Immunobilization of Luciferase From the Firefly Luciola mingrelica - Catalytic Properties and Stability of the Immobilized Enzyme

Firefly (Luciola mingrelica ) luciferase (Photinus luciferin 4-monooxygenase (ATP-hydrolysing); Photinus luciferin: oxygen 4-oxidoreductase (decarboxylating, ATP-hydrolysing), EC 1.13.12.7) has been immobilized on albumin and polyacrylamide gel, on AH-, CH- and CNBr-Sepharose 4B as well as on Ultragel, Ultradex and cellophane film activated by cyanogen bromide. Only immobilization on cyanogen bromide-activated polysaccharide carriers resulted in highly active immobilized luciferase. Kinetic properties of immobilized luciferase hardly differed from those of the soluble enzyme. The inactivation rate constants of soluble and immobilized luciferase were measured at pH 5.5-9.0 and 25 degree C as well as at pH 7.8 and 20-40 degree C. The Delta H super(not equal) and Delta S super(not equal) values for inactivation of soluble and immobilized luciferases were obtained. A 1000-fold stabilization effect was noted for the luciferase immobilized on CNBr-Sepharose 4B at pH 7.5 and 25 degree C. A stabilization mechanism for the immobilized luciferase is discussed.