Highly Stable, Cold-Active Aldehyde Dehydrogenase from the Marine Antarctic Flavobacterium sp. PL002

Abstract

Stable aldehyde dehydrogenases (ALDH) from extremophilic microorganisms constitute efficient catalysts in biotechnologies. In search of active ALDHs at low temperatures and of these enzymes from cold-adapted microorganisms, we cloned and characterized a novel recombinant ALDH from the psychrotrophic Flavobacterium PL002 isolated from Antarctic seawater. The recombinant enzyme (F-ALDH) from this cold-adapted strain was obtained by cloning and expressing of the PL002 aldH gene (1506 bp) in Escherichia coli BL21(DE3). Phylogeny and structural analyses showed a high amino acid sequence identity (89%) with Flavobacterium frigidimaris ALDH and conservation of all active site residues. The purified F-ALDH by affinity chromatography was homotetrameric, preserving 80% activity at 4 °C for 18 days. F-ALDH used both NAD+ and NADP+ and a broad range of aliphatic and aromatic substrates, showing cofactor-dependent compensatory KM and kcat values and the highest catalytic efficiency (0.50 µM−1 s−1) for isovaleraldehyde. The enzyme was active in the 4–60 °C-temperature interval, with an optimal pH of 9.5, and a preference for NAD+-dependent reactions. Arrhenius plots of both NAD(P)+-dependent reactions indicated conformational changes occurring at 30 °C, with four(five)-fold lower activation energy at high temperatures. The high thermal stability and substrate-specific catalytic efficiency of this novel cold-active ALDH favoring aliphatic catalysis provided a promising catalyst for biotechnological and biosensing applications.

Antarctic aldehyde dehydrogenase from Flavobacterium PL002 as a potent catalyst for acetaldehyde determination in wine

“…This high stability of this cold-active enzyme to temperatures above 25 °C commonly used in industrial applications was also observed for other ALDHs originating from Antarctic habitats, such as the recently described F-ALDH from Flavobacterium PL002 39 and the homologous enzyme from the psychrotrophic marine F. frigidimaris ( Cytophaga sp ) 41 .…”

“…1 ). Substrate specificity of S2-ALDH was different from that of the aldehyde dehydrogenase from the same Antarctic microorganism which preferred isovaleraldehyde among the aliphatic aldehyde substrates 39 , and also different from that of the commercially available mesophilic aldehyde dehydrogenase from yeast 52 and human 53 . Thus, the preference for acetaldehyde and the different substrate specificity indicated S2-ALDH as a promising component of bioelectronic e-tongues, i.e.…”

“…3 C, inset) corresponded to a bi-phasic plot with a calculated activation energy shift from E a 10–30 °C = 64.77 J/K mol to E a 30–35 °C = 18.72 J/K mol, suggesting a conformational change that occurred at 30 °C. This temperature effect on enzymes’ tertiary and quaternary structure entrained a wider energy shift and a lower activation energy at low temperature in the case of S2-ALDH as compared to that of the F-ALDH from the same host (from E a 10–30 °C = 76 J/K mol to E a 30–35 °C = 19 J/K mol) that occurred at the same temperature threshold (30 °C) 39 . These data suggested similar overall intramolecular interactions involved in catalysis for the two cold-active ALDHs and similar activation energy required at higher temperatures, while a favored catalysis (lower E a ) at low temperatures in the case of S2-ALDH.…”

“…These corroborated data confirmed the utilization of the novel cold-active ALDH as an effective biocatalyst for monitoring acetaldehyde in wine, with extensive applicative potential in biosensing. To further explore this potential, immobilization of S2-ALDH will be conducted based on crosslinking with glutaraldehyde, as demonstrated for the homologous enzyme originating from the same bacterial species 39 and Ni-histidine affinity for developing enzymatic biosensors for detection of aldehydes in alcoholic beverages and other relevant industrial applications.…”

“…Alternatively, cold-active enzymes were actively screened for a wide range of applications in 32 – 36 , being active at low temperatures and presenting different substrate specificity as compared to enzymes from mesophilic and thermophilic counterparts 37 , 38 . Our group has previously reported the production and characterization of a novel cold-active recombinant aldehyde dehydrogenase from the Antarctic Flavobacterium PL002, highlighting its applicatve potential for the electrochemical detection of benzaldehyde in pharmaceutical ingredients 15 , 39 , 40 . However, to date, limited data on cold-active oxidoreductases is available for developing catalysts for industrial applications 36 , 41 .…”