Electrosynthesis and electroanalysis using Clostridium sporogenes

Abstract

A polarographic approach was developed for studying the biochemical physiology and biotechnological utility of the proteolytic anaerobe Clostridium sporogenes. Methyl viologen-mediated reductions were assessed in both analytical and preparative modes, using a dropping mercury electrode and a mercury pool electrode, respectively. Using these assays, we observed an active MV-linked NAD(P) reductase in toluene-permeabilised cells of this organism. Since this enzyme could be of use in the regeneration of pyridine nucleotide cofactors used in enzymatic biotransformations, the kinetics and stability of the enzymatic activity were determined. Reduced MV could also be oxidised by exogenous D- or L-proline in both intact and toluene-permeabilised cells, via the rudimentary electron transport chain and proline reductase enzyme possessed by this organism. The polarographic approach provided a novel, continuous assay for proline reductase activity. The proline reductase activity was greatest in the mid-to-late exponential phase of growth in batch culture. The reductive carboxylation of acetyl phosphate to yield pyruvate was also studied in toluene-permeabilised cells, and the coulombic yield calculated. Whilst acetyl phosphate was reduced stoichiometrically, pyruvate formation accounted for only some 10–15% of the charge passed.