Immobilization of Ene Reductase in Polyvinyl Alcohol Hydrogel.

In this study, ene reductase (ER) was entrapped in polyvinyl alcohol hydrogel, adsorbed on montmorillonite and immobilized covalently on glutaraldehyde activated 3-aminopropyl-functionalized silica gel. Although protein recovery yields were at least 85% for adsorption and covalent immobilization, only the encapsulated ER showed activity. The activity of free and entrapped ER preparations was measured by following NADPH-dependent reduction of 2-cyclohexen-1-one. The both protein recovery and activity recovery yields were calculated as 100% when 1 mg protein was used for immobilization. The both free and entrapped ER preparations showed the same optimum pH and temperature as 7.0 and 30 °C, respectively. The entrapped ER showed 34.4-fold more thermal stability than that of the free ER at 30 °C. Michaelis-Menten constant and maximum velocity values were 0.25 mM and 1.2 U/mg protein, respectively for the free ER towards 2-cyclohexen-1-one. The corresponding values were 1.5 mM and 0.9 U/mg protein for the entrapped ER. The results of time-course reduction of 2-cyclohexen-1-one showed that the entrapped ER catalyzed the reaction as effectively as the free ER. The entrapped ER remained 85% of its initial activity after 10 reused cycles.