A Novel Nano-Sensor Based on Rhodamine-β-Isothiocyanate – Doped Silica Nanoparticle for pH Measurement

A novel and simple enzymatic method for the determination of trace cyanide in marine fish is presented. This method is based on the conversion of metal-cyanide complexes by a fungal enzyme extract containing cyanide hydratase (E.C. 4.2.1.66; CyHT) and formamidase (E.C. 3.5.1.49) into formate and ammonia. The formate produced in the sample pretreated with the fungal enzymes was measured by adding formate dehydrogenase (E.C. 1.2.1.2; FDH) and excess NAD⁺. The NADH formed accordingly was monitored at 340nm. The cyanide calibration curve was found to be linear in the range of 10–100µM, and the detection limit was 1.1µM (0.0286ppm). The proposed biotest was successfully applied to the determination of trace cyanide in a tropical marine food fish species (Russells Snapper (Lutjamus russellii)) which had been exposed to cyanide.     

CO2 fixation by [WIVO(S2C2(CN)2)2]2?: functional model for the tungsten-formate dehydrogenase of Clostridium thermoaceticum

(NEt₄)₂[W^IVO(S₂C₂(CN)₂)₂] (1), isolated by reaction of Na₂ WO₄, Na₂S₂C₂(CN)₂ (Na₂mnt) in acidified (pH5.5) aqueous medium in the presence of excess of sodium dithionite and NEt₄Br, reduces CO₂/HCO ₃ ⁻ (pH 7.5) to yield HCOO⁻ and (NEt₄)₂[W^VIO₂(S₂C₂(CN)₂)₂] (2) mimicking tungsten-formate dehydrogenase (W-FDH) activity. (1) reacts with Na₂MoO₄ in acidic medium to produce [Mo^IvO(S₂C₂(CN)₂)₂]²⁻ implicating the displacement of tungsten by molybdenum from the cofactor complex in W-FDH.     

New insights from X-ray photoelectron spectroscopy into the chemistry of covalent enzyme immobilization, with glutamate dehydrogenase (GDH) on silicon dioxide as an example

A three-step process for immobilization of glutamate dehydrogenase (GDH) on the surface of silicon dioxide has been studied by X-ray photoelectron spectroscopy (XPS). The enzyme layer was deposited on the silicon dioxide surface after first exposing the surface to 3-aminopropyltriethoxysilane (3-APTS) and reacting the silylated surface with glutaraldehyde (GA). Fine XPS analysis, performed after each step of the chemical procedure, revealed unknown details of the step-by-step construction of the enzyme layer under different experimental conditions.     

Synthesis of lactate derivatives via reductive radical addition to α-oxyacrylates

Lactate derivatives are important synthetic precursors to a variety of pharmaceutical products. Previously reported methods to prepare lactates require multiple steps or have limited scopes. Herein, we report a Ni-catalyzed reductive addition of a variety of alkyl iodides to α-oxyacrylates to afford substituted lactates. Exploring the scope of radical acceptors reveals that electron-deficient alkenes, ranging from cyclohexenone to para-caboxystyrene, undergo efficient coupling with alkyl iodides. This method represents an alternative strategy access lactate derivatives.     

Structural insights of a cellobiose dehydrogenase enzyme from the basidiomycetes fungus Termitomyces clypeatus

Filamentous fungi secrete various oxidative enzymes to degrade the glycosidic bonds of polysaccharides. Cellobiose dehydrogenase (CDH) (E.C.1.1.99.18) is one of the important lignocellulose degrading enzymes produced by various filamentous fungi. It contains two stereo specific ligand binding domains, cytochrome and dehydrogenase - one for heme and the other for flavin adenine dinucleotide (FAD) respectively. The enzyme is of commercial importance for its use in amperometric biosensor, biofuel production, lactose determination in food, bioremediation etc. Termitomyces clypeatus, an edible fungus belonging to the basidiomycetes group, is a good producer of CDH. In this paper we have analyzed the structural properties of this enzyme from T. clypeatus and identified a distinct carbohydrate binding module (CBM) which is not present in most fungi belonging to the basidiomycetes group. In addition, the dehydrogenase domain of T. clypeatus CDH exhibited the absence of cellulose binding residues which is in contrast to the dehydrogenase domains of CDH of other basidiomycetes. Sequence analysis of cytochrome domain showed that the important residues of this domain were conserved like in other fungal CDHs. Phylogenetic tree, constructed using basidiomycetes and ascomycetes CDH sequences, has shown that very surprisingly the CDH from T. clypeatus, which is classified as a basidiomycetes fungus, is clustered with the ascomycetes group. A homology model of this protein has been constructed using the CDH enzyme of ascomycetes fungus Myricoccum thermophilum as a template since it has been found to be the best match sequence with T. clypeatus CDH. We also have modelled the protein with its substrate, cellobiose, which has helped us to identify the substrate interacting residues (L354, P606, T629, R631, Y649, N732, H733 and N781) localized within its dehydrogenase domain. Our computational investigation revealed for the first time the presence of all three domains - cytochrome, dehydrogenase and CBM - in the CDH of T. clypeatus, a basidiomycetes fungus. In addition to discovering the unique structural attributes of this enzyme from T. clypeatus, our study also discusses the possible phylogenetic status of this fungus.     

