The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the short-chain dehydrogenase/ reductase (SDR) superfamily was identified in the extremely thermophilic, halotolerant gram-negative eubacterium Thermus thermophilus HB27. The T. thermophilus ADH gene (adh Tt ) was heterologously overexpressed in Escherichia coli, and the protein (ADH Tt ) was purified to homogeneity and characterized. ADH Tt is a tetrameric enzyme consisting of identical 26,961-Da subunits composed of 256 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to ϳ73°C and a 30-min half-inactivation temperature of ϳ90°C, as well as good tolerance to common organic solvents. ADH Tt has a strict requirement for NAD(H) as the coenzyme, a preference for reduction of aromatic ketones and ␣-keto esters, and poor activity on aromatic alcohols and aldehydes. This thermophilic enzyme catalyzes the following reactions with Prelog specificity: the reduction of acetophenone, 2,2,2-trifluoroacetophenone, ␣-tetralone, and ␣-methyl and ␣-ethyl benzoylformates to (S)-(؊)-1-phenylethanol (>99% enantiomeric excess [ee]), (R)-␣-(trifluoromethyl)benzyl alcohol (93% ee), (S)-␣-tetralol (>99% ee), methyl (R)-(؊)-mandelate (92% ee), and ethyl (R)-(؊)-mandelate (95% ee), respectively, by way of an efficient in situ NADH-recycling system involving 2-propanol and a second thermophilic ADH. This study further supports the critical role of the D37 residue in discriminating NAD(H) from NADP(H) in members of the SDR superfamily.Alcohol dehydrogenases (ADHs) are of interest for the synthesis of the (S) or (R) enantiomers of alcohols from prochiral ketones (11). Since their early application in asymmetric synthesis using horse liver and yeast ADHs (13) and Thermoanaerobacter brockii ADH (ADH Tb ) (15), screening efforts have been directed at various species of microorganisms, which has resulted in new ADHs that have distinctive substrate specificity, good efficiency, and high enantioselectivity. Representative examples of enzymes from mesophilic microorganisms are the NADP-dependent (R)-specific ADH from Lactobacillus brevis (RADH Lb ), which is active on aryl ketones and whose crystal structure has recently been solved (29), the NAD-dependent (S)-specific 1-phenylethanol dehydrogenase from the denitrifying bacterium strain EbN1 (PED), which was characterized and crystallized (10), and the NAD-dependent ADH from Leifsonia sp. strain S749 (ADH Ls ), which was found to be active on (R)-sec alcohols, aryl ketones, aldehydes, and keto esters and whose gene has recently been cloned for protein expression in Escherichia coli (12). These enzymes are homotetrameric and belong to the short-chain dehydrogenase/ reductase (SDR) superfamily (14), which is characterized by ϳ250-residue subunits, a Gly motif in the coenzyme-binding regions, and a catalytic triad formed by the highly conserved residues Tyr, Lys, and Ser, to which an Asn residue has been added based on the proposal of Filling et al. (2), which was...

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Self-assembling proteins forming amyloid fibrils are promising candidates for the fabrication of biomaterials, due to the chemical and mechanical stability of their structures. Among potential applications, their use as platforms for enzyme immobilization is rapidly gathering attention. In this work, we demonstrate that the production of the enzyme glutathione-S-transferase (GST) fused to the class I hydrophobin Vmh2 from Pleurotus ostreatus represents an invaluable tool for the development of self-immobilizing enzymes useful for high throughput analyses. The proposed immobilization strategy is versatile since it can be applied, in principle, to every recombinant protein able to refold from Escherichia coli inclusion bodies. A GST based biosensor has been developed to quantify toxic compounds, such as the pesticides molinate and captan, in aqueous environmental samples. The main advantages of this sensor include simplicity and speed of preparation, high sensitivity, reusability, and accuracy. Biotechnol. Bioeng. 2017;114: 46-52. © 2016 Wiley Periodicals, Inc.

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Steroids are a class of hormones improperly used in livestock as growth-promoting agents. Due to their high risk for human health, the European Union (EU) has strictly forbidden the administration of all natural and synthetic steroid hormones to food-producing animals, and the development of new rapid detection methods are greatly encouraged. This work reports a novel fluorescence polarization assay, ready to use, capable of detecting 17β-estradiol directly in milk samples with a low limit of detection of <10 pmol. It is based on the coupling of monospecific antibodies against 17β-estradiol and fluorophores, capable of modulating the fluorescence polarization emission on the basis of the specific binding of antibodies to fluorescence-labeled 17β-estradiol derivative. The successful detection of 17β-estradiol has disclosed the development of an efficient method, easily extensible to any food matrix and having the potential to become a milestone in food quality and safety.

The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh2 gene was heterologously overexpressed in Escherichia coli, and the resulting protein (SaADH2) was purified to homogeneity and both biochemically and structurally characterized. The crystal structure of the SaADH2 NADH-bound form reveals that the enzyme is a tetramer consisting of identical 27,024-Da subunits, each composed of 255 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 80 °C and a 30-min half-inactivation temperature of ∼88 °C. It also shows good tolerance to common organic solvents and a strict requirement for NAD(H) as the coenzyme. SaADH2 displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and α-ketoesters, but is poorly active on aliphatic, cyclic and aromatic alcohols, showing no activity on aldehydes. Interestingly, the enzyme catalyses the asymmetric reduction of benzil to (R)-benzoin with both excellent conversion (98 %) and optical purity (98 %) by way of an efficient in situ NADH-recycling system involving a second thermophilic ADH. The crystal structure of the binary complex SaADH2-NADH, determined at 1.75 Å resolution, reveals details of the active site providing hints on the structural basis of the enzyme enantioselectivity.