title: Highly efficient Michael-type addition of acetaldehyde to β-nitrostyrenes by whole resting cells of Escherichia coli expressing 4-oxalocrotonate tautomerase.
abstract: A novel whole cell system based on recombinantly expressed 4-oxalocrotonate tautomerase (4-OT) was developed and shown to be an effective biocatalyst for the asymmetric Michael addition of acetaldehyde to β-nitrostyrenes. Optimal ratio of substrates (2mM β-nitrostyrenes and 20mM acetaldehyde) and biocatalyst of 5 g of cell dry weight of biocatalyst per liter was determined. Through further bioprocess improvement by sequential addition of substrate 10mM nitrostyrene biotransformation was achieved within 150 min. Excellent enantioselectivity (>99% ee) and product yields of up to 60% were obtained with β-nitrostyrene substrate. The biotransformation product, 4-nitro-3-phenyl-butanal, was isolated from aqueous media and further transformed into the corresponding amino alcohol. The biocatalyst exhibited lower reaction rates with p-Cl-, o-Cl- and p-F-β-nitrostyrenes with product yields of 38%, 51%, 31% and ee values of 84%, 88% and 94% respectively. The importance of the terminal proline of 4-OT was confirmed by two proline enriched variants and homology modeling.
authors.affiliation: Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, P.O. Box No. 23, 11010 Belgrade, Serbia.
authors.forename: Tanja
authors.initials: T
authors.lastname: Narancic
authors.forename: Jelena
authors.initials: J
authors.lastname: Radivojevic
authors.forename: Predrag
authors.initials: P
authors.lastname: Jovanovic
authors.forename: Djordje
authors.initials: D
authors.lastname: Francuski
authors.forename: Miljan
authors.initials: M
authors.lastname: Bigovic
authors.forename: Veselin
authors.initials: V
authors.lastname: Maslak
authors.forename: Vladimir
authors.initials: V
authors.lastname: Savic
authors.forename: Branka
authors.initials: B
authors.lastname: Vasiljevic
authors.forename: Kevin E
authors.initials: KE
authors.lastname: O'Connor
authors.forename: Jasmina
authors.initials: J
authors.lastname: Nikodinovic-Runic
mesh_terms: D000079:Acetaldehyde / Q000378:metabolism*; D001483:Base Sequence; D055162:Biocatalysis; D001711:Biotransformation; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D007535:Isomerases / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011392:Proline / Q000378:metabolism; D013343:Styrenes / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D013343:Styrenes; C011955:beta-nitrostyrene; D011392:Proline; C021918:4-oxalocrotonate tautomerase; D007535:Isomerases; D000079:Acetaldehyde
keywords: 4-Oxalocrotonate tautomerase; Biocatalyst; Michael addition; Nitrostyrene; Whole cell