High Substrate Concentrations Research Articles

Properties and kinetic behavior of free and immobilized laccase from Oudemansiella canarii: Emphasis on the effects of NaCl and Na2SO4 on catalytic activities

Studies have highlighted the great potential of Oudemansiella canarii laccase in degrading synthetic dyes for reducing their toxicity. Immobilization of enzymes improves usability in degradation processes and the present work succeeded in immobilizing this laccase onto MANAE-agarose. Immobilization improved pH, thermal, and storage stabilities. Both, free and immobilized enzymes presented Michaelis-Menten kinetics with the substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) with Km values of 0.056 ± 0.003 and 0.195 ± 0.022 mM, respectively. Immobilization increased Vmax 1.27-fold. NaCl caused incomplete (hyperbolic) inhibition, which was satisfactorily described by the one-substrate one-modifier mechanism. Immobilization reduced the maximal inhibition by NaCl from 80.2 to 55.7 %. The effect of Na2SO4 was predominantly stimulation, but inhibition of the free enzyme occurred at high substrate concentrations. Stimulation of the immobilized enzyme by Na2SO4 was much more pronounced. It strongly depended on the substrate concentration and was much stronger (up to 300 %) at low substrate concentrations. The combined effects of substrate and sulfate on the immobilized laccase could be satisfactorily described by the one-substrate one-modifier mechanism. The modified response of the immobilized O. canarii laccase to NaCl and Na2SO4 considerably favors its use as a tool in bioremediation processes because environmental contamination by salts frequently represents a strong operational challenge.

Unlocking full potential of bamboo waster: Efficient co-production of xylooligosaccharides, lignin, and glucose through low-dosage mandelic acid hydrolysis with alkaline processing

Mandelic acid (MA), a natural and environmentally friendly organic acid, demonstrates high selectivity and efficiency in hydrolyzing hemicellulose, making it an excellent candidate for xylooligosaccharides (XOS) production at low acid dosages. Despite its potential, the application of MA for XOS production has not been evaluated. The study first investigated the effectiveness of MA in hydrolyzing hemicellulose in bamboo into XOS. Under optimized conditions (50 mM MA, 180 °C, 45 min), a high XOS yield of 65.9 % was achieved, with a total xylobiose and xylotriose yield of 43.5 %. Subsequent alkaline pretreatment enabled 92.1 % lignin removal from MA-pretreated bamboo. The recovered lignin exhibited a high purity of 95.2 % and retained fundamental structure and functional groups of native lignin. The resulting residue displayed enhanced crystallinity and accessibility, with reduced hydrophobicity and surface area lignin compared to untreated bamboo. At high substrate concentration of 20 %, cellulase hydrolysis resulted in a glucose conversion efficiency of 83.9 %. Overall, this integrated strategy offered an efficient approach for the co-production of valuable XOS, lignin, and glucose from bamboo. The efficient energy utilization and economic viability further highlight the potential of this method for large-scale industrial applications, making it an attractive option for biomass valorization.

The Role of Spacer Length in Macrocyclization Reactions Under Confinement

We studied the influence of the distance of olefin metathesis catalysts from the inner surface of a mesoporous support on macrocyclization and Z‐selectivity under confinement. For these purposes, the cationic molybdenum imido alkylidene N‐heterocyclic carbene (NHC) catalysts [Mo(N‐(2‐tBu‐C6H4)(1‐mesityl‐3‐(3‐trimethoxysilylprop‐1‐yl)‐imidazol‐2‐ylidene)(CHCMe2Ph)(MeCN)Br+ B(ArF)4‐] Mo2, [Mo(N‐(2‐tBu‐C6H4)(1‐mesityl‐3‐(3‐trimethoxysilylprop‐1‐yl)‐imidazol‐2‐ylidene)(CHCMe2Ph)(MeCN)OTf+ B(ArF)4‐] Mo3, [Mo(N‐(2,6‐Me2‐C6H3)(1‐mesityl‐3‐(3‐trimethoxysilylprop‐1‐yl)‐imidazol‐2‐ylidene)(CHCMe2Ph)(MeCN)Br+ B(ArF)4‐] Mo5 and [Mo(N‐(2,6‐iPr2‐C6H3)(1‐mesityl‐3‐(3‐trimethoxysilylprop‐1‐yl)‐imidazol‐2‐ylidene)(CHCMe2Ph)(MeCN)+Br B(ArF)4‐] Mo7 (B(ArF)4 = tetrakis[3,5‐bis(trifluoromethyl)phenyl]borate), all containing a trimethoxysilylpropyl tether, were selectively immobilized inside the mesopores of SBA‐15. Under confinement, both macro(mono)cyclization (MMC) and Z‐selectivity were higher than in solution but lower than with catalysts directly bound to the surface of the mesoporous supports. These findings are in agreement with existing theoretical models on substrate and product distribution in mesopores, which suggest that the highest substrate concentration is found at the pore wall and that it increases with decreasing pore diameter.

