Synthesis and DFT calculations of metal(II) oxime complexes bearing cysteine as coligand and investigation of their biological evolutions in vitro and in silico.

New complexes with the formula of [ML(Cys)(H2O)2] were obtained as a result of the reaction between the oxime ligand [HL: 4-(4-bromophenylaminoisonitrosoacetyl)biphenyl], cysteine (Cys), and the metal(II) salts (Mn, Ni, Co, Zn, Cu). The newly synthesized compounds were characterized using conventional techniques such as molar conductance, magnetic measurements, elemental analysis, infrared spectroscopy, and thermal analysis (TGA/DTA). Based on the conductivity measurements in DMF, it was determined that the complexes were non-electrolytes. The TGA/DTA analysis was performed to examine the thermal stability and degradation behavior of all samples, and results demonstrated that metal oxides or sulfides formed as a result of the decompositions. In conjunction with other data obtained, the elemental analysis confirmed the octahedral coordination of the complexes with deprotonated oxime (O, O-donor) and amino acid (N, S-donor) ligands and two coordinated waters. The compounds' optimized geometries, molecular electrostatic potential diagrams, and frontier molecular orbitals were computed at the DFT/B3LYP level using the 6-311 G(d,p) and LANL2DZ basis sets. The antibacterial and DNA cleavage activities of all synthesized compounds were also screened, and molecular docking simulations were performed. According to the results of molecular docking studies conducted with three different proteins, the best interaction was found to be between HL-1HNJ with a binding energy of -9.5 kcal/mol. The stability of the HL-1HNJ complex was also verified by a molecular dynamics simulation performed for 50 ns.Communicated by Ramaswamy H. Sarma.

Bioelectrocatalytic CO2 Reduction by Mo-Dependent Formylmethanofuran Dehydrogenase.

Massive efforts are invested in developing innovative CO2 -sequestration strategies to counter climate change and transform CO2 into higher-value products. CO2 -capture by reduction is a chemical challenge, and attention is turned toward biological systems that selectively and efficiently catalyse this reaction under mild conditions and in aqueous solvents. While a few reports have evaluated the effectiveness of isolated bacterial formate dehydrogenases as catalysts for the reversible electrochemical reduction of CO2 , it is imperative to explore other enzymes among the natural reservoir of potential models that might exhibit higher turnover rates or preferential directionality for the reductive reaction. Here, we present electroenzymatic catalysis of formylmethanofuran dehydrogenase, a CO2 -reducing-and-fixing biomachinery isolated from a thermophilic methanogen, which was deposited on a graphite rod electrode to enable direct electron transfer for electroenzymatic CO2 reduction. The gas is reduced with a high Faradaic efficiency (109±1 %), where a low affinity for formate prevents its electrochemical reoxidation and favours formate accumulation. These properties make the enzyme an excellent tool for electroenzymatic CO2 -fixation and inspiration for protein engineering that would be beneficial for biotechnological purposes to convert the greenhouse gas into stable formate that can subsequently be safely stored, transported, and used for power generation without energy loss.

Characterization of ferredoxins from the thermophilic, acetogenic bacterium Thermoanaerobacter kivui.

A major electron carrier involved in energy and carbon metabolism in the acetogenic model organism Thermoanaerobacter kivui is ferredoxin, an iron-sulfur-containing, electron-transferring protein. Here, we show that the genome of T. kivui encodes four putative ferredoxin-like proteins (TKV_c09620, TKV_c16450, TKV_c10420 and TKV_c19530). All four genes were cloned, a His-tag encoding sequence was added and the proteins were produced from a plasmid in T. kivui. The purified proteins had an absorption peak at 430 nm typical for ferredoxins. The determined iron-sulfur content is consistent with the presence of two predicted [4Fe4S] clusters in TKV_c09620 and TKV_c19530 or one predicted [4Fe4S] cluster in TKV_c16450 and TKV_c10420 respectively. The reduction potential (Em ) for TKV_c09620, TKV_c16450, TKV_c10420 and TKV_c19530 was determined to be -386 ± 4 mV, -386 ± 2 mV, -559 ± 10 mV and -557 ± 3 mV, respectively. TKV_c09620 and TKV_c16450 served as electron carriers for different oxidoreductases from T. kivui. Deletion of the ferredoxin genes led to only a slight reduction of growth on pyruvate or autotrophically on H2 + CO2 . Transcriptional analysis revealed that TKV_c09620 was upregulated in a ΔTKV_c16450 mutant and vice versa TKV_c16450 in a ΔTKV_c09620 mutant, indicating that TKV_c09620 and TKV_c16450 can replace each other. In sum, our data are consistent with the hypothesis that TKV_c09620 and TKV_c16450 are ferredoxins involved in autotrophic and heterotrophic metabolism of T. kivui.

