Μmol Min Research Articles

Effective UV–visible-infrared light-driven photothermocatalytic dry reforming of methane on Ni/Ni-MgO caused by a novel synergetic effect and photoactivation

Light-driven photothermocatalytic dry reforming of methane (DRM) is a promising technology which can efficiently convert two greenhouse gases of CO2 and CH4 into fuels and realize effective solar energy storage simultaneously. It is greatly challenging to achieve high fuel production rate with a relatively low light intensity and restrain the coking side-reactions that cause catalyst deactivation. Herein, a novel nanocomposite of Ni nanoparticles (NPs) loaded on Ni-doped MgO (Ni/Ni-MgO) was prepared. Very high production rate (r) of H2 and CO (82.78 mmol min−1 g−1 and 98.05 mmol min−1 g−1) as well as a light-to-fuel efficiency (η, 28.8%) are obtained with concentrated UV–vis IR irradiation at a relatively low light intensity (78.8 kw m−2). Ni/Ni-MgO displays excellent durability due to its very low coking rate of 2.46 × 10−3 gc h−1 g−1catalyst, reduced by 11.6 times as compared to that of a reference sample of Ni NPs loaded on MgO (Ni/MgO). This is attributed to a synergetic effect between Ni NPs and Ni-MgO, in which the active oxygen of Ni-doped MgO participates in the oxidation of carbon species as one of the rate-determining steps of DRM, thus facilitating DRM on Ni NPs and greatly improving coking resistance. A photoactivation is found to greatly reduce activation energy and thus enhances catalytic activity due to the substantial facilitation of carbon oxidation by light irradiation.

Direct Conversion of Ethane to Oxygenates, Ethylene, and Hydrogen in a Noncatalytic Biphasic Plasma Microreactor

We selectively upgrade ethane (C2H6) to ethanol (C2H5OH), methanol (CH3OH), and acetic acid (CH3COOH) in a catalyst-free, continuous, argon/water biphasic plasma microreactor. The water (H2O) evaporates and electron- dissociates into OH· radicals. OH· recombines with alkyl radicals, produced via electron dissociation of ethane, to generate the oxygenates that absorb into H2O. A plasma-assisted path, reminiscent of the low-temperature thermocatalytic ethane steam reforming, leads to significant H2 coproduction. The gaseous stream also comprises CO2 and C2H4. Up to 1.3 and 1 μmol min–1 of liquid C2H5OH and CH3OH are attained, respectively. Compared to CO2-assisted ethane plasma conversion, which produces many oxygenates with low selectivity, the carbon selectivity can range from >70% C2H5OH, CH3OH, and CH3COOH to 60% C2H4. The low carbon footprint, electrified, modular, intensified process using a reactive evaporation and separation plasma could pave the way for the valorization of underutilized shale gas resources in remote areas.

The Fe2P/NiCoP phosphating compound supported by nickel foam functions as a superior bi-functional catalyst to speed up the overall water splitting

In this work, we designed a bifunctional Fe2P/NiCoP/NF catalyst with a self supporting structure based on nickel foam (NF) as a conducting matrix. For both the hydrogen evolution reaction (HER) and the oxygen evolution reaction (OER), this catalyst showed improved bifunctional catalytic function. CoFe precursor are co-phosphated with NF, and this causes the NF surface to take part in the reaction, forming a spherical composite material composed of NiCoP particles and Fe2P particles. In addition, it was found that NF provides good conductivity and stability in the electrocatalytic test, and NiCoP and Fe2P have an apparent synergistic impact, which has a superior catalytic function on both HER and OER. OER overpotential of 261 mV and HER overpotential of 125 mV, respectively, are displayed by the Fe2P/NiCoP/NF catalyst with current densities of 50 mA cm−2 and 10 mA cm−2 separately. Fe2P/NiCoP/NF further performed exceptionally well throughout the overall water splitting process. With just 1.43 V, it can attain 10 mA cm−2 and maintain stability for 50 h. The H2 production in the total water splitting process can reach 4.35 μmol min−1. As a result, the self-supporting bifunctional electrocatalyst composed of Fe2P/NiCoP/NF can be effective and long-lasting for overall water splitting.

Ambient Carbon-Neutral Ammonia Generation via a Cyclic Microwave Plasma Process.

