Department of Chemistry, Kyung Hee University, Seoul 130-701, Republic of Korea.
Anal Biochem. 2011 Dec 15;419(2):205-10. doi: 10.1016/j.ab.2011.08.022. Epub 2011 Aug 18.
A new efficient immobilization method that enables oriented immobilization of biologically active proteins was developed based on concepts of active site masking and kinetic control. Taq DNA polymerase was immobilized covalently on mixed self-assembled monolayers (SAMs) of ω-carboxylated thiol and ω-hydroxylated thiol through amide bonds between the protein and the carboxyl group in SAMs. Activity of the immobilized enzyme as large as 70% of solution-phase enzyme was achieved by masking the active site of the Taq DNA polymerase prior to the immobilization. In addition, the number of immobilization bonds was controlled by optimizing the carboxyl group concentration in the mixed monolayer. The maximum activity of immobilized Taq DNA polymerase was achieved at 5% of 12-mercaptododecanoic acid. The activity observed with protected immobilized enzyme was approximately 20 times higher than that observed with randomly immobilized enzyme. The maximum activity was acquired at a 1:1 DNA/enzyme masking ratio, immobilization pH 8.3, and within 10 min of reaction time. This concept of the active site masking and kinetic control of the number of covalent bonds between proteins and the surface can be generally applicable to a broad range of proteins to be immobilized on the solid surface with higher activity.
一种新的高效固定化方法，基于活性位点掩蔽和动力学控制的概念，实现了生物活性蛋白质的定向固定化。通过蛋白质与 SAM 中羧基之间的酰胺键，Taq DNA 聚合酶被共价固定在ω-羧基化硫醇和 ω-羟基化硫醇的混合自组装单层（SAM）上。在固定化之前，通过掩蔽 Taq DNA 聚合酶的活性位点，实现了固定化酶高达 70%的溶液相酶活性。此外，通过优化混合单层中羧基的浓度来控制固定化键的数量。在 12-巯基十二烷酸的 5%时，固定化 Taq DNA 聚合酶的最大活性达到。用受保护的固定化酶观察到的活性比随机固定化酶观察到的活性高约 20 倍。最大活性出现在 1:1 DNA/酶掩蔽比、固定化 pH8.3 和 10min 的反应时间内。这种活性位点掩蔽和动力学控制蛋白质与表面之间共价键数量的概念可以广泛应用于将各种蛋白质固定在固体表面上，以获得更高的活性。