Investigation of interaction between dexamethasone/pheniramine and trypsin by fluorescence, UV-vis, CD, and molecular docking.

Antihistamines and glucocorticoids are commonly used to treat allergy symptoms and the inflammatory conditions. In present study, the in-vitro binding interactions a glucocortikoid, dexamethasone/an antihistamine, pheniramine with TSN (TSN) secreted from pancreas to small intestine for protein digestion were investigated by fluorescence emission spectroscopy (FES), UV-Vis spectroscopy, synchronous fluorescence spectroscopy (SFS), CD spectroscopy, FT-IR and molecular modeling methods. Also, the effect of these drugs on the catalytic activity of trypsin (TSN) was determined. The fluorescence quenching experiments indicated that each drugs quenched the intrinsic fluorescence of TSN with their increased concentrations. The results of SFS and UV-Vis spectra proved the interaction of dexamethasone and pheniramine with TSN. CD spectra showed that the secondary structure of enzyme was altered in the presence of the drugs. All these spectroscopy results were validated and explained by molecular docking and molecular dynamic simulation (MD) studies. The IC50 values were determined as 0.0049 mM and 0.0038 mM for dexamethasone and pheniramine, respectively. So, both drugs have inhibition effect on the catalytic activity of TSN. The results of this study can provide valuable information in the field of pharmacokinetics and pharmacodynamics.Communicated by Ramaswamy H. Sarma.

Enzymatic and Microbial Electrochemistry: Approaches and Methods.

The coupling of enzymes and/or intact bacteria with electrodes has been vastly investigated due to the wide range of existing applications. These span from biomedical and biosensing to energy production purposes and bioelectrosynthesis, whether for theoretical research or pure applied industrial processes. Both enzymes and bacteria offer a potential biotechnological alternative to noble/rare metal-dependent catalytic processes. However, when developing these biohybrid electrochemical systems, it is of the utmost importance to investigate how the approaches utilized to couple biocatalysts and electrodes influence the resulting bioelectrocatalytic response. Accordingly, this tutorial review starts by recalling some basic principles and applications of bioelectrochemistry, presenting the electrode and/or biocatalyst modifications that facilitate the interaction between the biotic and abiotic components of bioelectrochemical systems. Focus is then directed toward the methods used to evaluate the effectiveness of enzyme/bacteria-electrode interaction and the insights that they provide. The basic concepts of electrochemical methods widely employed in enzymatic and microbial electrochemistry, such as amperometry and voltammetry, are initially presented to later focus on various complementary methods such as spectroelectrochemistry, fluorescence spectroscopy and microscopy, and surface analytical/characterization techniques such as quartz crystal microbalance and atomic force microscopy. The tutorial review is thus aimed at students and graduate students approaching the field of enzymatic and microbial electrochemistry, while also providing a critical and up-to-date reference for senior researchers working in the field.

Investigation of binding interaction behavior between antiemetic drugs and Trypsin by spectroscopy and molecular docking.

Antiemetic drugs are used to control excessive vomiting and nausea and generally absorbed through gastrointestinal tract. In present study, the in-vitro binding interactions two of the antiemetic drugs (dimenhydrinate and ondansetron) between Trypsin (Tsn) secreted from pancreas to small intestine for protein digestion were investigated by fluorescence emission spectroscopy (FES), UV-VIS spectroscopy, synchronous fluorescence spectroscopy (SFS), FT-IR spectroscopy and molecular modeling methods. Also, the effect of these drugs on the catalytic activity of Tsn was determined. The fluorescence quenching experiments indicated that each drugs quenched the intrinsic fluorescence of Tsn with their increased concentrations. The results of SFS and UV-VIS spectra proved the interaction of dimenhydrinate and ondansetron with Tsn. FT-IR spectra showed that the secondary structure of enzyme was altered in the presence of the drugs. All these spectroscopy results were validated and explained by molecular docking studies. Both drugs have inhibition effect on the catalytic activity of Tsn and the IC50 values were determined as 2.6 × 10-4 M and 6.4 × 10-4 M for dimenhydrinate and ondansetron, respectively. Docking results revealed that the hydrogen bond interaction of dimenhydrinate with active-site residue Ser195 and ondansetron with active-site residues His57 and Ser195 hydrogen bonds might be cause the inhibition of enzyme activity. The results of this study can provide valuable information in the field of pharmacokinetics and pharmacodynamics.