Exploring the influence of conserved lysine69 on the catalytic activity of the helicobacter pylori shikimate dehydrogenase: A combined QM/MM and MD simulations

Shikimate dehydrogenase (SDH) catalyzes the reversible, NADPH-dependent reduction of 3-dehydroshikimate to shikimate, involved in the shikimate pathway. This pathway has emerged as an important target for the development of antimicrobial agent. Structural and functional analyses suggest that the conserved Lys69 plays an important role in the catalytic activity of Helicobacter pylori (H. pylori) SDH. However, the detailed mechanism how mutation of Lys69 affects the catalytic activity of H. pylori SDH remains unclear. Here, two-layered ONIOM-based quantum mechanics/molecular mechanics (QM/MM) calculation and molecular dynamics (MD) simulations were performed to explore the role of Lys69 in the H. pylori SDH. Our results showed that in addition to act as a catalytic base, the conserved Lys69 plays an additional, important role in the maintenance of the substrate shikimate in the active site, facilitating the catalytic reaction between the cofactor NADP⁺ and shikimate. Mutation of Lys69 triggers the movement of shikimate away from the active site of SDH, thereby disrupting the catalytic activity. This result can advance our understanding the catalytic mechanism of SDH family, which may benefit of the rational design of SDH inhibitors.     

Reagentless amperometric formaldehyde-selective biosensors based on the recombinant yeast formaldehyde dehydrogenase

Novel formaldehyde-selective amperometric biosensors were developed based on NAD(+)- and glutathione-dependent formaldehyde dehydrogenase isolated from a gene-engineered strain of the methylotrophic yeast Hansenula polymorpha. Electron transfer between the immobilized enzyme and a platinized graphite electrode was established using a number of different low-molecular free-diffusing redox mediators or positively charged cathodic electrodeposition paints modified with Os-bis-N,N-(2,2'-bipyridil)-chloride ([Os(bpy)(2)Cl]) complexes. Among five tested Os-containing redox polymers of different chemical structure and properties, complexes of osmium-modified poly(4-vinylpyridine) with molecular mass of about 60 kDa containing diaminopropyl groups were selected. The positively charged cathodic paint exhibited the best electron-transfer characteristics. Moreover, the polymer layers simultaneously served as a matrix for keeping the negatively charged low-molecular cofactors, glutathione and NAD(+), in the bioactive layer. Additionally, covering the enzyme/polymer layer with a negatively charged Nafion membrane significantly decreased cofactors leakage and simultaneously enhanced the sensor' stability. The developed sensors revealed a high selectivity to formaldehyde (FA) and a low cross-sensitivity to other substances (such as, e.g. butyraldehyde, propionaldehyde, acetaldehyde, methylglyoxal). The maximum current value was 34.2+/-0.72 microA/mm(2) (3.05 mm diameter electrode) and the apparent Michaelis-Menten constant (K(M)(app)) derived from the FA calibration curves was 120+/-5mM with a linear detection range for FA up to 20mM. The best observed sensitivity for reagentless sensor was 1.8 nA microM(-1) (358 Am(-2)M(-1)). The developed sensors had a good operational and storage stability. The laboratory prototype of the sensor was applied for FA testing in some real samples of pharmaceutical (formidron), disinfectant (descoton forte) and industrial product (formalin). A good correlation was revealed between the concentration values measured using the developed FdDH-based sensor, an enzymatic method and standard chemical methods of FA determination.     

Glucose-induced heat production, insulin secretion and lactate production in isolated Wistar rat pancreatic islets

Transplantation of pancreatic islets is efficient in improving the metabolic control and quality of life and in preventing severe hypoglycemia in patients with brittle type 1 diabetes mellitus. More accurate methods to assess islet viability would be extremely useful in designing target interventions for islet cytoprotection and in reducing the number of islets required to achieve insulin independence. Here we report on an application of calorimetry to evaluate the metabolic response of pancreatic islets to glucose stimulation. A significant increase in metabolic heat was produced by islet samples when consecutively subjected to 2.8 and 16.3 mmol L⁻¹ glucose. Under these glucose concentrations, 1000 islets released average heat values of 9.16 ± 0.71 mJ and 14.90 ± 1.21 mJ over 50 min, respectively. Additionally, the glucose stimulation indexes were 1.67 ± 0.30 for insulin, 1.72 ± 0.13 for heat and 2.91 ± 0.50 for lactate, raising the important possibility of substituting the secreted insulin index/ratio by the index/ratio of the heat released in the evaluation of Langerhans islets viability for transplantation. Altogether, our results demonstrate the applicability of calorimetry to assess the quality of isolated pancreatic islets and to study vital islet functions.