ABSTRACT
Latest solutions in biotechnologies and biosensing targeted cold-active extremozymes. Analysis of acetaldehyde as a relevant quality indicator of wine is one example of application that could benefit from using low-temperatures operating catalysts. In search of novel aldehyde dehydrogenases (ALDH) with high stability and activity at low temperatures, the recombinant S2-ALDH from the Antarctic Flavobacterium PL002 was obtained by cloning and expression in Escherichia coli BL21(DE3). Structural and phylogenetic analyses revealed strong protein similarities (95%) with psychrophilic homologs, conserved active residues and structural elements conferring enzyme flexibility. Arrhenius plot revealed a conformational shift at 30 °C, favoring catalysis (low activation energy) at lower temperatures. In addition to a broad substrate specificity with preference for acetaldehyde (Km = 1.88 mM), this enzyme showed a high tolerance for ethanol (15%) and several salts and chelators (an advantage for wine analysis), while being sensitive to mercury (I50 = 1.21 µM). The neutral optimal pH (7.5) and the stability up to 40 °C and after lyophilization represent major assets for developing S2-ALDH-based sensors. An enzymatic electrochemical assay was developed for acetaldehyde detection in wines with proven accuracy in comparison with the reference spectrophotometric method, thus evidencing the potential of S2-ALDH as effective biocatalyst for industry and biosensing.
Subject(s)
Mercury , Wine , Acetaldehyde/metabolism , Aldehyde Dehydrogenase/metabolism , Antarctic Regions , Catalysis , Chelating Agents , Escherichia coli/genetics , Escherichia coli/metabolism , Ethanol/metabolism , Flavobacterium/genetics , Flavobacterium/metabolism , Phylogeny , SaltsABSTRACT
Herein we present highly stable ferromagnetic nickel nanowires modified with streptavidin (NiNW-STR). This versatile functionalized nanomaterial works as an excellent biosensing platform for the immobilization of a wide range of biotinylated molecules. Moreover, these NWs can be employed in magnetic-based assays. Different proofs-of-concept such as streptavidin-biotin assays and capture of single and double stranded DNA were successfully carried out, corroborating NiNW-STR usefulness. Moreover, repeatability and stability studies were also effectively performed.
