Pressure-induced covalent immobilization of enzymes onto solid surface

Here in, we demonstrate a rapid method for covalent immobilization of enzymes onto activated polystyrene microtiter plate by application of pressure. Thus, when HRP, taken in an activated well of a polystyrene microtiter plate, is subjected to an optimum pressure of 2.0 × 105 Pa for an optimum time of 25 min in a closed chamber, it shows more than 5-fold increase in absorbance value compared to control experiment carried out without applying pressure. Pressure-induced immobilized HRP shows better thermal and storage stability with respect to free enzyme. Reusability study shows that immobilized enzyme retained almost full activity after three successive uses; however activity falls to half after ten cycles of repeated use. The Michaelis–Menten constant (Km) and maximum reaction velocity (Vmax) for pressure-induced immobilized HRP are found to be 55.3 μM and 0.957 mM/min, respectively, whereas for free enzyme the corresponding values are 34.7 μM and 1.089 mM/min. The method is found to be equally effective for immobilization of other enzymes including glucose oxidase, alkaline phosphatase and invertase. As no thermal incubation is required, pressure-induced immobilization could be used for immobilization of heat sensitive biomolecules useful for preparation of biosensors, biochips and protein-microarrays.