A novel reactor methodology was developed for chemical looping ammonia synthesis processes using microwave plasma for pre-activation of the stable dinitrogen molecule before reaching the catalyst surface. Microwave plasma-enhanced reactions benefit from higher production of activated species, modularity, quick startup, and lower voltage input than competing plasma-catalysis technologies. Simple, economical, and environmentally benign metallic iron catalysts were used in a cyclical atmospheric pressure synthesis of ammonia. Rates of up to 420.9 μmol min-1 g-1 were observed under mild nitriding conditions. Reaction studies showed that both surface-mediated and bulk-mediated reaction domains were found to exist depending on the time under plasma treatment. The associated density functional theory (DFT) calculations indicated that a higher temperature promoted more nitrogen species in the bulk of iron catalysts but the equilibrium limited the nitrogen converion to ammonia, and vice versa. Generation of vibrationally active N2 and, N2+ ions is associated with lower bulk nitridation temperatures and increased nitrogen contents versus thermal-only systems. Additionally, the kinetics of other transition metal chemical looping ammonia synthesis catalysts (Mn and CoMo) were evaluated by high-resolution time-on-stream kinetic analysis and optical plasma characterization. This study sheds new light on phenomena arising in transient nitrogen storage, kinetics, effect of plasma treatment, apparent activation energies, and rate-limiting reaction steps.

Biohybrid membranes for organophosphate pesticides degradation: Hyperactivation of immobilized phosphotriesterase by surfactants

The aim of this work is to develop a hyperactivated biocatalytic membrane (BM) for the degradation of organophosphate pesticides (OPs), combining an enzyme-loaded membrane with cationic (cetyl trimethyl ammonium bromide, CTAB) or anionic (sodium dodecyl sulfate, SDS) surfactants. To analyze the effect of surfactants on the catalytic performance of BM, a systematic study of enzyme activity, stability, change in the secondary structure, protein aggregate formation, and kinetic parameters ( KM, Kcat, Kcat/KM) was performed. The results showed that the activity of the immobilized phosphotriesterase toward the pesticide paraoxon was greatly enhanced in the presence of SDS and CTAB (90% and 80%, respectively). Circular dichroism, dynamic light scattering, electrophoresis, and kinetics studies made it clear that surfactants affect catalytic performance by either changing the secondary structure and aggregation state of the enzyme (CTAB) or increasing the affinity of the enzyme for the substrate (SDS). Despite the strong improvement in free enzyme activity (2.6μmol min−1mg−1 versus 4.9 and 4.6μmol min−1mg−1 with SDS and CTAB, respectively), a smaller enhancement effect was observed with the immobilized enzyme, which may be attributed to the increased stiffness of the enzyme due to immobilization. Nevertheless, stable BMs with doubled specific activity were prepared that remained almost constant across various reaction cycles. Moreover, tests in a biocatalytic membrane reactor showed a 96% conversion at one-third of the residence time compared to the literature. The proposed strategy is a real advance in improving the activity/stability of BMs, which is one of the main drawbacks of this technology.

Extraordinary Catalytic Performance of Nickel Half‐Metal Clusters for Light‐Driven Dry Reforming of Methane

AbstractGlobal warming and the energy shortage due to the burning of fossil fuels are two major challenges. A strategy of light‐driven catalytic dry reforming of methane (DRM) using inexhaustible solar energy has provided an effective approach to tackle these challenges. Loaded metallic Ni nanoparticles have been identified as promising catalysts as alternatives to noble metals for DRM. However, they suffer from quick deactivation caused by severe coking due to thermodynamically inevitable side‐reactions. Herein, a unique nickel half‐metal catalyst of monolayer nickel clusters stabilized by alumina is reported, with 100% atomic utilization efficiency and extraordinary catalytic performance for light‐driven DRM, dramatically different from its counterpart of conventional metallic Ni nanoparticles. It has extremely high specific H2 and CO production rates of 8572.96 and 9614.26 mmol min−1 gNi−1, more than one order of magnitude higher than those of Ni nanoparticles. No detectable coke is formed due to the alteration of kinetic processes for DRM, resulting in the inhibition of coke deposition. It is discovered that light not only significantly enhances catalytic activity, but also enables quick thermocatalytic deactivation to be durable due to the oxidation of carbon species and the desorption of strongly adsorbed CO2 and CO being dramatically promoted by light.

