Functional characterization of tobacco (Nicotiana benthamiana) serotonin N-acetyltransferases (NbSNAT1 and NbSNAT2)

Nicotiana benthamiana (tobacco) is an important dicotyledonous model plant; however, no serotonin N-acetyltransferases (SNATs) have been characterized in tobacco. In this study, we identified, cloned, and characterized the enzyme kinetics of two SNAT genes from N. benthamiana, NbSNAT1 and NbSNAT2. The substrate affinity (Km) and maximum reaction rate (Vmax) for NbSNAT1 were 579 µM and 136 pkat/mg protein for serotonin, and 945 µM and 298 pkat/mg protein for 5-methoxytryptamine, respectively. Similarly, the Km and Vmax values for NbSNAT2 were 326 µM and 26 pkat/mg protein for serotonin, and 872 µM and 92 pkat/mg protein for 5-methoxytryptamine, respectively. Moreover, we found that NbSNAT1 and NbSNAT2 localized to chloroplasts, similar to SNAT proteins from other plant species. The activities of the NbSNAT proteins were not affected by melatonin feedback inhibition in vitro. Finally, transgenic tobacco plants overexpressing either NbSNAT1 or NbSNAT2 did not exhibit increased melatonin levels, possibly due to the expression of catabolic enzymes. Generating transgenic tobacco plants with downregulated NbSNAT expression would provide further insight into the functional role of melatonin in tobacco plants.

pH-Based Control Strategies for the Nitrification of High-Ammonium Wastewaters

Aquatic nitrogen pollution is one of the most urgent environmental issues requiring prevention and mitigation. Large quantities of high-ammonium wastewaters are generated by several industrial sectors, such as fertilizer and anaerobic-digestion plants. Nitrification of these wastewaters is commonly carried out, either to remove nitrogen or produce liquid fertilizers. Standard control methodologies for the efficient nitrification of high-ammonium wastewaters to produce liquid fertilizers have not yet been established and are still within their early stages of development. In this paper, novel pH-based control algorithms are presented that maintain operation at the microbial maximum reaction rate (υmax) in batch and continuous reactors. Complete conversion of ammonium to nitrate was achieved in a batch setup, and a conversion of 93% (±1%) was achieved in a continuously-stirred-tank-reactor. The unparalleled performance and affordability of the control schemes proposed offer a steppingstone to the future of sustainable fertilizer production.

The effect of sludge retention time (SRT) on the Nitrifier typical kinetics at ambient temperature under the low ammonia density

Sludge retention time (SRT) regulation is one of the essential management techniques for refined control of the main-sidestream treatment process under the low ammonia density. It is indispensable to understand the effect of SRTs changes on the Nitrifier kinetics to obtain the functional separation of the Nitrifier and the refined control of the nitrification process. In this study, Nitrifier was cultured with conditions of 35 ± 0.5 °C, pH 7.5 ± 0.2, DO 5.0 ± 0.5 mg-O/L, and SRTs was controlled for 40 d, 20 d, 10 d, and 5 d. The net growth rate (), decay rate (), specific growth rate (), the yield of the Nitrifier (), temperature parameter (), and inhibition coefficient () have been measured and extended with the SRT decreases. Instead, the half-saturation coefficient () decreased. In addition, the limited value of pH inhibition occurs (), and the pH of keeping 5% maximum reaction rate () was in a relatively stable state. The trade of kinetics may be induced by the species structure of Nitrifier changed. The Nitrosomonas proportion was increased, and the Nitrospira used to be contrary with the SRT decreasing. It is a match for the functional separation of Nitrifier when SRTs was 20 d at ambient temperature under the low ammonia density. The kinetics of ammonia-oxidizing organism (AOO) and nitrite-oxidizing organism (NOO) in Nitrifier under different SRT conditions should be measured respectively to the refined control of the partial nitrification process in the future study. HIGHLIGHT The Nitrifier typical kinetics used to be affected notably by way of SRTs changes. The species structure of the Nitrifier was recognized beneath distinctive SRTs. The change of Nitrifier kinetics with SRTs used to be estimated by the species structure changes.

