초록
<P>Optimization and scaling up of the Baeyer-Villiger oxidation of 3,3,5-trimethyl-cyclohexanone to trimethyl-ε-caprolactones (CHLs) were studied to demonstrate this technology on a 100 L pilot plant scale. The reaction was catalyzed by a cyclohexanone monooxygenase from <I>Thermocrispum municipale</I> that utilizes the costly redox cofactor nicotinamide adenine dinucleotide phosphate (reduced form), which was regenerated by a glucose dehydrogenase (GDH). As a first stage, different cyclohexanone monooxygenase formulations were tested: cell-free extract, whole cells, fermentation broth, and sonicated fermentation broth. Using broth resulted in the highest yield (63%) and required the least biocatalyst preparation effort. Two commercial glucose dehydrogenases (GDH-105 and GDH-01) were evaluated, resulting in similar performances. Substrate dosing rates and biocatalyst loadings were optimized. On a 30 mL scale, the best conditions were found when 30 mM h<SUP>-1</SUP> dosing rate, 10% (v/v) cyclohexanone monooxygenase broth, and 0.05% (v/v) of glucose dehydrogenase (GDH-01) liquid enzyme formulation were applied. These same conditions (with oxygen instead of air) were applied on a 1 L scale with 92% conversion, achieving a specific activity of 13.3 U g<SUB>cell wet weight (cww)</SUB><SUP>-1</SUP>, a space time yield of 3.4 g<SUB>CHL</SUB> L<SUP>-1</SUP> h<SUP>-1</SUP>, and a biocatalyst yield of 0.83 g<SUB>CHL</SUB> g<SUB>cww</SUB><SUP>-1</SUP>. A final 100 L demonstration was performed in a pilot plant facility. After 9 h, the reaction reached 85% conversion, 12.8 U g<SUB>cww</SUB><SUP>-1</SUP>, a space time yield of 2.7 g L<SUP>-1</SUP> h<SUP>-1</SUP>, and a biocatalyst yield of 0.60 g<SUB>CHL</SUB> g<SUB>cww</SUB><SUP>-1</SUP>. The extraction of product resulted in 2.58 kg of isolated final product. The overall isolated CHL yield was 76% (distal lactone 47% and proximal lactone 53%).</P><BR>[FIG OMISSION]</BR>