Heterogeneous doping via charge carrier transport improves Photoelectrochemical H2O oxidative H2O2 synthesis

Photoelectrochemical (PEC) water splitting into H2O2 and H2 has attracted significant attention due to its low cost and sustainability. However, slow charge carrier transport and water oxidation kinetics limit the selectivity for H2O2 production and solar conversion efficiency. Herein, we propose a heterogeneous doping approach that combines surface gradient doping with bulk doping to improve the charge carrier transport in the photoelectrode. Inducing gradient Nb and homogeneous Mo doping into BiVO4 photoanode (G-Nb/Mo:BVO) can promote charge separation and carrier transfer, leading to the high selectivity for H2O2 generation and suppression of O2 production. Consequently, the heterogeneously doped G-Nb/Mo:BVO photoanode presents an average Faraday efficiency (FE) of over 80% for H2O2 production in a wide potential of 0.6–1.8 VRHE with the maximum FE of 83.7% at 1.2 VRHE under AM 1.5G illumination. More importantly, H2O2 production rate can reach 1.23 μmol min−1 cm−2 at 1.23 VRHE, representing the best H2O2 production rate reported for the photoelectrodes. Density functional theory calculations prove that Mo and Nb co-doping increases the reactivity of BiVO4 and improves 2e− water oxidation reaction activity and selectivity. This work demonstrates that heterogeneous doping provides a cost-effective strategy to break performance trade-offs by improving charge carrier separation and transport in light absorbers, and modulating the selectivity and activity of water oxidation reaction to generate H2O2, which can be extended to other photocatalytic reactions.

Self-assembled membranes modulate the active site of carbon fiber paper to boost the two-electron water oxidation reaction

Two-electron water oxidation (2e-WOR) is an attractive method for the electrosynthesis of hydrogen peroxide (H2O2). However, its practical application is limited by the viable 2e-WOR catalysts that can ensure high selectivity and high H2O2 yield. In this paper, a highly active 2e-WOR electrocatalyst was prepared by modifying carbon fiber paper (CFP) with a self-assembled membrane (SAMs). The SAMs modified CFP can achieve an H2O2 yield of 79.8 μmol min−1 cm−2 and an H2O2 selectivity of 82.5% at a low potential (2.1 V vs. RHE). The DFT calculation reveals that SAMs modification can modulate the *OH binding energy of the active site on CFP, thus leading to a significantly improved selectivity and production rate of H2O2. Moreover, the 2e-WOR performance is found to be independent of the functional tail groups of SAMs because the electron transfer during the 2e-WOR occurs through direct electron transfer via the carbon atoms adjacent to the O=C-M on carbon fiber rather than the well-known electron tunneling through SAMs. This study provides a new strategy for surface modification of 2e-WOR carbon-based catalysts and opens a new way for efficient electrosynthesis of H2O2.

Photo-Fenton process applied for the treatment of industrial wastewaters containing diclofenac: optimization with low iron ions concentrations and without pH control

Diclofenac (DCF) can cause several adverse effects in the environment and it should be removed from industrial pharmaceutical wastewaters. Advanced oxidation processes (AOPs) are promising methods for the DCF degradation. But, in many cases, AOPs require acidic pH. However, at this condition, DCF precipitates, which may hinder its oxidation. Thus, in this work, some AOP were studied for the DCF degradation, especially the photo-Fenton process, applying the experimental design technique (Doehlert matrix), operating without and with pH control (between 6.5 and 7.0). As independent variables, the initial ferrous ion concentration ([Fe2+]) and the molar addition rate of H2O2 (FH2O2) were evaluated. Empirical models were proposed and optimized conditions were determined without ([Fe2+] = 0.27 mmol L−1 and FH2O2 = 1.64 mmol min−1) and with pH control ([Fe2+] = 1.0 mmol L−1 and FH2O2 = 1.64 mmol L−1), with the following predicted mineralization percentages: 93% and 68%, respectively. So, photo-Fenton process without pH control presented the best performances. Furthermore, at this condition, iron concentration respects the limit value established by the Brazilian environmental legislation. That is, in this condition, additional processes, in order to remove iron ions, would not be necessary, that is very interesting for applications on an industrial scale.

