Stability enhancement of an O2-tolerant NAD+-reducing [NiFe]-hydrogenase by a combination of immobilisation and chemical modification

• Immobilisation of soluble hydrogenase from Ralstonia eutropha by adsorptive and covalent attachment with high yields and high residual activity.
• Enhancement of enzyme half-life in aqueous medium at optimum conditions from 5 to 32 h by combination of immobilisation and chemical modification.
• Mutlifold enhancement of stability under conditions relevant to technical application.

The oxygen-tolerant, NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 is a promising catalyst for cofactor regeneration in enzyme-catalysed reduction processes. The technical use of the isolated enzyme, however, is limited by its relatively low stability under operational conditions such as agitation, elevated temperature or addition of co-solvents. The maximum half-life at a reaction temperature of 35 °C and pH 8.0 was only 5.3 h. In order to enhance the stability of the enzyme, it was immobilised onto the anionic resin Amberlite™ FPA54. At an immobilisation yield of 93.4% for adsorptive and 100% for covalent attachment, corresponding activities of 48.9 and 39.3%, respectively, were obtained. Covalent binding always yielded superior stabilisation. At elevated temperature and under agitation, stabilisation was further increased by modification of the covalently bound SH with methoxy-poly(ethylene) glycol (mPEG). A comparable effect was not achieved when SH modification was performed before immobilisation. In stationary aqueous solution, half-lives of up to 161 h at 25 °C and 32 h at 35 °C were obtained. In presence of the technically relevant co-solvents DMSO, DMF, 2-propanol and [EMIM][EtSO4] half-lives of 14–29 h can now be achieved.

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