Stabilization of formate dehydrogenase from Candida boidinii through liposome-assisted complexation with cofactors

The activity of formate dehydrogenase from Candida boidinii (CbFDH) was stabilized at 60 °C through interaction with its cofactor (NAD+ or NADH) in the liposomal aqueous phase. The activity of 8.0 μM free CbFDH without liposomal encapsulation progressively decreased at 60 °C in Tris buffer of pH 8.5 following the first-order kinetics. Free CbFDH without cofactor showed the half-life of enzyme activity t1/2 of 3.5 min, while t1/2 increased to 22 and 236 min with 15 mM NAD+ and 4.5 mM NADH, respectively. Turbidity measurements revealed that the free CbFDH and CbFDH/NAD+ became their aggregate-prone states at 60 °C. For the liposomal CbFDH/cofactor systems, the cofactor-induced stabilization of CbFDH was also observed. Typically, the liposomal 6.0 μM CbFDH/4.3 mM NAD+ showed significantly large t1/2 of 36 min compared to the corresponding free CbFDH/NAD+ (t1/2 = 8.9 min). Mixing of free CbFDH/NAD+ with the enzyme-free liposomes resulted in the insufficient interaction between liposomes and CbFDH showing t1/2 of 14 min. The results obtained demonstrate that the lipid membrane assists the formation of highly thermostable enzyme–cofactor complex through stabilizing the structure of the liposome-encapsulated CbFDH.