Improved photocatalytic oxidation of ciprofloxacin by NiS-coupled WO3 nanorods synthesized by solvothermal method under visible light

Photocatalytic oxidation of antibiotics over semiconducting nanostructured photocatalysts is stared as a capable procedure for preventing possible antimicrobial resistance. Accordingly, plenteous research suggestions for photocatalyst design and trials are investigated to realize this approach. Moreover, photostability and reusability are important factors for sustainable utilization. Herein, tungsten trioxide (WO3) nanorods were grown by a solvothermal route in the presence of double soft templates, followed by incorporating nickel sulfide (NiS) nanoparticles on their surface to have NiS/WO3 nanocomposite photocatalysts. These formed heterojunctions were analyzed via several tools. The NiS incorporation has widened the visible-light harvesting owing to the bandgap reduction from 2.82 eV for pure WO3 to 2.40 eV at 9 wt% of NiS. The mesoporous surface has not been meaningfully impacted by incorporating NiS with a value of the surface area between 163 and 189 m2 g-1. The photooxidation of ciprofloxacin (CiP) over the formed photocatalysts were done under visible-light irradiation. The 2.0 g L-1 of 9% NiS/WO3 has completely oxidized 30.2 μmol CiP at a 1.38 μmol min−1. This progressive NiS/WO3 p-n heterojunction has also implied reusability for five repetitive cycles. This excellent photoactivity is ascribed to the charge transfer by the S-scheme mechanism and the synergistic upshot of a particular NiS expanse.

Aldose reductase inhibition alleviates diabetic cardiomyopathy and is associated with a decrease in myocardial fatty acid oxidation

BackgroundCardiovascular diseases, including diabetic cardiomyopathy, are major causes of death in people with type 2 diabetes. Aldose reductase activity is enhanced in hyperglycemic conditions, leading to altered cardiac energy metabolism and deterioration of cardiac function with adverse remodeling. Because disturbances in cardiac energy metabolism can promote cardiac inefficiency, we hypothesized that aldose reductase inhibition may mitigate diabetic cardiomyopathy via normalization of cardiac energy metabolism.MethodsMale C57BL/6J mice (8-week-old) were subjected to experimental type 2 diabetes/diabetic cardiomyopathy (high-fat diet [60% kcal from lard] for 10 weeks with a single intraperitoneal injection of streptozotocin (75 mg/kg) at 4 weeks), following which animals were randomized to treatment with either vehicle or AT-001, a next-generation aldose reductase inhibitor (40 mg/kg/day) for 3 weeks. At study completion, hearts were perfused in the isolated working mode to assess energy metabolism.ResultsAldose reductase inhibition by AT-001 treatment improved diastolic function and cardiac efficiency in mice subjected to experimental type 2 diabetes. This attenuation of diabetic cardiomyopathy was associated with decreased myocardial fatty acid oxidation rates (1.15 ± 0.19 vs 0.5 ± 0.1 µmol min−1 g dry wt−1 in the presence of insulin) but no change in glucose oxidation rates compared to the control group. In addition, cardiac fibrosis and hypertrophy were also mitigated via AT-001 treatment in mice with diabetic cardiomyopathy.ConclusionsInhibiting aldose reductase activity ameliorates diastolic dysfunction in mice with experimental type 2 diabetes, which may be due to the decline in myocardial fatty acid oxidation, indicating that treatment with AT-001 may be a novel approach to alleviate diabetic cardiomyopathy in patients with diabetes.

A putative cytotoxic serine protease from Salmonella typhimurium UcB5 recovered from undercooked burger

A putative virulence exoprotease designated as UcB5 was successfully purified from the bacterium Salmonella typhimurium to the electrophoretic homogeneity with 13.2-fold and 17.1% recovery by hydrophobic, ion-exchange, and gel permeation chromatography using Phenyl-Sepharose 6FF, DEAE-Sepharose CL-6B, and Sephadex G-75, respectively. By applying SDS-PAGE, the molecular weight was confirmed at 35 kDa. The optimal temperature, pH, and isoelectric point were 35 °C, 8.0, 5.6 ± 0.2, respectively. UcB5 was found to have a broad substrate specificity against almost all the tested chromogenic substrates with maximal affinity against N-Succ-Ala-Ala-Pro-Phe-pNA achieving Km of 0.16 mM, Kcat/Km of 3.01 × 105 S−1 M−1, and amidolytic activity of 28.9 µmol min−1 L−1. It was drastically inhibited by TLCK, PMSF, SBTI, and aprotinin while, DTT, β-mercaptoethanol, 2,2′-bipyridine, o-phenanthroline, EDTA, and EGTA had no effect, which suggested a serine protease-type. Also, it has shown a broad substrate specificity against a broad range of natural proteins including serum proteins. A cytotoxicity and electron microscopy study revealed that UcB5 could cause subcellular proteolysis that finally led to liver necrosis. For this, future research should focus on using a combination of external antiproteases and antimicrobial agents for the treatment of microbial diseases instead of using drugs alone.