Optimization of fermentation parameters for high-activity inulinase production and purification from Rhizopus oryzae by Plackett-Burman and Box-Behnken.

The aim of study was to optimize fermentation parameters for inulinase production from Rhizopus oryzae by a statistical approach and to carry out purification of inulinase. Five isolated fungal strains were screen out inulin degradation by using Lugol's iodine solution. R. oryzae exhibited maximum zone of clearance around the colony and was used as an inulinase producer. The effect of carbon sources (inulin, glucose, maltose, sucrose, lactose, onion peel, stevia root, wheat bran) as medium component and fermentation parameters (temperature (25-45 °C), initial pH (4-7), time (3-7 days)) on inulinase production was investigated by Plackett-Burman Design. Wheat Bran (WB), temperature, pH, and incubation time were found to be significant for the production of inulinase (P < 0.05). Furthermore, Box-Behnken Design was employed to optimize fermentation conditions. The maximum experimental results for inulinase activity and specific activity were 348.36 EU/mL and 3621.78 EU/mg, respectively. The results were obtained at 5 days of incubation time, 35 °C of incubation temperature, initial pH of 5.5, and 2% (w/v) WB. Also, inulinase was purified by using ammonium sulfate precipitation, gel filtration chromatography with 2.19-fold and its molecular weight was found as 89.12 kDa. The optimal pH and temperature of the purified enzyme were 4.0 and 60 °C, respectively. Furthermore, the purified enzyme showed excellent stability at 60 °C. In conclusion, the present study offers cost-effective method to produce inulinase from Rhizopus oryzae. Also, it can be suggested that the purified inulinase has strong potential for usage in production of fructose syrup and other industrial areas.

Covalent immobilization of trypsin on polyvinyl alcohol-coated magnetic nanoparticles activated with glutaraldehyde.

Magnetic nanoparticles were coated with polyvinyl alcohol and activated with glutaraldehyde for trypsin immobilization. The prepared magnetic nanoparticles were characterized by transmission electron microscopy, fourier transform infrared spectroscopy, thermal gravimetric analysis, zeta potential meter and vibrating sample magnetometer. Free and immobilized trypsin showed optimum activity at pH 6.0, 30 °C and pH 7.0, 40 °C, respectively. Immobilized trypsin was more stable than the free enzyme at 40 °C. After immobilization, Km of the immobilized trypsin increased, however, Vmax value was almost the same with free trypsin. According to the results, the immobilized trypsin retained 50 % of its initial activity, whereas free trypsin retained 19 % of its initial activity after 12-days at 4 °C. Immobilized trypsin sustained 56 % of its initial activity after eight times of successive reuse. The performance of the immobilized trypsin was evaluated by digestion of cytochrome c. The peptide fragments in digest solution were determined by using MALDI-TOF mass spectrometry. Immobilized trypsin showed effective proteolytic activity in shorter time (15 min) than free trypsin (24 h). Hence, immobilized trypsin on the polyvinyl alcohol coated magnetic nanoparticles could be promising biocatalyst for large-scale proteomics studies and practical applications.

Stability evaluation of 6-phosphogluconate dehydrogenase immobilized on amino-functionalized magnetic nanoparticles.

In this study, 6-phosphogluconate dehydrogenase was covalently immobilized onto the N-2-aminoethyl-3-aminopropyltriethoxysilane (APTES) modified core-shell Fe3O4@SiO2 magnetic nanoparticles (ASMNPs) using glutaraldehyde (GA). Immobilization of 6PGDH on ASMNPs was confirmed using fourier transform-infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) analysis. The NADP+ conversion ratio, the reusability, thermal, and storage stability of the immobilized 6PGDH were determined and compared with those of the free enzyme. The maximum retention of enzyme activity reached to 96% when the enzyme was immobilized on ASMNPs activated with monomer form of GA. Although the thermal stability of free and immobilized enzymes was similar, at 30 °C, the immobilized 6PGDH showed the improved thermal stability at 40 °C and 50 °C compared with free 6PGDH. While the free 6PGDH only converted 33% of NADP+ in reaction medium upon 480 s, the immobilized 6PGDH performed 56% conversion of NADP+ at same time. The immobilized 6PGDH retained 62% of its initial activity up to the fifth cycle and 35% of its initial activity after 22 days of storage at 4 °C.

