Enhanced Thermal Stability of NADH/NAD⁺ through Tethering to Silica Nanoparticles

The poor thermal stability of the cofactor β-nicotinamide adenine dinucleotide (NAD⁺) in industrial settings can be a limiting factor in the expansion of biosynthetic approaches to chemical production. In this work, we report that the half-life of SiNP-tethered NAD⁺ when stored in solution at 37 °C, and subsequently catalyzed by glycerol-3-phosphate dehydrogenase from E. coli (EcG3PD) at ambient temperature, is increased 11-fold to over 500 h, compared with 34.5 h for free NAD⁺. Similarly, the half-life for the degradation of the tethered NAD⁺ stored at 100 °C was 5 h compared with 0.3 h for free NAD⁺ corresponding to a 15-fold enhancement in the retention of activity of tethered NAD⁺. Kinetic analysis indicates that activity loss of NAD⁺ is similar to that of the normal hydrolysis mechanism, with the difference likely being due to steric effects and access to labile bonds. We also demonstrated that the retention of reactivity of heat-treated EcG3PD adsorbed to the surface NAD⁺-functionalized particles was improved compared to freely diffusing EcG3PD and NAD⁺ at ambient temperature, consistent with our previous work which showed a surface-localized enzyme/substrate interaction resulting in a concentrating effect. These results demonstrate the great potential for the long-term use of tethered NAD⁺ and enzymes, even at high operational temperatures, in biocatalytic applications.