Protein inorganic hybrid nanoflowers of a microbial carbonic anhydrase as efficient tool for the conversion of CO2 into value added product

AbstractBACKGROUNDCarbonic anhydrase (CA) is one of the most widely distributed enzymes among plants, animals, and microorganisms, and it has an enormous ability to capture carbon dioxide (CO2). However, the real‐time application of CA is still a challenge due to its low operational stability, its difficulty in recovery from the reaction medium, and its poor durability.RESULTSThe synthesis of insoluble protein inorganic hybrid structures at nanoscale was proven as quite useful to catalyze enzymatic biotransformation. Here, CA nanoflowers (CANF) were synthesized with the self‐assembly of metal phosphate and CA. The synthesis of CANF was performed using 0.2 mg mL−1 protein and 2.0 mM CuSO4 at 4 °C under mild shaking conditions. The CANF exhibited optimum activity at pH 7.5 and a temperature of 40°C. The synthesized CANF were used for CO2 conversion under optimized conditions and their kinetic parameters were studied using p‐NPA hydrolysis. The Vmax and Km of CANF were 185.18 μmol min−1 mL−1 and 4.72 mM, compared with those of free CA, 166.66 μmol min−1 mL−1 and 5.12 mM, respectively. The stability of CANF has improved remarkably. The CANF made of metal ions and protein showed higher stability and enzyme activity than free enzymes. Furthermore, the CANF showed good reusability due to their mechanical properties and monodispersity. The production of CaCO3 by CANF was 1.71‐fold higher than that by free CA.CONCLUSIONThe newly formed CANF showed flower‐like morphology with good catalytic activity. This study demonstrated that CANF technology has a bright future in the conversion of CO2 into CaCO3. © 2023 Society of Chemical Industry (SCI).

Single atomic Ru in TiO2 boost efficient electrocatalytic water oxidation to hydrogen peroxide

Electrocatalytic two-electron water oxidation affords a promising approach for distributed production of H2O2 using electricity. However, it suffers from the trade-off between the selectivity and high production rate of H2O2 due to the lack of suitable electrocatalysts. In this study, single atoms of Ru were controllably introduced into titanium dioxide to produce H2O2 through an electrocatalytic two-electron water oxidation reaction. The adsorption energy values of OH intermediates could be tuned by introducing Ru single atoms, offering superior H2O2 production under high current density. Notably, a Faradaic efficiency of 62.8% with an H2O2 production rate of 24.2 μmol min−1 cm−2 (>400 ppm within 10 min) was achieved at a current density of 120 mA cm−2. Consequently, herein, the possibility of high-yield H2O2 production under high current density was demonstrated and the importance of regulating intermediate adsorption during electrocatalysis was evidenced.

Catalytic Oxidation of n-Decane, n-Hexane, and Propane over Pt/CeO2 Catalysts.

Pt species with different chemical states and structures were supported on CeO2 by solution reduction (Pt/CeO2-SR) and wet impregnation (Pt/CeO2-WI) and investigated in catalytic oxidation of n-decane (C10H22), n-hexane (C6H14), and propane (C3H8). Characterization by X-ray diffraction, Raman spectroscopy, X-ray photoelectron spectroscopy, H2-temperature programming reduction, and oxygen temperature-programmed desorption showed that Pt0 and Pt2+ existed on Pt nanoparticles of the Pt/CeO2-SR sample, which promoted redox, oxygen adsorption, and activation. On Pt/CeO2-WI, Pt species were highly dispersed on CeO2 as the Pt-O-Ce structure, in which surface oxygen decreased significantly. The Pt/CeO2-SR catalyst presents high activity in oxidation of C10H22 with a rate of 0.164 μmol min-1 m-2 at 150 °C. The rate increased with oxygen concentration. Moreover, Pt/CeO2-SR presents high stability on feed stream containing 1000 ppm C10H22 at gas hour space velocity = 30,000 h-1 as low as 150 °C for 1800 min. The low activity and stability of Pt/CeO2-WI were probably related to its low availability of surface oxygen. In situ Fourier transform infrared results showed that the adsorption of alkane occurred through the interaction with Ce-OH. The adsorption of C6H14 and C3H8 was much weaker than that of C10H22, which resulted in the decrease in activity for C6H14 and C3H8 oxidation of Pt/CeO2 catalysts.

