Process development for the electroenzymatic synthesis of (R)-methylphenylsulfoxide by use of a 3-dimensional electrode.

The electrogeneration of hydrogen peroxide via reduction of dissolved oxygen was carried out in a three-dimensional electrochemical cell using graphite grains as cathode material and combined with the enantioselective oxidation of thioanisole to (R)-methylphenylsulfoxide catalyzed by Chloroperoxidase from Caldariomyces fumago. The relevant parameters for process development (e.g., conductivity of the biotransformation medium) were identified and optimized, leading to an efficient electroenzymatic process with a space-time yield of 104 g x L(-1) x d(-1). The isolated product was produced on gram scale (1.2 g, purity >98%, ee > 98.5%).

Indirect electrochemical reduction of nicotinamide coenzymes.

Nicotinamide coenzymes nicotinamide adenine dinucleotide (NAD(+)) and nicotinamide adenine dinucleotide phosphate (NADP(+)) were electrochemically reduced to NADH and NADPH, respectively. As direct reduction of nicotinamide coenzymes leads to inactive by-products, an indirect method using (pentamethylcyclopentadienyl-2,2'-bipyridine aqua) rhodium (III) as the mediator, was applied. A phosphate buffer solution, pH 8, with 1-10 mM NAD(P)(+) and 2.5-200 microM mediator, was pumped through a glassy carbon packed bed cathode. Virtually all the NAD(P)(+) was reduced to NAD(P)H in the cell. No sign of mediator loss due to side-reactions was detected though the mediator molecules shuttled hundreds of times between the oxidised and the reduced form. Adsorption of mediator molecules on the surface of the carbon cathode was found to be important for the reduction process. Due to strong adsorption, only minute amounts of mediator were consumed.

Process strategies to enhance pyruvate production with recombinant Escherichia coli: from repetitive fed-batch to in situ product recovery with fully integrated electrodialysis.

Using the pyruvate production strain Escherichia coli YYC202 ldhA::Kan different process alternatives are studied with the aim of preventing potential product inhibition by appropriate product separation. This strain is completely blocked in its ability to convert pyruvate into acetyl-CoA or acetate, resulting in acetate auxotrophy during growth in glucose minimal medium. Continuous experiments with cell retention, repetitive fed-batch, and an in situ product recovery (ISPR) process with fully integrated electrodialysis were tested. Although the continuous approach achieved a high volumetric productivity (QP) of 110 g L(-1) d(-1), this approach was not pursued because of long-term production strain instabilities. The highest pyruvate/glucose molar yield of up to 1.78 mol mol(-1) together with high QP 145 g L(-1) d(-1) and high pyruvate titers was achieved by the repetitive fed-batch approach. To separate pyruvate from fermentation broth a fully integrated continuous process was developed. In this process electrodialysis was used as a separation unit. Under optimum conditions a (calculated) final pyruvate titer of >900 mmol L(-1) (79 g L(-1)) was achieved.