Optimization of cell harvesting and assay procedures for reductive biotransformations in obligate anaerobes

Caffeate reductase is an enoate reductase which is produced by the acetogenic, obligate anaerobe, Acetobacterium woodii. The function of the enzyme appears to be the redn. of arom. enoates, which are utilized as alternative electron acceptors to carbon dioxide, the usual electron acceptor for this organism. The substrate specificity of this enzyme seems to differ from that of other enoate reductases. Therefore, caffeate reductase may be useful for biotransformations which cannot be achieved at present. The objective of this work was to develop an assay for caffeate reductase so that the substrate range and characteristics of the enzyme could be investigated. Using cells harvested from a chemostat culture, an anaerobic harvesting procedure and an assay for caffeate reductase in whole cells were developed. An electron donor was essential for the redn. of caffeate, and for this fructose was used. The optimum buffer pH was 7.0 and the optimum caffeate concn. was 2.5 mM. At higher caffeate concns. a decrease in redn. rate was obsd. The substrate range of the enzyme appeared to be fairly wide, as cinnamaldehyde and cinnamyl alc. were reduced, as well as cinnamate, p-hydroxycinnamate and caffeate. [on SciFinder (R)]