Reversible, selective immobilization of nuclease P₁ from a crude enzyme solution on a weak base anion resin activated by polyethylenimine

Abstract The reversible immobilization of crude nuclease P₁ fermented by Penicllium citrinum and used without further purification was performed via adsorption on a weak base anion resin activated by polyethylenimine (PEI). The immobilization conditions, including PEI concentration, the amount of support, the immobilization time, and the reusability of the PEI-activated resin were investigated. The results have shown that the PEI-activated resin has the capability of selectively adsorbing nuclease P₁. Subsequently, the functional properties of the immobilized nuclease P₁ were studied and compared to those of the free enzyme. The apparent K_m value for immobilized nuclease P ₁ on the activated resin (15.31 g L^-1) was about 4.41-fold higher than that of the free enzyme (3.47 g L^-1), and the apparent V_max value of the immobilized enzyme (530 U g^-1) was about 3.9-fold less than that of the free enzyme (2082 U mL^-1). The optimum temperature was observed to be 70 C, 15 C higher than that of the free enzyme. The optimum pH was the same for both free and immobilized nuclease P₁ (pH 5.0). The apparent activation energies (E_a) of the free and immobilized nuclease P₁ were 163.09 kJ mol^-1 and 156.32 kJ mol^-1, respectively, implying that the catalytic efficiency of the immobilized enzyme was restricted by mass-transfer rather than kinetic limitations.