Chicken tallow, a low-cost feedstock for the two-step lipase-catalysed synthesis of biolubricant

Biolubricants are oleochemical esters that are non-toxic to the environment, and they are preferred because of their high-performance activities in various automobile and industrial fields. In this study, chicken tallow has been explored as an alternative to a non-renewable source, which acts as a low-cost feedstock for synthesizing biolubricants in a two-step hydro esterification process catalysed by Candida rugosa lipase (CRL). The enzymatic hydrolysis of chicken tallow is optimized at different time intervals at 40 °C, and the maximum release of 160.2 µmol min−1 free fatty acids (FFAs) is observed at 100 min. The hydrolysed FFAs are characterized by the gas chromatography-mass spectrometry (GC-MS) analysis that has depicted the fatty acid methyl esters (FAMEs) composition of the tallow oil. The recovered FFA is then enzymatically esterified with trimethylolpropane (TMP) to produce TMP esters. Optimization studies are performed to explore the effect of factors such as time, temperature, amount of the enzyme, and TMP: FFA molar ratio for the esterification process. Thus, the optimal conditions for achieving a maximal hydroxyl group conversion of 60% during TMP ester production include incubation time (120 min), temperature (35 °C), lipase concentration (3% w/w), and TMP: FFA molar ratio (1:4). Fourier transform infrared spectroscopy (FT-IR), and nuclear magnetic resonance (NMR) studies confirm the synthesized TMP esters. Moreover, physicochemical properties such as viscosity, cloud point (CP), and calorific value (CV) are found to be 36 mm2/s, 7159.14 cal/g, and 20 °C, respectively.

Biochemical characterization and insights into the potency of the acidic Aspergillus niger NRC114 purified α-galactosidase in removing raffinose family oligosaccharides from soymilk yogurt

BackgroundBecause humans lack α-galactosidase, foods containing certain oligosaccharides from the raffinose family, such as soybeans and other legumes, may disrupt digestion and cause flatulence.ResultsAspergillus niger NRC114 α-galactosidase was purified using protein precipitation, gel filtration, and ion exchange chromatography steps, which resulted in a 123-fold purification. The purified enzyme was found to be 64 kDa using the SDS-PAGE approach. The optimum pH and temperature of the purified α-galactosidase were detected at pH 3.5 and 60 ºC, respectively. The pure enzyme exhibited potent acidic pH stability at pH 3.0 and pH 4.0 for 2 h, and it retained its full activity at 50 ºC and 60 ºC for 120 min and 90 min, respectively. The enzyme was activated using 2.5 mM of K+, Mg2+, Co2+, or Zn2+ by 14%, 23%, 28%, and 11%, respectively. The Km and Vmax values of the purified enzyme were calculated to be 0.401 µM and 14.65 μmol min−1, respectively. The soymilk yogurt showed an increase in its total phenolic content and total flavonoids after enzyme treatment, as well as several volatile compounds that were detected and identified using GC–MS analysis. HPLC analysis clarified the enzymatic action in the hydrolysis of raffinose family oligosaccharides.ConclusionThe findings of this study indicate the importance of A. niger NRC114 α-galactosidase enzyme for future studies, especially its applications in a variety of biological fields.

A Novel, Highly Potent NADPH-Dependent Cytochrome P450 Reductase from Waste Liza klunzingeri Liver.

The use of marine enzymes as catalysts for biotechnological applications is a topical subject. Marine enzymes usually display better operational properties than their animal, plant or bacterial counterparts, enlarging the range of possible biotechnological applications. Due to the fact that cytochrome P450 enzymes can degrade many different toxic environmental compounds, these enzymes have emerged as valuable tools in bioremediation processes. The present work describes the isolation, purification and biochemical characterization of a liver NADPH-dependent cytochrome P450 reductase (CPR) from the marine fish Liza klunzingeri (LkCPR). Experimental results revealed that LkCPR is a monomer of approximately 75 kDa that is active in a wide range of pH values (6-9) and temperatures (40-60 °C), showing the highest catalytic activity at pH 8 and 50 °C. The activation energy of the enzyme reaction was 16.3 kcal mol-1 K-1. The KM values for cytochrome C and NADPH were 8.83 μM and 7.26 μM, and the kcat values were 206.79 s-1 and 202.93 s-1, respectively. LkCPR displayed a specific activity versus cytochrome C of 402.07 µmol min-1 mg1, the highest activity value described for a CPR up to date (3.2-4.7 times higher than the most active reported CPRs) and showed the highest thermostability described for a CPR. Taking into account all these remarkable catalytic features, LkCPR offers great potential to be used as a suitable biocatalyst.

