Recombinant Escherichia coli whole-cell biocatalysts harboring either a Baeyer-Villiger monooxygenase or ferulic acid decarboxylase were employed in organic-aqueous two-phase bioreactor systems. The feasibility of the bioproduction of water-insoluble products, viz., lauryl lactone from cyclododecanone and 4-vinyl guaiacol from ferulic acid were examined. Using hexadecane as the organic phase, 10∼16 g of lauryl lactone were produced in a 3-l bioreactor that operated in a semicontinuous mode compared to 2.4 g of product in a batch mode. For the decarboxylation of ferulic acid, a new recombinant biocatalyst, ferulic acid decarboxylase derived from Bacillus pumilus, was constructed. Selected solvents as well as other parameters for in situ recovery of vinyl guaiacol were investigated. Up to 13.8 g vinyl guaiacol (purity of 98.4%) were obtained from 25 g of ferulic acid in a 2-l working volume bioreactor by using octane as organic phase. These selected examples highlight the superiority of the two-phase biotransformations systems over the conventional batch mode.
The gene encoding an (S)-specific NAD-dependent alcohol dehydrogenase (RE-ADH) was isolated from the genomic DNA of Rhodococcus erythropolis DSM 43297. The nucleotide sequence of 1,047 bp, coding for 348 amino acids, was cloned in Escherichia coli cells and successfully expressed. The subunit molecular mass as deduced from the amino acid sequence was determined to be 36.026 kDa. The recombinant enzyme exhibited high thermostability, which facilitated its purification by heat treatment, followed by two column-chromatography steps. RE-ADH shows high similarity to several zinc-containing medium-chain alcohol dehydrogenases. All zinc ligands seem to be conserved except one of the catalytic zinc ligands, where Cys is probably substituted by Asp. A similarity of 84% with a phenylacetaldehyde reductase from Corynebacterium sp. ST-10 was determined. Biochemical properties such as thermostability and substrate specificity of the two enzymes were compared.
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