Agmatine Improves Oxidative Stress Profiles in Rat Brain Tissues Induced by Sodium Azide

Introduction: The brain is highly susceptible to oxidative damage due to excessive oxygen tension, a high concentration of oxidizable substrates, and low antioxidant capacity. Consequently, oxidative stress is linked to several brain disorders and neurodegeneration. Sodium azide is a cytochrome oxidase inhibitor that promotes neurodegeneration by enhancing the release of excitotoxins and inducing oxidative stress through the peroxidation of membrane lipids. This process results in the release of intra-mitochondrial Ca+2 and H2O2 (ROS Dependent-Ca+2 release). Agmatine, a biogenic amine, is also referred to as a free radical scavenger, protecting the brain from membrane collapse, apoptosis, and mitochondrial swelling. Objective: This study was designed to identify the antioxidative effects of agmatine on sodium azide- induced oxidative stress in brain tissues. Methodology: Twenty-four male albino Wistar rats were allocated into two groups: a control group receiving water and a test group administered sodium azide (5 mg/kg, intraperitoneally) for a duration of 14 days. Subsequently, the animals were further subdivided and treated for an additional two weeks with either water or agmatine (100 mg/kg). Behavioral assessments were performed onehour post-agmatine administration, and brain homogenates were prepared for biochemical analyses. Results: The agmatine-treated group exhibited a significant increase (P<0.01) in both the number of entries and the time spent in the light box and the open arms of the light/dark transition box and elevated plus maze tests, respectively. Additionally, agmatine administration significantly enhanced (P<0.01) the total number of squares crossed in the open field test. Biochemical assessments revealed that agmatine treatment significantly reduced (P<0.01) the levels of reactive oxygen species and malondialdehyde. Moreover, it significantly increased (P<0.01) the levels of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) and glutathione compared to the control group. Conclusion: The present study revealed that agmatine has substantial effects on oxidative and antioxidant enzyme levels in sodium azide-induced oxidative stress. Agmatine-treated rats exhibited decreased reactive oxygen species levels and improvements in behavioral impairments resulting from sodium azide administration.

Boosting Fructosyl Transferase's Thermostability and Catalytic Performance for Highly Efficient Fructooligosaccharides (FOS) Production.

Achieving enzymatic food processing at high substrate concentrations can significantly enhance production efficiency; however, related studies are notably insufficient. This study focused on the enzymatic synthesis of fructooligosaccharides (FOS) at high temperature and high substrate concentration. Results revealed that increased viscosity and limited substrate solubility in high-concentration systems could be alleviated by raising the reaction temperature, provided it aligned with the enzyme's thermostability. Further analysis of enzyme thermostability in real sucrose solutions demonstrates that the enzyme's thermostability was remarkedly improved at higher sucrose concentrations, evidenced by a 10.3 °C increase in melting temperature (Tm) in an 800 g/L sucrose solution. Building upon these findings, we developed a novel method for enzymatic FOS synthesis at elevated temperatures and high sucrose concentrations. Compared to existing commercial methods, the initial transglycosylation rate and volumetric productivity for FOS synthesis increased by 155.9% and 113.5%, respectively, at 65 °C in an 800 g/L sucrose solution. This study underscores the pivotal role of substrate concentration, incubation temperature, and the enzyme's actual status in advancing enzyme-catalyzed processes and demonstrates the potential of enzymatic applications in enhancing food processing technologies, providing innovative strategies for the food industry.

Understanding Cytochrome P450 Enzyme Substrate Inhibition and Prospects for Elimination Strategies.

