title: Aldehyde oxidoreductase as a biocatalyst: Reductions of vanillic acid.
abstract: Aldehyde oxidoreductase (carboxylic acid reductase) catalyzes the Mg(2+), ATP and NADPH dependent reduction of carboxylic acids to their corresponding aldehydes. The identification of the gene from Nocardia sp. NRRL 5646 and its expression in E. coli BL21-CodonPlus(®)(DE3)-RP/pHAT305 provided an avenue to develop a biocatalyst for reduction of carboxylic acids. In addition to aromatic acids, the recombinant carboxylic acid reductase also accepts several aliphatic mono, di and tri carboxylic acids as substrates. A recently identified Nocardia sp., phosphopantetheinyl transferase gene (npt) enhanced the activity of carboxylic acid reductase. Coexpression of car and npt in E. coli BL21-CodonPlus(®)(DE3)-RP/pPV2.83 resulted in a purified recombinant carboxylic acid reductase with improved specific activity of 2.2U/mg protein. The utility of the recombinant carboxylic acid reductase as a biocatalyst has been demonstrated using vanillic acid as substrate. E. coli BL21-CodonPlus(®)(DE3)-RP/pHAT305 expressing Car reduced 50% of vanillic acid to vanillin in 10h. E. coli BL21-CodonPlus(®)(DE3)-RP/pPV2.83 resting cells expressing Car and Npt reduced 90% of vanillic acid to vanillin in 6h. Enhanced, in vivo cofactor NADPH regeneration by glucose dehydrogenase (gdh) was accomplished using E. coli BL21-CodonPlus(®)(DE3)-RP/pPV2.85, that carried car, npt, and gdh. Resting cell reactions using E. coli BL21-CodonPlus(®)(DE3)-RP/pPV2.85 with in situ product removal by XAD-2 resin efficiently reduced 5g/L of vanillic and benzoic acids within 2h.
authors.affiliation: Center for Biocatalysis and Bioprocessing, 2501 Crosspark Road, Suite C100, Oakdale Research Park, University of Iowa, Iowa City, IA 52242-5000, United States.
authors.forename: Padmesh
authors.initials: P
authors.lastname: Venkitasubramanian
authors.forename: Lacy
authors.initials: L
authors.lastname: Daniels
authors.forename: Shuvendu
authors.initials: S
authors.lastname: Das
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Lamm
authors.forename: John P N
authors.initials: JP
authors.lastname: Rosazza