Development of Voltammetric Glucose-6-phosphate Biosensors Based on the Immobilization of Glucose-6-phosphate Dehydrogenase on Polypyrrole- and Chitosan-Coated Fe3O4 Nanoparticles/Polypyrrole Nanocomposite Films.

Polypyrrole (PPy) and PPy-containing chitosan-coated Fe3O4 have been electrochemically polymerized on pencil graphite electrodes (PGEs). After the resulting electrodes were characterized by SEM-EDS analysis, glucose-6-phosphate dehydrogenase (G6PD) was immobilized onto these electrodes via glutaraldehyde. The biosensors prepared for the chronopotentiometric detection of glucose-6-phosphate (G6P) at 0.25 mAcm-2 were studied and optimized at different parameters such as the pH of supporting electrolyte, the temperature, and NADP+ and G6P concentrations related with the analytical performance of the biosensors. PPy/G6PD (BS-1) and CS/Fe3O4-PPy/G6PD (BS-2) biosensors showed a broad linear response in the concentration range 0.025-0.25 mM and 0.0025-0.05 mM, and their detection limits for G6P and the RSD values were determined as 0.008 mM and 0.002 mM and 3.80% and 4.60% after 15 times usage, respectively. The interference study with various major blood components such as urea, glucose, and cysteine was performed to evaluate the selectivity of the biosensors. The proposed BS-2 biosensor showed almost free response from available interferences in blood serum with a recovery of 91 to 110%. The developed biosensors could be used in the G6P level measurement of medical samples.

Pyrene hydrogel for promoting direct bioelectrochemistry: ATP-independent electroenzymatic reduction of N2.

Enzymatic bioelectrocatalysis often requires an artificial redox mediator to observe significant electron transfer rates. The use of such mediators can add a substantial overpotential and obfuscate the protein's native kinetics, which limits the voltage of a biofuel cell and alters the analytical performance of biosensors. Herein, we describe a material for facilitating direct electrochemical communication with redox proteins based on a novel pyrene-modified linear poly(ethyleneimine). This method was applied for promoting direct bioelectrocatalytic reduction of O2 by laccase and, by immobilizing the catalytic subunit of nitrogenase (MoFe protein), to demonstrate the ATP-independent direct electroenzymatic reduction of N2 to NH3.

Creating a Low-Potential Redox Polymer for Efficient Electroenzymatic CO2 Reduction.

Increasing greenhouse gas emissions have resulted in greater motivation to find novel carbon dioxide (CO2 ) reduction technologies, where the reduction of CO2 to valuable chemical commodities is desirable. Molybdenum-dependent formate dehydrogenase (Mo-FDH) from Escherichia coli is a metalloenzyme that is able to interconvert formate and CO2 . We describe a low-potential redox polymer, synthesized by a facile method, that contains cobaltocene (grafted to poly(allylamine), Cc-PAA) to simultaneously mediate electrons to Mo-FDH and immobilize Mo-FDH at the surface of a carbon electrode. The resulting bioelectrode reduces CO2 to formate with a high Faradaic efficiency of 99±5 % at a mild applied potential of -0.66 V vs. SHE.

The In Vivo Potential-Regulated Protective Protein of Nitrogenase in Azotobacter vinelandii Supports Aerobic Bioelectrochemical Dinitrogen Reduction In Vitro.

Nitrogenase, the only enzyme known to be able to reduce dinitrogen (N2) to ammonia (NH3), is irreversibly damaged upon exposure to molecular oxygen (O2). Several microbes, however, are able to grow aerobically and diazotrophically (fixing N2 to grow) while containing functional nitrogenase. The obligate aerobic diazotroph, Azotobacter vinelandii, employs a multitude of protective mechanisms to preserve nitrogenase activity, including a "conformational switch" protein (FeSII, or "Shethna") that reversibly locks nitrogenase into a multicomponent protective complex upon exposure to low concentrations of O2. We demonstrate in vitro that nitrogenase can be oxidatively damaged under anoxic conditions and that the aforementioned conformational switch can protect nitrogenase from such damage, confirming that the conformational change in the protecting protein can be achieved solely by regulating the potential of its [2Fe-2S] cluster. We further demonstrate that this protective complex preserves nitrogenase activity upon exposure to air. Finally, this protective FeSII protein was incorporated into an O2-tolerant bioelectrosynthetic cell whereby NH3 was produced using air as a substrate, marking a significant step forward in overcoming the crippling limitation of nitrogenase's sensitivity toward O2.