Cytochrome P450 (CYP450) enzymes, which are widely distributed and pivotal in various biochemical reactions, catalyze diverse processes such as hydroxylation, epoxidation, dehydrogenation, dealkylation, nitrification, and bond formation. These enzymes have been applied in drug metabolism, antibiotic production, bioremediation, and fine chemical synthesis. Recent research revealed that CYP450 catalytic kinetics deviated from the classic Michaelis-Menten model. A notable substrate inhibition phenomenon that affects the catalytic efficiency of CYP450 at high substrate concentrations was identified. However, the substrate inhibition of various reactions catalyzed by CYP450 enzymes have not been comprehensively reviewed. This review describes CYP450 substrate inhibition examples and atypical Michaelis-Menten kinetic models, and provides insight into mechanisms of these enzymes. We also reviewed 3D structure and dynamics of CYP450 with substrate binding. Outline methods for alleviating substrate inhibition in CYP450 and other enzymes, including traditional fermentation approaches and protein engineering modifications. The comprehensive analysis presented in this study lays the foundation for enhancing the catalytic efficiency of CYP450 by deregulating substrate inhibition.

Selective switching hydrogenation products of 5-hydroxymethylfurfural at high substrate concentrations by regulating Pd-MgO interactions

Controlling the selective activation of one or some functional groups as desired is still a challenge in hydrogenation, especially at high substrate concentrations. Herein, Pd-MgO interfaces over Al2O3 were finely regulated for efficiently selective hydrogenation of furan ring in 5-hydroxymethylfurfural (HMF) to produce 5-hydroxymethyltetrahydro-2-furaldehyde (5-HMTHFF) or total hydrogenation to 2,5-bis(hydroxymethyl)tetrahydrofuran (BHMTHF), also inhibiting side reaction. The Pd-MgO/Al2O3 with 12.0 wt% of MgO selectively hydrogenates the furan rings with a 5-HMTHFF yield of 82.4 % while that with 2.0 wt% of MgO totally hydrogenates HMF with a DHMTHF yield of 96.2 % at high substrate concentrations (400 mM). Characterizations and DFT calculations demonstrated that oligomeric MgO species suppress the agglomeration of Pd nanoparticles and strengthen HMF adsorption, consequently promoting total hydrogenation. Furthermore, Pdδ+-MgO1-x sites between the crystallized MgO and Pd metal favored tilted adsorption on the interface and weakened the activation of the CO bond, consequently selectively producing 5-HMTHFF.

Effects of different substrate ratios on the enrichment of anammox bacteria at low substrate concentration

Anammox bacteria (AnAOB) can be easily enriched under high temperatures and high substrate concentrations, while the application of the mainstream anammox process in low substrate municipal sewage is still relatively uncommon. Therefore, this study investigated the enrichment of AnAOB under conditions of low ammonia nitrogen and nitrite concentration at 25 °C. Results showed that using inoculated aerobic sludge, four ASBRs (R1, R2, R3 and R4) were successfully initiated with different influent substrate (NO2−-N/NH4+-N) ratios of 1.2, 1.32, 1.4 and 1.5, respectively, with reactor start-up times were 162, 150, 120 and 134 days, respectively. The values of ΔNO2--N/ΔNH4+-N in reactors were stable at 1.17, 1.32, 1.43 and 1.53 respectively. The increase in influent substrate ratios resulted in improved TN removal rates and accelerated consumption of chemical oxygen demand (COD) during the initial start-up stage. The maximum TN removal rates achieved in the four reactors were 76.09%, 79.24%, 82.82% and 82.63%, respectively. The color of sludge gradually changes from yellowish-brown to reddish-brown. Furthermore, the surface of sludge exhibited a porous mineral structure, with crater-like cavities. The dominant anammox species in the system was identified as Candida Brocadia (3.04%). According to qPCR, the abundance of hzsB in the system is 1.65 × 1012 copies/g VSS, confirming the effective enrichment of AnAOB.

