The co-immobilization of ketoreductase (KRED) and glucose dehydrogenase (GDH) on highly cross-linked agarose (sepharose) was studied. Immobilization of these two enzymes was performed via affinity interaction between His-tagged enzymes (six histidine residues on the N-terminus of the protein) and agarose matrix charged with nickel (Ni2+ ions). Immobilized enzymes were applied in a semicontinuous flow reactor to convert the model substrate; α-hydroxy ketone. A series of biotransformation reactions with a substrate conversion of >95% were performed. Immobilization reduced the requirement for cofactor (NADP+) and allowed the use of higher substrate concentration in comparison with free enzymes. The immobilized system was also tested on bulky ketones and a significant enhancement in comparison with free enzymes was achieved.
Butanol production from glycerol was investigated through Clostridium pasteurianum entrapped into polyvinyl alcohol particles. Using an optimized system, batch and repeated batch fermentations with free and entrapped cells were performed, respectively. In both systems, glycerol samples of different purity were tested. In repeated batch fermentations, process time decreased from 19.5 to 2.7 hours and butanol productivity increased 6.3 times (3.08 g.L-1 .h-1) compared with the free-cell process, using pure glycerol. In the case of glycerol from biodiesel production, butanol productivity of 2.90 and 1.76 g.L-1 .h-1 was achieved from glycerol 01 and glycerol 02, respectively. No cell growth or solvent production was observed when the same glycerols were used in free-cell fermentations.
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