Liposomal Encapsulation of Yeast Alcohol Dehydrogenase with Cofactor for Stabilization of the Enzyme Structure and Activity

Yeast alcohol dehydrogenase (YADH) with its cofactor nicotinamide adenine dinucleotide (NAD+) could be stably encapsulated in liposomes composed of POPC (1‐palmitoyl‐2‐oleoyl‐ sn‐glycero‐3‐ phosphocholine). The YADH‐ and NAD+‐containing liposomes (YADH‐NADL) were 100 nm in mean diameter. The liposomal YADH and NAD+ concentrations were 2.3 mg/mL and 3.9 mM, respectively. A synergistic effect of the liposomal encapsulation and the presence of NAD+ was examined on the thermal stability of YADH at 45 and 50 °C. The enzyme stability of the YADH‐NADL was compared to the stabilities of the liposomal YADH (YADHL) containing 3.3 mg/mL YADH without NAD+ as well as the free YADH with and without NAD+. Free YADH was increasingly deactivated during its incubation at 45 °C for 2 h with decrease of the enzyme concentration from 3.3 to 0.01 mg/mL because of the dissociation of tetrameric YADH into its subunits. At that temperature, the coexistence of free NAD+ at 3.9 mM improved the stability of free YADH at 2.3 mg/mL through forming their thermostable complex, although the stabilization effect of NAD+ was lowered at 50 °C. The turbidity measurements for the above free YADH solution with and without NAD+ revealed that the change in the enzyme tertiary structure was much more pronounced at 50 °C than at 45 °C even in the presence of NAD+. This suggests that YADH was readily deactivated in free solution due to a decrease in the inherent affinity of YADH with NAD+. On the other hand, both liposomal enzyme systems, YADH‐NADL and YADHL, showed stabilities at both 45 and 50 °C much higher than those of the above free enzyme systems, YADH/NAD+ and YADH. These results imply that the liposome membranes stabilized the enzyme tertiary and thus quaternary structures. Furthermore, the enzyme activity of the YADH‐NADL showed a stability higher than that of the YADHL with a more remarkable effect of NAD+ at 50 °C than at 45 °C. This was considered to be because even at 50 °C the stabilization effect of lipid membranes on the tertiary and quaternary structures of the liposomal YADH allowed the enzyme to form its thermostable complex with NAD+ in liposomes.