Photocatalytic ozonation in an immersion rotary body reactor for the removal of micro-pollutants from the effluent of wastewater treatment plants

Carrier-bound titanium dioxide catalysts were used in a photocatalytic ozonation reactor for the degradation of micro-pollutants in real wastewater. A photocatalytic immersion rotary body reactor with 36 cm disk diameter was used, which was irradiated using UV-A LEDs. The rotating disks were covered with catalysts based on stainless steel grids coated with titanium dioxide. The dosing of ozone was carried out through the liquid phase via an external enrichment and a supply system transverse to the flow direction. The influence of irradiation power and ozone dose on the degradation rate for photocatalytic ozonation was investigated. In addition, the performance of the individual processes photocatalysis and ozonation were studied. The degradation kinetics of the parent compounds were determined by LC-MS/MS. First-order kinetics were determined for photocatalysis and photocatalytic ozonation. A maximum reaction rate of the reactor was determined, which could be achieved by both photocatalysis and photocatalytic ozonation. At a dosage of 0.4 mg /mg DOC, the maximum reaction rate could be achieved using 75% of the irradiation power used for sole photocatalysis, allowing increases in the energetic efficiency of photocatalytic wastewater treatment processes. The process of photocatalytic ozonation is suitable to remove a wide spectrum of micro-pollutants from wastewater. HIGHLIGHT within the work, reaction rates for the degradation of micropollutants in real wastewater matrix are presented. due to the number of investigated pollutants as well as the practical investigation conditions, a more precise evaluation of the use of photocatalysis and photocatalytic ozonation for wastewater treatment is possible.

Synthesis, structure, and photo-Fenton activity of PrFeO3-TiO2 mesoporous nanocomposites

Porous nanocomposites based on PrFeO3-TiO2 were synthesized using the glycine-nitrate combustion method with different values of mass content of TiO2 (0–7.5 %) and subsequent heat treatment in air. The results of X-ray phase analysis and Raman spectroscopy confirmed the presence of ultradispersed TiO2, structurally close to that of anatase. The morphology, specific surface area, and porous structure of the obtained powders were characterized by scanning electron microscopy and adsorption-structural analysis, the results of which showed that the samples had a foam-like mesoporous structure.The specific surface area and the average pore size were in the ranges of 7.6–17.8 m2/g and 7.2–15.2 nm, respectively, and varied depending on the TiO2 content. The optical properties of the nanocomposites were studied by UV-visible diffuse reflection spectroscopy, the energy of the band gap was calculated as 2.11–2.26 eV. The photocatalytic activity of PrFeO3‑TiO2 nanocomposites was investigated in the process of photo-Fenton-like degradation of methyl violet under the action of visible light. It was shown that the maximum reaction rate constant was 0.095 min-1, which is ten times higher than the value for the known orthoferrite-based analogs. The obtained photocatalysts were also characterized by their high cyclic stability. Based on the studies carried out, the obtained porous PrFeO3-TiO2 nanocomposites can be considered to be apromising basis for photocatalysts applied in advanced oxidative processes of aqueous media purification from organic pollutants.

Selective and predicable amine conjugation sites by kinetic characterization under excess reagents

The site selectivity for lysine conjugation on a native protein is difficult to control and characterize. Here, we applied mass spectrometry to examine the conjugation kinetics of Trastuzumab-IgG (Her-IgG) and α-lactalbumin under excess linker concentration ([L]0) based on the modified Michaelis–Menten equation, in which the initial rate constant per amine (kNH2 = Vmax/NH2/KM) was determined by the maximum reaction rate (Vmax/NH2) under saturated accessible sites and initial amine–linker affinity (1/KM). Reductive amination (RA) displayed 3–4 times greater Vmax/NH2 and a different panel of conjugation sites than that observed for N-hydroxysuccinimide ester (NHS) chemistry using the same length of polyethylene glycol (PEG) linkers. Moreover, faster conversion power rendered RA site selectivity among accessible amine groups and a greater tunable range of linker/protein ratio for aldehyde-linkers compared to those of the same length of NHS-linkers. Single conjugation with high yield or poly-conjugations with site homogeneity was demonstrated by controlling [L]0 or gradual addition to minimize the [L]0/KM ratio. Formaldehyde, the shortest aldehyde-linker with the greatest 1/KM, exhibited the highest selectivity and was shown to be a suitable probe to predict conjugation profile of aldehyde-linkers. Four linkers on the few probe-predicted hot spots were elucidated by kinetically controlled RA with conserved drug efficacy when conjugated with the payload. This study provides insights into controlling factors for homogenous and predictable amine bioconjugation.

