 氯 - 2,4 - 二硝基苯的固定饱和浓度来进行动力学测量。反应动力学遵循稳态平衡行为。溶液中游离酶的比活性比固定化酶高约4倍，我们测得固定化酶的表观K'(m)(GSH)值为1.0 +/- 0.3。最大速度V'(max)与固定化酶浓度呈线性比例，但幅度比溶液中的低约20倍。结果表明，约25%的酶以无活性构象或受阻取向与催化位点结合。最后，介绍了水合作用和暴露于变性剂对固定化酶活性的影响。
