KM Values Research Articles

ENKIE: A package for predicting enzyme kinetic parameter values and their uncertainties.

Relating metabolite and enzyme abundances to metabolic fluxes requires reaction kinetics, core elements of dynamic and enzyme cost models. However, kinetic parameters have been measured only for a fraction of all known enzymes, and the reliability of the available values is unknown. The ENzyme KInetics Estimator (ENKIE) uses Bayesian Multilevel Models to predict value and uncertainty of KM and kcat parameters. Our models use five categorical predictors and achieve prediction performances comparable to deep learning approaches that use sequence and structure information. They provide calibrated uncertainty predictions and interpretable insights into the main sources of uncertainty. We expect our tool to simplify the construction of priors for Bayesian kinetic models of metabolism. Code and Python package are available at https://gitlab.com/csb.ethz/enkie and https://pypi.org/project/enkie/. Supplementary data are available at Bioinformatics online.

Measurement of the Taylor Microscale and the Effective Magnetic Reynolds Number in the Solar Wind With Cluster

AbstractWe use magnetic field data from the Cluster mission to estimate the value of the Taylor microscale and the effective magnetic Reynolds number in the interplanetary solar wind. Turbulent cascades can be characterized by the spatial scale at which dissipation begins to impact the local energy transfer, estimated by the Taylor microscale, as well as the separation between the injection and dissipation scales, estimated by the effective magnetic Reynolds number. Estimating the Taylor microscale requires measurements of the autocorrelation function at small separations. The Cluster spacecraft have exceptionally sensitive search coil magnetometers with high time resolution, making them ideal for measuring the Taylor microscale. We obtain a value of km; smaller than most previous measurements. We interpret this value as being smaller due to the higher time resolution, enabling the curvature of the autocorrelation function to be measured closer to the origin, giving a more accurate measurement. Combining the Taylor Microscale's computed value with concurrent correlation length measurements, we obtain a value of for the effective magnetic Reynolds number, which compares well to other observations. The four spacecraft of Cluster also allow directions transverse to the flow to be surveyed. The small separations (7 km) of Clusters 3 and 4 show that the Taylor microscale may vary as a function of direction to the mean magnetic field direction. The observed differences are small, requiring more observations to confirm this anisotropy.

Multipartite Fluorogenic Sensors for Monitoring Tyrosine Phosphatase Activity.

Fluorogenic substrates are essential tools for studying the activity of many enzymes including the protein tyrosine phosphatases (PTPs). Here, we have taken the first step toward the development of genetically encodable sensors for PTP activity using fluorescent and fluorogen-activating proteins. The Fluorescence-Activating and absorption Shifting Tag (FAST) is a small protein that becomes fluorescent upon binding to a small molecule dye. We demonstrate that FAST protein can be used as a sensor for PTP-mediated dephosphorylation of phosphorylated dye molecules. Phosphorylated 4-hydroxybenzylidene rhodanine (pHBR) is not able to bind to the FAST protein and induce fluorescence, but provides a sensitive assay for PTP activity, readily detecting 100 pM concentrations of PTP1B in the presence of FAST with a kcat value of 19 ± 1 s-1 and a KM value of 93 ± 3 µM. In addition, while phosphorylation of the C-terminal peptide of split GFP does not result in appreciable change in fluorescence of the reconstituted protein, phosphorylation of the C-terminal peptide of the split FAST protein abrogates fluorescence. Upon PTP-mediated dephosphorylation of the C-terminal peptide, the ability of the N- and C-terminal components to form a fluorescent complex with the small molecule dye is restored, leading to fluorescence.

Non-additivity of the functional properties of individual P450 species and its manifestation in the effects of alcohol consumption on the metabolism of ketamine and amitriptyline

To explore functional interconnections between multiple P450 enzymes and their manifestation in alcohol-induced changes in drug metabolism, we implemented a high-throughput study of correlations between the composition of the P450 pool and the substrate saturation profiles (SSP) of amitriptyline and ketamine demethylation in a series of 23 individual human liver microsomes preparations from donors with a known history of alcohol consumption. The SSPs were approximated with linear combinations of three Michaelis-Menten equations with globally optimized KM (substrate affinity) values. This analysis revealed a strong correlation between the rate of ketamine metabolism and alcohol exposure. For both substrates, alcohol consumption caused a significant increase in the role of the low-affinity enzymes. The amplitudes of the kinetic components and the total rate were further analyzed for correlations with the abundance of 11 major P450 enzymes assessed by global proteomics. The maximal rate of metabolism of both substrates correlated with the abundance of CYP3A4, their predicted principal metabolizer. However, except for CYP2D6 and CYP2E1, responsible for the low-affinity metabolism of ketamine and amitriptyline, respectively, none of the other potent metabolizers of the drugs revealed a positive correlation. Instead, in the case of ketamine, we observed negative correlations with the abundances of CYP1A2, CYP2C9, and CYP3A5. For amitriptyline, the data suggest inhibitory effects of CYP1A2 and CYP2A6. Our results demonstrate the importance of functional interactions between multiple P450 species and their decisive role in the effects of alcohol exposure on drug metabolism.

