Regulating Flavor Compound Synthesis and Cofactor Recycling in Heterogeneous Enzymatic Reactions by Mixtures of Bacterial Cell-Free Extracts

Enzymatic cofactor recycling and generation of acetic acid, 3-methylbutanal, and 3-methylbutanol were evaluated using a mixture of cell-free extracts of Gluconobacter oxydans and Streptococcus lactis var. maltigenes. Ethanol was used as a substrate by cell-free extracts of G. oxydans to produce acetic acid and to generate reduced nicotinamide adenine dinucleotide from nicotinamide adenine dinucleotide. Leucine was converted by S. lactis var. maltigenes cell-free extracts to 3-methylbutanal and 3-methylbutanol and to regenerate nicotinamide adenine dinucleotides. Optimum pH to form 3-methylbutanal by the combined cellfree extracts was about 6.8 and that for 3-methylbutanol was 6.4. At 30C, 3-methylbutanol reached a maximum of 73 ppm after 30 h but declined slightly at 48 h. Concentrations of 3-methylbutanol increased gradually over 48 h to a maximum of 56 ppm. Rates of production of these compounds at 12.8C were about half that at 32C. Concentrations of 3-methylbutanol were increased to about those of 3-methylbutanal by adjusting the concentrations of substrate, nicotinamide adenine dinucleotide, cell-free extract, and pH so as to produce large amounts of acetic acid and reduced nicotinamide adenine dinucleotide.