Gao En-Feng, Kang Kyung Lhi, Kim Jeong Hee
J Nanosci Nanotechnol. 2014 Jun;14(6):4626-31. doi: 10.1166/jnn.2014.8203.
Retaining biological activity of a protein after immobilization is an important issue and many studies reported to enhance the activity of proteins after immobilization. We recently developed a new immobilization method of enzyme using active-site protection and minimization of the cross-links between enzyme and surface with a DNA polymerase as a model system. In this study, we extended the new method to an enzyme with a small mono-substrate using alkaline phosphatase (AP) as another model system. A condition to apply the new method is that masking agents, in this case its own substrate needs to stay at the active-site of the enzyme to be immobilized in order to protect the active-site during the harsh immobilization process. This could be achieved by removal of essential divalent ion, Zn2+ that is required for full enzyme activity of AP from the masking solution while active-site of AP was protected with p-nitrophenyl phosphate (pNPP). Approximately 40% of the solution-phase activity was acquired with active-site protected immobilized AP. In addition to protection active-site of AP, the number of immobilization links was kinetically controlled. When the mole fraction of the activated carboxyl group of the linker molecule in self-assembled monolayer (SAM) of 12-mercaptododecanoic acid and 6-mercapto-1-ethanol was varied, 10% of 12-mercaptododecanoic acid gave the maximum enzyme activity. Approximately 51% increase in enzyme activity of the active-site protected AP was observed compared to that of the unprotected group. It was shown that the concept of active-site protection and kinetic control of the number of covalent immobilization bonds can be extended to enzymes with small mono-substrates. It opens the possibility of further extension of the new methods of active-site protection and kinetic control of immobilization bond to important enzymes used in research and industrial fields.
固定化后保持蛋白质的生物活性是一个重要问题，许多研究报道了提高固定化后蛋白质活性的方法。我们最近开发了一种新的酶固定化方法，以DNA聚合酶为模型系统，利用活性位点保护和最小化酶与表面之间的交联。在本研究中，我们将该新方法扩展到以碱性磷酸酶（AP）为另一个模型系统的单底物小分子酶。应用该新方法的一个条件是，掩蔽剂，在这种情况下是其自身的底物，需要保留在待固定化酶的活性位点上，以便在苛刻的固定化过程中保护活性位点。这可以通过从掩蔽溶液中去除AP全酶活性所需的必需二价离子Zn2+来实现，同时用对硝基苯磷酸酯（pNPP）保护AP的活性位点。活性位点受保护的固定化AP获得了约40%的溶液相活性。除了保护AP的活性位点外，还对固定化连接的数量进行了动力学控制。当改变12-巯基十二烷酸和6-巯基-1-乙醇自组装单层（SAM）中连接分子活化羧基的摩尔分数时，12-巯基十二烷酸的10%给出了最大酶活性。与未保护组相比，活性位点受保护的AP的酶活性提高了约51%。结果表明，活性位点保护和共价固定化键数量的动力学控制概念可以扩展到单底物小分子酶。这为将活性位点保护和固定化键动力学控制的新方法进一步扩展到研究和工业领域中使用的重要酶开辟了可能性。
