A FRET Approach to Detect Paraoxon among Organophosphate Pesticides Using a Fluorescent Biosensor

The development of faster, sensitive and real-time methods for detecting organophosphate (OP) pesticides is of utmost priority in the in situ monitoring of these widespread compounds. Research on enzyme-based biosensors is increasing, and a promising candidate as a bioreceptor is the thermostable enzyme esterase-2 from Alicyclobacillus acidocaldarius (EST2), with a lipase-like Ser–His–Asp catalytic triad with a high affinity for OPs. This study aimed to evaluate the applicability of Förster resonance energy transfer (FRET) as a sensitive and reliable method to quantify OPs at environmentally relevant concentrations. For this purpose, the previously developed IAEDANS-labelled EST2-S35C mutant was used, in which tryptophan and IAEDANS fluorophores are the donor and the acceptor, respectively. Fluorometric measurements showed linearity with increased EST2-S35C concentrations. No significant interference was observed in the FRET measurements due to changes in the pH of the medium or the addition of other organic components (glucose, ascorbic acid or yeast extract). Fluorescence quenching due to the presence of paraoxon was observed at concentrations as low as 2 nM, which are considered harmful for the ecosystem. These results pave the way for further experiments encompassing more complex matrices.

Metabolic Efficiency of Sugar Co-Metabolism and Phenol Degradation in Alicyclobacillus acidocaldarius for Improved Lignocellulose Processing

Substrate availability plays a key role in dictating metabolic strategies. Most microorganisms consume carbon/energy sources in a sequential, preferential order. The presented study investigates metabolic strategies of Alicyclobacillus acidocaldarius, a thermoacidophilic bacterium that has been shown to co-utilize glucose and xylose, as well as degrade phenolic compounds. An existing metabolic model was expanded to include phenol degradation and was analyzed with both metabolic pathway and constraint-based analysis methods. Elementary flux mode analysis was used in conjunction with resource allocation theory to investigate ecologically optimal metabolic pathways for different carbon substrate combinations. Additionally, a dynamic version of flux balance analysis was used to generate time-resolved simulations of growth on phenol and xylose. Results showed that availability of xylose along with glucose did not predict enhanced growth efficiency beyond that of glucose alone, but did predict some differences in pathway utilization and byproduct profiles. In contrast, addition of phenol as a co-substrate with xylose predicted lower growth efficiency. Dynamic simulations predicted co-consumption of xylose and phenol in a similar pattern as previously reported experiments. Altogether, this work serves as a case study for combining both elementary flux mode and flux balance analyses to probe unique metabolic features, and also demonstrates the versatility of A. acidocaldarius for lignocellulosic biomass processing applications.

Highly Sensitive Detection of Chemically Modified Thio-Organophosphates by an Enzymatic Biosensing Device: An Automated Robotic Approach

Pesticides represent some of the most common man-made chemicals in the world. Despite their unquestionable utility in the agricultural field and in the prevention of pest infestation in public areas of cities, pesticides and their biotransformation products are toxic to the environment and hazardous to human health. Esterase-based biosensors represent a viable alternative to the expensive and time-consuming systems currently used for their detection. In this work, we used the esterase-2 from Alicyclobacillus acidocaldarius as bioreceptor for a biosensing device based on an automated robotic approach. Coupling the robotic system with a fluorescence inhibition assay, in only 30 s of enzymatic assay, we accomplished the detection limit of 10 pmol for 11 chemically oxidized thio-organophosphates in solution. In addition, we observed differences in the shape of the inhibition curves determined measuring the decrease of esterase-2 residual activity over time. These differences could be used for the characterization and identification of thio-organophosphate pesticides, leading to a pseudo fingerprinting for each of these compounds. This research represents a starting point to develop technologies for automated screening of toxic compounds in samples from industrial sectors, such as the food industry, and for environmental monitoring.

Crystal Structures of Putative Flavin Dependent Monooxygenase from Alicyclobacillus Acidocaldarius

Flavin dependent monooxygenases catalyze various reactions to play a key role in biological processes, such as catabolism, detoxification, and biosynthesis. Group D flavin dependent monooxygenases are enzymes with an Acyl-CoA dehydrogenase (ACAD) fold and use Flavin adenine dinucleotide (FAD) or Flavin mononucleotide (FMN) as a cofactor. In this research, crystal structures of Alicyclobacillus acidocaldarius protein formerly annotated as an ACAD were determined in Apo and FAD bound state. Although our structure showed high structural similarity to other ACADs, close comparison of substrate binding pocket and phylogenetic analysis showed that this protein is more closely related to other bacterial group D flavin dependent monooxygenases, such as DszC (sulfoxidase) and DnmZ and Kijd3 (nitrososynthases).

Linear enzyme cascade for the production of (–)-iso-isopulegol

Biocatalysis has developed enormously in the last decade and now offers solutions for the sustainable production of chiral and highly functionalised asset molecules. Products generated by enzymatic transformations are already being used in the food, feed, chemical, pharmaceutical and cosmetic industry, and the accessible compound panoply is expected to expand even further. In particular, the combination of stereo-selective enzymes in linear cascade reactions is an elegant strategy toward enantiomeric pure compounds, as it reduces the number of isolation and purification steps and avoids accumulation of potentially unstable intermediates. Here, we present the set-up of an enzyme cascade to selectively convert citral to (–)-iso-isopulegol by combining an ene reductase and a squalene hopene cyclase. In the initial reaction step, the ene reductase YqjM from Bacillus subtilis selectively transforms citral to (S)-citronellal, which is subsequently cyclised exclusively to (–)-iso-isopulegol by a mutant of the squalene hopene cyclase from Alicyclobacillus acidocaldarius (AacSHC). With this approach, we can convert citral to an enantiopure precursor for isomenthol derivatives.

Effective Adsorption of Patulin from Apple Juice by Using Non-Cytotoxic Heat-Inactivated Cells and Spores of Alicyclobacillus Strains

Patulin (PAT) is a major threat to many food products, especially apple and apple products, causing human health risks and economic losses. The aim of this study was to remove PAT from apple juice by using the heat-inactivated (HI) cells and spores of seven Alicyclobacillus strains under controlled conditions. The HI cells and spores of seven strains adsorbed PAT effectively, and the HI cells and spores of Alicyclobacillus acidocaldarius DSM 451 (A51) showed maximum PAT adsorption capacity of up to 12.6 μg/g by HI cells and 11.8 μg/g by HI spores at 30 °C and pH 4.0 for 24 h. Moreover, the PAT adsorption process followed the pseudo-first order kinetic model and the Freundlich isotherm model; thermodynamic parameters revealed that PAT adsorption is a spontaneous exothermic physisorption process. The results also indicated that PAT adsorption is strain-specific. The HI cells and spores of Alicyclobacillus strains are non-cytotoxic, and the bioadsorption of PAT did not affect the quality of the juice. Furthermore, the cell wall surface plays an important role in the adsorption process.