Characterization of free and immobilized (S)-aminotransferase for acetophenone production
Enzyme immobilization often improves process economics, but changes in kinetic properties may also occur. The immobilization of a recombinant thermostable (S)-aminotransferase was made by entrapment on calcium alginate-3% (w/v)-and tested with (S)-(-)-(α)-methylbenzylamine for acetophenone production. The best immobilization results were obtained for beads of concentration of 10 mg of spray-dried cells (containing recombinant (S)-aminotransferase) per milliliter of sodium alginate bead. As a result of immobilization, the properties of immobilized spray-dried cells differed from the properties of free spray-dried cells. V m for the immobilized enzyme was between 0.08 and 0.09 mM/min, while the V m for free enzyme was 0.06-0.07 mM/min. K m values differed for immobilized and free spray-dried cells by a factor of between 3 and 5 for (S)-(-)-(α)- methylbenzylamine (6.05 mM for immobilized, 1.78 mM for free) and pyruvate (5.0 mM for immobilized, 1.01 mM for free) at 55°C. Optimum pH values were 7.7 and 8.1 for the free spray-dried cells and the immobilized formulation, respectively. The maximum activity for free spray-dried cells was measured at 55°C, whereas for immobilized ones, it was at 60°C. Activation and deactivation energy values for free spray-dried cells were 15.13 and 41.73 kcal/mol, while those for immobilized spray-dried cells were 8.86 and 48.88 kcal/mol, respectively. Overall, as a result of immobilization, an increase in V m was measured for the (S)-aminotransferase by 28 to 33% with respect to free enzyme; K m increased by a factor of three- to fivefold and had a shift of 5°C in optimum temperature, and the activation energy was 41% lower than the activation energy of free (S)-aminotransferase. © 2007 Springer-Verlag.
Applied Microbiology and Biotechnology
Characterization of free and immobilized (S)-aminotransferase for acetophenone production.
Applied Microbiology and Biotechnology,
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