Exploration of catalytic activities of Caesalpinia bonduc (L.) Roxb. ash on green-oxidation of small organic substrates: an effective way to reduce the use of harsh chemicals

In this modern era, large-scale industrial use of harsh chemicals is an alarming problem as it can cause serious environmental damage. Among industrial pollutants, harsh chemical oxidizing agents are one of the leading chemicals that can cause severe environmental threats. These harmful harsh chemicals are practically omnipresent in industries and are used to oxidize numerous small organic substrates. Replacement of these harsh chemicals with some plant-based products might be helpful to safeguard our environmental damage. In this study, Caesalpinia bonduc (L.) Roxb. ash is used as a catalyst to activate aerial oxygen for promoting the oxidation of 3,5-di-tert-butylcatechol (3,5-DTBC) and o-aminophenol (OAPH) like small organic substrates. Detailed kinetic studies for these catalytic oxidation reactions have been performed spectrophotometrically, which confirms that catalytic reactions follow Michaelis–Menten kinetics. Several enzyme-kinetics plots such as the Michaelis–Menten plot, Lineweaver–Burk plot, Hanes–Woolf plot, and Eadie–Hofstee plot have been used to determine the maximum rate achieved by the reaction (Vmax) and Michaelis constant (KM) like kinetic parameters. The average values of Vmax and KM for catalytic oxidation of 3,5-DTBC are (8.6669 ± 0.6519) × 10–5 M S−1 and (21.4289 ± 0.4741) × 10–4 M, respectively. Similarly, the average values of Vmax and KM for catalytic oxidation of OAPH are (2.1781 ± 0.2071) × 10–5 M S−1 and (20.1062 ± 1.2690) × 10–3 M, respectively. The mineral composition of Caesalpinia bonduc (L.) Roxb. ash has also been evaluated by using inductively coupled plasma optical emission spectroscopy (ICP-OES).Graphical

Recombinant production of Paenibacillus wynnii β-galactosidase with Komagataella phaffii

BackgroundThe β-galactosidase from Paenibacillus wynnii (β-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low KM value) compared to industrially used β-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, β-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce β-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the β-gal-Pw in K. phaffii either extracellularly or intracellularly.ResultsFirstly, 11 different signal peptides were tested for extracellular production of β-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of β-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular β-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric β-galactosidase activity of 7537 ± 66 µkatoNPGal/Lculture was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkatoNPGal/gDCW/h was achieved when using the GAP promoter for β-gal-Pw production compared to the AOX1 promoter. After partial purification, a β-gal-Pw enzyme preparation with a total β-galactosidase activity of 3082 ± 98 µkatoNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter).ConclusionThis study showed that the β-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular β-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.

Identification of a robust bacterial pyranose oxidase that displays an unusual pH dependence

Pyranose oxidases are valuable biocatalysts, yet only a handful of bacterial pyranose oxidases are known. These bacterial enzymes exhibit noteworthy distinctions from their extensively characterized fungal counterparts, encompassing variations in substrate specificity and structural attributes. Herein a bacterial pyranose oxidase from Oscillatoria princeps (OPOx) was biochemically characterized in detail. In contrast to the fungal pyranose oxidases, OPOx could be well expressed in Escherichia coli as soluble, fully flavinylated and active oxidase. It was found to be highly thermostable (melting temperature >90 ⁰C) and showed activity on glucose, exhibiting an exceptionally low KM value (48 μM). Elucidation of its crystal structure revealed similarities with fungal pyranose oxidases, such as being a tetramer with a large central void leading to a narrow substrate access tunnel. In the active site, the FAD cofactor is covalently bound to a histidine. OPOx displays a relatively narrow pH optimum for activity with a sharp decline at relatively basic pH values which is accompanied with a drastic change in its flavin absorbance spectrum. The pH-dependent switch in flavin absorbance features and oxidase activity was shown to be fully reversible. It is hypothesized that a glutamic acid helps to stabilize the protonated form of the histidine that is tethered to the FAD. OPOx presents itself as a valuable biocatalyst as it is highly robust, well-expressed in E. coli, shows low KM values for monosaccharides and has a peculiar pH dependent “on-off switch”.

