Abstract
Glycerol dehydrogenase (GlyDH) which oxidizes glycerol to the value-added chemical, 1,3-dihydroxyacetone, is of interest due to the oversupply of glycerol as a by-product of the biodiesel industry. To exploit the enzymatic oxidation of glycerol industrially, silica coated magnetic Fe3O4 nanoparticles were prepared and then activated with an amino-silane reagent for covalent immobilization of GlyDH via a glutaraldehyde linkage. At the optimal glutaraldehyde concentration of 0.05% (v/v), an enzyme loading of up to 57.5 mg/g-nanoparticles was achieved with 81.1% of the original activity retained. Reaction kinetic analysis indicated that the immobilized GlyDH had almost the same Michaelis-Menten constants for both NAD+ and glycerol as the free GlyDH did. However, after immobilization the turnover number kcat of the GlyDH decreased from 164 s−1 to 113 s−1, and the reaction was 1.3-fold less sensitive to inhibition by DHA, which could compensate the decrease in kcat. The immobilized GlyDH was also less sensitive to changes in pH and temperature, and showed a 5.3-fold improvement in thermal stability at 50°C. Furthermore, excellent reusability was observed such that 10 cycles of re-use only led to 9% loss of enzyme activity.
Acknowledgements
The authors thank support from the National Natural Science Foundation of China (Grant No. 20728607, 20706054, 20976180), 973 Program (2009CB724705) and 863 Project (2008AA10Z302).
Declaration of interest:
The authors report no declarations of interest.