Kinetic characterization of two neuraminic acid synthases and evaluation of their application potential

Neuraminic acid synthases are an important yet underexplored group of enzymes. Thus, in this research, we performed a detailed kinetic and stability analysis and a comparison of previously known neuraminic acid synthase from Neisseria meningitidis, and a novel enzyme, PNH5, obtained from a metagenomic library. A systematic analysis revealed a high level of similarity of PNH5 to other known neuraminic acid synthases, except for its pH optimum, which was found to be at 5.5 for the novel enzyme. This is the first reported enzyme from this family that prefers an acidic pH value. The effect of different metal cofactors on enzyme activity, i.e. Co2+, Mn2+ and Mg2+, was studied systematically. The kinetics of neuraminic acid synthesis was completely elucidated, and an appropriate kinetic model was proposed. Enzyme stability study revealed that the purified enzyme exhibits changes in its structure during time as observed by differential light scattering, which cause a drop in its activity and protein concentration. The operational enzyme stability for the neuraminic acid synthase from N. meningitidis is excellent, where no activity drop was observed during the batch reactor experiments. In the case of PNH5, some activity drop was observed at higher concentration of substrates. The obtained results present a solid platform for the future application of these enzymes in the synthesis of sialic acids.Key points• A novel neuraminic acid synthase was characterized.• The effect of cofactors on NeuS activity was elucidated.• Kinetic and stability characterization of two neuraminic acid synthases was performed.Graphical

Efficient bioremediation of multiple steroid hormones by halotolerant 17β-hydroxysteroid dehydrogenase derived from moderately halophilic Pontibacillus chungwhensis HN14.

Steroid hormones exhibit potent endocrine disrupting activity and have been shown to disrupt the equilibrium of aquatic ecosystems and pose a threat to public health through their persistent and carcinogenic effects. Pontibacillus chungwhensis HN14, a moderately halophilic bacterium with the capacity to effectively degrade various polycyclic aromatic hydrocarbons and other organic pollutants, was previously isolated. Additionally, the strain HN14 showed strong environmental adaptability under various environmental stress conditions. In this study, the steroid degradation by strain HN14 was studied for the first time. We demonstrated that strain HN14 could degrade estradiol (E2) to maintain the growth of the strain and could convert E2 to estrone. Additionally, the efficient substrate degradation efficiency of P. chungwhensis HN14 under high salinity and high substrate concentration conditions was demonstrated. Furthermore, a 17β-hydroxysteroid dehydrogenase, 17β-HSD(HN14), was identified in strain HN14. Comparative analysis reveals that 17β-HSD(HN14) shares approximately 38% sequence identity with 17β-HSDx from Rhodococcus sp. P14. In addition, 100µg of purified 17β-HSD(HN14) could effectively convert about 40% of 0.25 mM of E2 within 1h period, with an enzyme activity of 17.5 U/mg, and catalyze the dehydrogenation of E2 and testosterone at the C-17 position. The characterization of purified enzyme properties reveals that 17β-HSD(HN14) exhibits exceptional structural robustness and enzymatic efficacy even under high salinity conditions of up to 20%. Overall, this study enhances our comprehension of steroid biodegradation in strain HN14 and contributes novel ideas and theoretical underpinnings for advancing bioremediation technologies targeting steroid pollution in high-saline environments.

Asymmetric Hydrogenation of Ketones by Simple Alkane-Diyl-Based Ir(P,N,O) Catalysts: A Comparative Study.

The development of new chiral ligands with simple and modular structure represents a challenging direction in the design of efficient homogeneous transition metal catalysts. Herein, we report on the asymmetric hydrogenation of prochiral ketones catalyzed by the iridium complexes of simple alkane-diyl-based P,N,O-type chiral ligands with a highly modular structure. The role of (i) the P-N and N-O backbone in the potentially tridentate ligands, (ii) the number, position and relative configuration of their stereogenic elements and (iii) the effect of their NH and OH subunits on the activity and enantioselectivity of the catalytic reactions are studied. The systematic variation in the ligand structure and the comparative catalytic experiments shed light on different mechanistic aspects of the iridium-catalyzed reaction. The catalysts containing the simple alkane-diyl-based ligands with central chirality provided high enantioselectivities (up to 98% ee) under optimized reaction conditions and proved to be active and selective even at very high substrate concentrations (100 mmol substrate/mL solvent).

Cluster-Level Heterostructure of PMo12/Cu for Efficient and Selective Electrocatalytic Hydrogenation of High-Concentration 5-Hydroxymethylfurfural.

