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 (NADI) 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 degrees 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 degrees 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 NADI was lowered at 50 degrees 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 degrees C than at 45 degrees 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 degrees 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 degrees C than at 45 degrees C. This was considered to be because even at 50 degrees 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.