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)-(-)-(alpha)-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)-(-)-(alpha)-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 degrees 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 degrees C, whereas for immobilized ones, it was at 60 degrees 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 degrees C in optimum temperature, and the activation energy was 41% lower than the activation energy of free (S)-aminotransferase.
Martin
Abraham R
AR
Department of Chemical Engineering, Michigan Technological University, Houghton, MI 49931, USA. armartin@mtu.edu
Shonnard
David
D
Pannuri
Sachin
S
Kamat
Sanjay
S
D000098:Acetophenones / Q000378:metabolism*; D000464:Alginates / Q000737:chemistry; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D020723:Glucuronic Acid / Q000737:chemistry; D006603:Hexuronic Acids / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology / Q000379:methods*; D007700:Kinetics; D010316:Particle Size; D010627:Phenethylamines / Q000378:metabolism; D000637:Transaminases / Q000737:chemistry / Q000378:metabolism*
D000098:Acetophenones; D000464:Alginates; D004800:Enzymes, Immobilized; D006603:Hexuronic Acids; D010627:Phenethylamines; D020723:Glucuronic Acid; D000637:Transaminases; C033198:1-phenethylamine; C038699:acetophenone