Immobilization adjusted clock reaction in the urea-urease-H⁺ reaction system

The bell-shaped reactivity-pH curve is the fundamental reason that the temporal programmable kinetic switch in clock reactions can be obtained in bio-competitive enzymatic reactions. In this work, urease was loaded on small resin particles through ionic binding. Experimental results reveal that the immobilization not only increased the stability of the enzyme and the reproducibility of the clock reaction, but also shifted the bell-shaped activity curve to lower pHs. The latter change enables the clock reaction to occur from an initial pH of 2.3, where the free enzyme had already lost its activity. Two mechanisms explain the influence of the immobilization on the clock reaction. Immobilization modified the pH sensitive functional groups on the enzyme, shifting the activity curve to a more acidic region, and reduced diffusion alters the enzyme dynamics.