Electrochemical hydrogenation of aldehyde molecules, exemplified by 5-hydroxymethylfurfural (HMF), offers a sustainable approach for synthesizing higher value-added alcohols. However, severe coupling side reactions impede its practical implementation at high concentrations. In this work, a cluster-level heterostructure of a PMo12/Cu catalyst is synthesized by loading Keggin-type phosphomolybdic acid (H3PMo12O40, PMo12) onto Cu nanowires. The catalyst exhibits high selectivity in electrocatalytic hydrogenation (ECH) of HMF to 2,5-bishydroxymethylfuran (BHMF) under an unprecedentedly high substrate concentration of 1.0 M. Under -0.3 V (vs RHE) with 1.0 M HMF, PMo12/Cu shows a Faradaic efficiency as high as 98% with an excellent productivity of 4.35 mmol cm-2 h-1 toward BHMF, much higher than those on the pristine Cu nanowires. Mechanism studies and density functional theory calculations demonstrate that the heterostructural interface of PMo12/Cu serves as an active reaction center for the ECH. The unique electronic properties and geometric structure promote the dissociative reduction of water molecules to generate H* and reduce HMF with a decreased reaction energy barrier, which is responsible for exceptional reactivity and selectivity.

Start-up and Recovery Performance of ANAMMOX Reactors Under Long-term Starvation

The application of ANAMMOX technology is constrained by sluggish growth and difficulty in enriching ANAMMOX bacteria. Long-term starvation of functioning bacteria due to limited substrate supply makes the steady operation of ANAMMOX reactors more difficult. Re-examining the start-up and recovery performance of the ANAMMOX reactor and identifying its resistance mechanism are important from the standpoint of long-term starvation. By inoculating nitrifying and denitrifying sludge under various operating circumstances, the ANAMMOX reactors were successfully started. Under various start-up procedures, the tolerance mechanism and recovery performance were examined. The outcomes demonstrated that the denitrifying sludge-inoculated reactor operated steadily with a high substrate concentration and low flow rate. After 85 days of operation, the removal efficiencies of NH4+-N, NO2--N, and total nitrogen reached 98.7%, 99.3%, and 89.3%, respectively. After 144 days of starvation and 30 days of recovery, the better nitrogen removal performance was achieved at a low substrate concentration and high flow rate, and the removal efficiencies were 99.8% （NH4+-N）, 99.8% （NO2--N）, and 93.6% （total nitrogen）. During the starvation, extracellular polymeric substances wrapped the ANAMMOX bacteria and kept them intact to resist long-term starvation stress. The expression of nirS, hzsA, and hdh genes ensured the synthesis of nitrite/nitric oxide oxidoreductase, hydrazine synthase, and hydrazine dehydrogenase to maintain ANAMMOX activity. There was no significant difference in the relative abundance of ANAMMOX bacteria before and after starvation recovery. Candidatus Kuenenia had better anti-hunger ability, and the relative abundance increased by more than 86% after 30 days of recovery, confirming its tolerance to long-term starvation.

Optimizing Continuous‐Flow Biocatalysis with 3D‐Printing and Inline IR Monitoring

AbstractEnzymatic biocatalysis typically generates less waste, uses less water, and minimizes energy consumption compared to traditional chemical methods. Efficient, cell‐free biosynthesis relies on the reuse of its valuable biocatalysts. Immobilization of enzymes on solid supports, such as enzyme carrier resins (ECRs), offers a reliable and widely deployed approach to maximize enzyme turnover in cell‐free biosynthesis. We focus on two major bottlenecks associated with optimizing cell‐free biocatalysis. First, we apply our lab's 3D‐printed labware to screen ECRs in 96‐well mini‐reactors to optimize enzyme immobilization conditions. Second, we introduce inline infrared spectroscopy to monitor bioreactor output and maximize enzyme productivity. Urease provides a model system for examining immobilization conditions and continuous assessment of biocatalyst performance. As required for the high substrate concentrations to improve process efficiency and minimize waste, urease was studied in unusually high concentrations of its substrate – molar concentrations of urea. The optimized reactor processed 3.24 L of 4.00 M urea at an average volumetric productivity of 13 g ⋅ L−1 ⋅ h−1 over 18 h and achieved an estimated productivity number of >17.4 kg urea processed per g of immobilized urease Type‐IX. This workflow can be generalized to most biocatalytic processes and could accelerate adoption of cell‐free biosynthesis for greater chemical sustainability.

