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Stability enhancement of an O2-tolerant NAD+-reducing [NiFe]-hydrogenase by a combination of immobilisation and chemical modification
- Herr, Nicole, Ratzka, Juliane, Lauterbach, Lars, Lenz, Oliver, Ansorge-Schumacher, Marion B.
- Journal of Molecular Catalysis. B, Enzymatic 2013 v.97 pp. 169-174
- Cupriavidus necator, agitation, aqueous solutions, catalysts, catalytic activity, dimethyl sulfoxide, enzyme stability, ethylene, ferredoxin hydrogenase, half life, isopropyl alcohol, pH, temperature
- 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 5.3h. 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 161h at 25°C and 32h at 35°C were obtained. In presence of the technically relevant co-solvents DMSO, DMF, 2-propanol and [EMIM][EtSO4] half-lives of 14–29h can now be achieved.