Derivation of volatile fatty acids concentration-independent optimum trace elements configuration and elucidation of optimization kinetics of biomethanization processes

Trace elements (TEs) requirements for improved volatile fatty acids (VFA) degradation during biomethanization depend on VFA concentration of a reactor and the temperature of the process. While temperature remains relatively constant, VFA concentrations change in the course of biomethanization and this implies that for efficient VFA degradation, different trace elements configurations (TEC) should be supplemented. While this is the most efficient approach, it is impractical and constitutes a challenge for the effective use of TEs in the optimization of biomethanization processes. To alleviate this challenge, we modelled the biomethanization efficiency of various VFA concentration-dependent (VCD) TEs configuration as scenarios and derived a TEs configuration that produced optimum biomethanization across a wider range of VFA concentrations. The study was carried out at 37oC using different concentrations of fixed VFA composition and TEs configurations as scenarios. Response surface model and desirability function were used to determine and compare the biomethanization efficiency of the scenarios, and to derive a VFA concentration-independent (VCI) TEs configuration. Michaelis-Menten kinetics for two parameters was used to ascertain that the mechanism by which TEs supplementation enhanced mesophilic biomethanization was through an increase in maximum reaction rate (MRR). However, the enhancement was accompanied by an insignificant decline in inverse affinity (IA).

CO2-Derived Carbon Capture Using Microalgae and Sodium Bicarbonate in a PhotoBioCREC Unit: Kinetic Modeling

By converting bicarbonates via Chlorella vulgaris photosynthesis, one can obtain valuable biofuel products and find a route toward carbon-derived fossil fuel conversion into renewable carbon. In this research, experiments were carried out in the PhotoBioCREC prototype under controlled radiation and high mixing conditions. Sodium bicarbonate (NaHCO3) was supplied as the inorganic carbon-containing species, at different concentrations, in the 18 to 60 mM range. Both the NaHCO3 concentrations and the organic carbon concentrations were quantified periodically during microalgae culture, with the pH being readjusted every day to the 7.00 level. It was found that sodium bicarbonate was converted with a selectivity up to 33.0% ± 2.0 by Chlorella vulgaris. It was also observed that the inorganic carbon conversion was 0.26 ± 0.09 day−1, while the maximum reaction rate constant for organic carbon formation was achieved with a 28 mM NaHCO3 concentration and displayed a 1.18 ± 0.05 mmole L−1 day−1 value.

Kinetic, Catalytic and Thermodynamic Properties of Immobilized Milk Clotting Enzyme on Activated Chitosan Polymer and its Application in Cheese Making

Milk clotting enzyme (MCE) from Bacillus circulans 25 was immobilized by covalent binding, ionic binding and entrapment methods using various carriers. MCE covalently immobilized on activated chitosan polymer with the bifunctional agent glutaraldehyde (Ch-MCE) exhibited highest immobilization yield (74.6 %). Comparing to the native MCE, Ch-MCE exhibited higher optimum pH, higher optimum reaction temperature, lower activation energy, higher half-life time, lower deactivation rate constant and higher energy for denaturation. After immobilization, maximum reaction rate, Michaelis-Menten constant, specificity constant, turnover number, and catalytic efficiency of the enzyme were significantly changed. Calculated thermodynamic parameters for denaturation (enthalpy, entropy and Gibbs free energy) confirmed that the catalytic properties of MCE were significantly improved after immobilization. Reusability tests showed that after 7 catalytic cycles, the Ch-MCE retained about 71 % of its activity confirming its suitability for industrial applications.

Inhibition of phospholipase A2 by Virasole derivation

Kinetics of phosphatidylcholine (PC) hydrolysis under the action of pancreatic phospholipase A2 IB, (EC 3.1.1.4, PLA2) in the presence of a lipophilic derivative of the antiviral drug Virazole 1-(3-((tert-butyldimethylsilyl)oxy)-4-hydroxy-5- (((4-methoxyphenyl)diphenylmethoxy)methyl)tetrahydrofuran-2-yl)-1H-1,2,4-triazole-3-carboxamide (Virazole2ЗГ) was studied. The both steps of phospholipolysis were quantitatively characterized: the binding of the enzyme to the lipid-water interface (Ks) and directly the catalytic act (Km) with the determination of the maximum reaction rate (Vmax). It was found that Virazole2ЗГ at a concentration of 0.5 μmol/ml does not affect the Ks value; on the contrary, the Michaelis constant, Km, increases by a factor of 1.8 along with the constancy of the parameter Vmax. Based on the constancy of the Ks values, it seems to be assumed that there is no inhibition of the disintegration of the enzyme-micelle complex in the presence of the effector under the studied reaction conditions. The kinetic parameters of the reaction (the increase in Km and the constancy of Vmax in the presence of Virazole2ЗГ) testify in favor of a moderate competitive inhibition of pancreatic PLA2, Ki = 65 mM, which indicates the possibility of searching for the biological activity of the anti-pancreatitis action in the series of pro-drugs of nucleoside nature.