Carboxylic acid reductases: Structure, catalytic requirements, and applications in biotechnology.

Biocatalysts have been gaining extra attention in recent decades due to their industrial-relevance properties, which may hasten the transition to a cleaner environment. Carboxylic acid reductases (CARs) are large, multi-domain proteins that can catalyze the reduction of carboxylic acids to corresponding aldehydes, with the presence of adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH). This biocatalytic reaction is of great interest due to the abundance of carboxylic acids in nature and the ability of CAR to convert carboxylic acids to a wide range of aldehydes essentially needed as end products such as vanillin or reaction intermediates for several compounds production such as alcohols, alkanes, and amines. This modular enzyme, found in bacteria and fungi, demands an activation via post-translational modification by the phosphopantetheinyl transferase (PPTase). Recent advances in the characterization and structural studies of CARs revealed valuable information about the dynamics, mechanisms, and unique features of the enzymes. In this comprehensive review, we summarize the previous findings on the phylogeny, structural and mechanistic insight of the domains, post-translational modification requirement, strategies for the cofactors regeneration, the extensively broad aldehyde-related industrial application properties of CARs, as well as their recent immobilization approaches.

Characterization of Carboxylic Acid Reductase from Mycobacterium phlei Immobilized onto Seplite LX120.

A multi-domain oxidoreductase, carboxylic acid reductase (CAR), can catalyze the one-step reduction of carboxylic acid to aldehyde. This study aimed to immobilize bacterial CAR from a moderate thermophile Mycobacterium phlei (MpCAR). It was the first work reported on immobilizing bacterial CAR onto a polymeric support, Seplite LX120, via simple adsorption. Immobilization time and protein load were optimized for MpCAR immobilization. The immobilized MpCAR showed optimal activity at 60 °C and pH 9. It was stable over a wide range of temperatures (10 to 100 °C) and pHs (4-11), retaining more than 50% of its activity. The immobilized MpCAR also showed stability in polar solvents. The adsorption of MpCAR onto the support was confirmed by Scanning Electron Microscopy (SEM), Fourier-Transform Infrared (FTIR) spectroscopy, and Brunauer-Emmett-Teller (BET) analysis. The immobilized MpCAR could be stored for up to 6 weeks at 4 °C and 3 weeks at 25 °C. Immobilized MpCAR showed great operational stability, as 59.68% of its activity was preserved after 10 assay cycles. The immobilized MpCAR could also convert approximately 2.6 mM of benzoic acid to benzaldehyde at 60 °C. The successfully immobilized MpCAR on Seplite LX120 exhibited improved properties that benefit green industrial processes.

Cyanobacterial aldehyde deformylating oxygenase: Structure, function, and potential in biofuels production.

The conversion of aldehydes to valuable alkanes via cyanobacterial aldehyde deformylating oxygenase is of great interest. The availability of fossil reserves that keep on decreasing due to human exploitation is worrying, and even more troubling is the combustion emission from the fuel, which contributes to the environmental crisis and health issues. Hence, it is crucial to use a renewable and eco-friendly alternative that yields compound with the closest features as conventional petroleum-based fuel, and that can be used in biofuels production. Cyanobacterial aldehyde deformylating oxygenase (ADO) is a metal-dependent enzyme with an α-helical structure that contains di­iron at the active site. The substrate enters the active site of every ADO through a hydrophobic channel. This enzyme exhibits catalytic activity toward converting Cn aldehyde to Cn-1 alkane and formate as a co-product. These cyanobacterial enzymes are small and easy to manipulate. Currently, ADOs are broadly studied and engineered for improving their enzymatic activity and substrate specificity for better alkane production. This review provides a summary of recent progress in the study of the structure and function of ADO, structural-based engineering of the enzyme, and highlight its potential in producing biofuels.