Structural basis for the transport and regulation mechanism of the Multidrug resistance-associated protein 2.

Multidrug resistance-associated protein 2 (MRP2) is an ATP-powered exporter important for maintaining liver homeostasis and a potential contributor to chemotherapeutic resistance. Deficiencies in MRP2 function are associated with Dubin-Johnson Syndrome and increased vulnerability to liver injury from cytotoxic drugs. Using cryogenic electron microscopy (cryo-EM), we determined the structures of human MRP2 in three conformational states: an autoinhibited state, a substrate-bound pre-translocation state, and an ATP-bound post-translocation state. These structures show that MRP2 functions through the classic alternating access model, driven by ATP binding and hydrolysis. Its cytosolic regulatory (R) domain serves as a selectivity gauge, wherein only sufficiently high concentrations of substrates can effectively compete with and disengage the R domain to initiate transport. Comparative structural analyses of MRP2 in complex with different substrates reveal how the transporter recognizes a diverse array of compounds, highlighting the transporter's role in multidrug resistance.

Problem-Based Learning for Calculating Kinetic Parameters from Michaelis-Menten Equation

In “Advanced Enzymology” at the University of Barcelona, we teach various methods for identifying uni-uni irreversible reactions. Kinetic constants such as maximum velocity (Vmax) and Michaelis constant (Km) are calculated using non-linear regression with the Michaelis-Menten equation. However, if a computer is not available, students must calculate these kinetic parameters using linear regressions with Lineweaver-Burk, Eadie-Hofstee, and Hanes-Woolf plots. Michaelis-Menten equation: v = (Vmax [S]) / (Km + [S]), Lineweaver-Burk equation: 1/v = (Km/Vmax) (1/[S]) + 1/Vmax, Eadie-Hofstee equation: v = – Km (v/[S]) + Vmax, Hanes-Woolf equation: [S]/v = (1/Vmax) [S] + (Km/Vmax). The objective of this work was that students explore various methods for calculating kinetic constants and determine the most effective approach. A problem was prepared with data on substrate concentrations ([S]) and reaction rates (v). Data points with lower substrate concentration, and also with lower velocities had higher errors. The Michaelis-Menten plot provided only an approximate estimation of the kinetic constants, as it is not a linear graph. The Lineweaver-Burk plot generated maximum velocities that were too low due to the higher errors for low substrate concentrations, rendering impossible values of the kinetic parameters. The Eadie-Hofstee plot provided better results, as the values of v/[S] compensated their errors. Furthermore, kinetic constants were obtained directly from the slope and intersection of the line equation. The Hanes-Woolf plot also produced correct values for kinetic parameters, since high substrate concentrations were less erroneous than lower ones. After completing this computer class, students were very satisfied and learned the different methods for obtaining kinetic parameters. Non-linear regression is undoubtedly the most accurate method for obtaining kinetic parameters values. However, in the absence of computers, the Eadie-Hofstee plot is the best and most commonly used plot in kinetic papers.

Evaluation of the trade-off between water use efficiency and nutrient use efficiency in two semiarid coniferous tree species growing on an organic amended metalliferous mine tailing substrate

We evaluated the ecophysiological responses of two semiarid coniferous tree species, Pinus halepensis and Tetraclinis articulata, growing on a nutrient-poor metalliferous mine tailings substrate to organic amendments (biochar and/or organic municipal waste). The trees were grown in mesocosms under irrigated conditions for 20 months. Then, a comprehensive characterization of soil and plant parameters (including stable isotopes) was carried out. Treatments containing municipal waste showed better soil fertility indicators (approximately 2-fold higher organic carbon and total nitrogen concentrations) and higher plant biomass (up to 5-fold higher) than unamended and only biochar treatments. Trees in most of the treatments exhibited leaf N/P ratios <14 indicating severe N limitation of plant growth. Metal uptake was below phytotoxic levels across all the treatments. Leaf δ13C values correlated positively with δ18O across treatments for both species indicating increasing water use efficiency with tighter stomatal regulation of water flux, and with T. articulata exhibiting tighter stomatal control (higher δ18O values) than P. halepensis. Trees in treatments containing only biochar did not differ in ecophysiological performance from those in the unamended treatments. In contrast, leaf stable isotopes revealed sharply increased of time-integrated photosynthetic activity (favoured by higher leaf N concentrations) combined with lower time-integrated stomatal conductance in the treatments containing municipal waste, indicating greatly enhanced water use efficiency in better nourished plants. Trade-offs between water use efficiency and nutrient (N and P) use efficiency were evident across treatments, with higher leaf nutrient concentrations associated with higher water use efficiency, at the cost of a lower nutrient use efficiency. These trade-offs were not impaired by the high metal concentrations of the tailings substrate, indicating that ecophysiological adjustments in response to changes in plant nutrient status promoted by the addition of organic amendments are critical for the adaptability of native tree species employed in the phytostabilisation of mine tailings.

