Control of Enzyme Activity by Introduction of Molecular Recognition Moiety

摘要: Regulation of enzyme activity by biding a ligand or protein, such as allosteric enzyme and signal transduction, is common in vivo. This enzyme function is attractive from a view of application for bio reactor, DDS, and bio sensor. Some research groups developed conjugation of enzyme and stimuli responsive polymer to control enzyme activity or specific binding by stimulus such as temperature, pH, and light for affinity separation, microfluidic protein analysis and capture [1-3]. However, in order to control the enzyme activity by molecular recognition, the problem is that how to modify the surface of the enzyme to create the binding site for another ligand and how to control the activity of the enzyme when the ligand is recognized. There are some previous studies controlling enzyme activity by genetic modification [4] or antigen-antibody complex reaction [5]. These approaches utilize conformational change of enzyme with genetic modification or protein-ligand interaction. However, the reaction mechanism is complicated and is mostly difficult to know, thus we must repeat the genetic modification process and change the position of binding site by trial and error approach. The approach for making molecular recognition enzyme in previous researches also lacks versatility.Our approach to develop novel functional enzyme is that molecular recognition moiety is conjugated only near active pocket of the mutant enzyme. In this research, Cytochrome P450cam was used as enzyme and biotin with spacer arm was used as molecular recognition moiety. This enzyme regulates its activity by utilizing avidin-biotin affinity interaction as the following. In the absence of …