Quantitative fluorescence resonance energy transfer-based immunoassay for activated complement C1s.

C1s activation is associated with the pathogenesis of various diseases, indicating the potential value of C1s activation detection in clinic. Here we aimed to establish fluorescence resonance energy transfer (FRET)-based immunoassay for the quantitative detection of activated C1s in serum. FRET-based fluorogenic peptides, sensitive to the enzymatic activity of activated C1s, were prepared and labeled with the fluorophore ortho-aminobenzoic acid (Abz) and quencher 2,4-dinitrophenyl (Dnp), and then were further selected depending on its Kcat/Km value. C1s in the samples was captured and separated using anti-C1s-conjugated magnetic microbeads. Next, enzymatic activity of activated C1s in samples and standards was examined using fluorescent quenched substrate assays. Limit of detection (LOD), accuracy, precision, and specificity of FRET-based immunoassay were also investigated. This method presented a linear quantification range for the enzymatic activity of activated C1s up to 10 μmol min-1 mL-1 and LOD of 0.096 μmol·min-1·mL-1 for serum samples. The recovery of the method was in the range of 90% ~ 110%. All CV values of the intra-analysis and inter-analysis of three levels in samples were less than 10%. The cross-reaction rates with C1r enzyme, MASP1, and MASP2 were less than 0.5%. No significant interferences were found with bilirubin (0.2 mg mL-1), Chyle (2000 FTU), and haemoglobin (5 mg mL-1), but anticoagulants (EDTA, citrate and heparin) inhibited the enzymatic ability of activated C1s. Thus, this established method can be used for the determination of active C1s in human serum samples in the concentration interval of 0.096-10.000 μmol min-1 mL-1. One anti-C1s-based FRET immunoassay for activated C1s detection in serum samples were established, and it will be useful to explore the role of C1s activation in the pathogenesis, diagnosis and treatment in complement-related diseases.

Towards the rate limit of heterologous biotechnological reactions in recombinant cyanobacteria

BackgroundCyanobacteria have emerged as highly efficient organisms for the production of chemicals and biofuels. Yet, the productivity of the cell has been low for commercial application. Cyanobacterial photobiotransformations utilize photosynthetic electrons to form reducing equivalents, such as NADPH-to-fuel biocatalytic reactions. These photobiotransformations are a measure to which extent photosynthetic electrons can be deviated toward heterologous biotechnological processes, such as the production of biofuels. By expressing oxidoreductases, such as YqjM from Bacillus subtilis in Synechocystis sp. PCC 6803, a high specific activity was obtained in the reduction of maleimides. Here, we investigated the possibility to accelerate the NAD(P)H-consuming redox reactions by addition of carbohydrates as exogenous carbon sources such as D-Glucose under light and darkness.ResultsA 1.7-fold increase of activity (150 µmol min−1 gDCW−1) was observed upon addition of D-Glucose at an OD750 = 2.5 (DCW = 0.6 g L−1) in the biotransformation of 2-methylmaleimide. The stimulating effect of D-Glucose was also observed at higher cell densities in light and dark conditions as well as in the reduction of other substrates. No increase in both effective photosynthetic yields of Photosystem II and Photosystem I was found upon D-Glucose addition. However, we observed higher NAD(P)H fluorescence when D-Glucose was supplemented, suggesting increased glycolytic activity. Moreover, the system was scaled-up (working volume of 200 mL) in an internally illuminated Bubble Column Reactor exhibiting a 2.4-fold increase of specific activity under light-limited conditions.ConclusionsResults show that under photoautotrophic conditions at a specific activity of 90 µmol min−1 gDCW−1, the ene-reductase YqjM in Synechocystis sp. PCC 6803 is not NAD(P)H saturated, which is an indicator that an increase of the rates of heterologous electron consuming processes for catalysis and biofuel production will require funnelling further reducing power from the photosynthetic chain toward heterologous processes.