Photodecarboxylative Ring Annulation of α‐ and β‐Functionalized Phthaloyl‐GABA Derivatives: Bioactive Pyrroloisoindolinones with High Quantum Efficiency

AbstractThe triplet‐sensitized (by the solvent acetone) as well as the direct (λex=300–320 nm) photochemical decarboxylation of N‐phthaloylated γ‐aminobutyric acid (GABA) derivatives are versatile and high‐yielding routes to benzopyrrolizidines via intramolecular electron transfer initiated decarboxylation followed by radical coupling. The ß‐mono‐ and ß,ß'‐disubstituted N‐phthaloyl GABA derivatives 7 a–7 g, respectively, were applied as substrates. Decarboxylative photocyclization yielded hydroxy benzopyrrolizidines 8 a–8 g in high chemical yields and with moderate diastereoselectivities from the ß‐monosubstituted substrates. The analogous α‐substituted GABA derivatives 11 a–11 c were also applied as potential substrates for memory of chirality effects. The reaction quantum yields of the photodecarboxylation reactions for the parent GABA derivative 13 and for the new substrates 7 h and 11 a were determined by the quantum yield determination system (QYDS) and showed a remarkable concentration dependency indicating aggregation at higher substrate concentrations. Inhibition studies on the atherogenic human serine hydrolase cholesterol esterase showed derivatives 8 a and 8 d to exhibit a hyperbolic mode of inhibition with moderate IC50 values of about 60–80 μM.

Facile Asymmetric Syntheses of Non-Natural Amino Acid (S)-Cyclopropylglycine by the Developed NADH-Driven Biocatalytic System

A self-sufficient bifunctional enzyme integrating reductive amination and coenzyme regeneration activities was developed and successfully employed to synthesize (S)-cyclopropylglycine with an improved reaction rate 2.1-fold over the native enzymes and a short bioconversion period of 6 h at a high substrate concentration of 120 g·L−1 and space–time yield of (S)-cyclopropylglycine up to 377.3 g·L−1·d−1, higher than that of any previously reported data. Additionally, (S)-cyclopropylglycine could be continuously synthesized for 90 h with the enzymes packed in a dialysis tube, providing 634.6 g of (S)-cyclopropylglycine with &gt;99.5% ee and over 95% conversion yield up to 12 changes. These results confirmed that the newly developed NADH-driven biocatalytic system could be utilized as a self-sufficient biocatalyst for industrial application in the synthesis of (S)-cyclopropylglycine, which provides a chiral center and cyclopropyl fragment for the frequent synthesis of preclinical/clinical drug molecules.

Optimizing light-driven ene-reductase reactions with g-C3N4 and electron mediators

In the realm of green chemistry and sustainable industrial processes, the integration of enzymes with photocatalytic materials offers a promising avenue for achieving efficient, light-driven catalysis while minimizing environmental impact. This study introduces an enzyme-photo-coupled catalytic system (EPCS) using graphitic carbon nitride (g-C3N4) as a photocatalyst and methyl viologen (MV2+) as an electron mediator to drive the reduction of 2-cyclohexen-1-one by the Old Yellow Enzyme (OYE), reducing the reliance on expensive and scarce bioelectronic carriers and demonstrating a sustainable alternative for biocatalytic applications. Furthermore, we explored the incorporation of a Pickering emulsion system to enhance mass transfer, especially in reactions involving highly concentrated substrates. This adaptation not only improves the yield of the EPCS at higher substrate concentrations but also offers a scalable and environmentally benign alternative to traditional photocatalytic systems.