Lipase Enzyme Kinetics with the Addition of Avocado Seed Extract as an Inhibitor in Olive Oil

Free fatty acids are compounds resulting from the hydrolysis of triglycerides catalyzed by the enzyme lipase. Free fatty acids harm health if they enter the body in large amounts. Avocado seed extract can reduce free fatty acid levels by inhibiting lipase activity. This research is a type of true-experimental research that aims to determine the effect of variations in substrate concentration on lipase enzyme activity with the addition of avocado seed extract and to determine the impact of the addition of avocado seed extract as an inhibitor on lipase enzyme kinetics. Lipase activity was determined based on the amount of oleic acid formed using Cu acetate pyridine reagent by Uv-Vis spectrophotometry at a wavelength of 715 nm. Lipase enzyme kinetics were determined using the Lineweaver-Burk curve equation, a transformed form of the Michaelis-Menten equation. The results showed that 60% substrate concentration was the most effective in reducing lipase enzyme activity by 60%. In addition, the addition of avocado seed extract proved to act as a competitive inhibitor, with the KM value decreasing from 0.014 M to 0.004 M and Vmaks remaining relatively stable at 23.2 µM. This shows that avocado seed extract effectively reduces lipase enzyme activity, which has implications for reducing FFA levels in olive oil. Thus, this study concludes that avocado seed extract has great potential as a natural inhibitor agent in reducing lipase enzyme activity.

PRODUCTION AND CHARACTERIZATION OF CELLULASES FROM ASPERGILLUS NIGER UNDER STATIC FERMENTATION

The ever-increasing uses of cellulase in various industries have made it popular among researchers. Annually, a large amount of fruit wastes go into vain, which is a great source of cellulases. The objective of this study is to use grape wastes as substrate for production of cellulases from Aspergillus niger using static fermentation. CMCase and FPase assays were performed to characterize the cellulases. The cellulases’ CMCase and FPase activities and stabilities were analyzed for optimum temperature and pH. The effect of substrate concentration and kinetic constants Km and Vmax, along with thermodynamic analysis, were determined. The effects of several metals on the activity of the enzyme were observed. The optimal temperatures were found as 40 °C and 50 °C for CMCase and FPase activity, respectively. CMCase activity shows stability at 20 °C-60 °C, FPase shows low thermal stability as its activity starts to decrease after 50 °C. CMCase and FPase both show maximum activity at pH 6, and maintain their stability at pH 6-7. The values of Km and Vmax obtained from Lineweaver and Burk plot for CMCase are 0.648 mM and 12.953 mM/min, and for FPase are 0.975 mM and 41.493 mM/min. The Arrhenius plot was used to calculate activation energy (Ea) as -19 kJ/mol, and enthalpy of reaction (ΔH) as 16.4 kJ/mol, while entropy ΔS -16.4 kJ/mol was obtained from the plot of ln(Vmax/T) versus the inverse of temperature (1/T). Most metals induce enzyme activities, whereas EDTA inhibits enzyme activities. The findings suggest A. niger has remarkable cellulase production potential from grape wastes in static fermentation, at optimum temperature and pH levels for achieving enzyme activity and stability.

Optimization of the production of a new milk clotting enzyme by Aspergillus flavus thermophile on agro-industrial residues medium and characterization of the enzyme

This study focused on the optimization of the production of a milk clotting protease by Aspergillus flavus, isolated from a thermal station. The fermentation was carried out on wheat bran as a medium, followed by a partial characterization of the enzyme. The fungal isolate was screened for protease production based on clear zone hydrolysis on skim milk agar, and then it was identified as belonging to the genus Aspergillus based on its morphological characteristics. This identification was confirmed by molecular analyzes with 100% similarity with the A. flavus strain. After 7 days of fermentation, the crude extract is recovered by simple filtration. The production and optimization of the milk coagulant protease using wheat bran and agro-industrial waste as a medium was carried out using a statistical method of experimental design based on the Plackett and Burman matrices. The results showed that the optimal fermentation medium consisted of wheat bran (4% w/w), incubated at a temperature of 30 °C, with a pH of 5.0 after 6 days of fermentation. The enzyme was partially purified by ammonium sulfate precipitation, yielding an estimated proteolytic activity of 2,985 U. The partially purified protease exhibits optimal activity at pH 5 with hemoglobin as a substrate and an optimal temperature of 50 °C. The Lineweaver-Burk plot showed a KM value of 4.83 g/L and a Vm of 1,560 U. This enzyme causes very rapid coagulation of fresh cow’s milk with an optimum at 45 °C, a pH 7 and a concentration of 50 mM of calcium.

Characterization of a Halostable Metalloprotease from the Halophilic Bacterium Bacillus clausii J1G-0%B

Protein plays a crucial role as a biocatalyst in various industries, particularly in breaking down proteins into amino acids. The demand for proteases capable of functioning under extreme conditions, such as high salinity, temperature, and pH, is increasing. To address this, the exploration of bacteria that produce stable proteases in such environments is essential. Bacillus clausii J1G-0%B, a halophilic bacterium isolated from Madura salt ponds, thrives in salinity levels of 0-20% NaCl. This study aims to obtain and characterize the protease produced by Bacillus clausii J1G-0%B, focusing on its activity and stability under extreme conditions. The research involved screening, production, and purification of the protease using ammonium sulfate fractionation and dialysis. Protease activity was measured using the Kunitz method, and protein content was determined using the Lowry method. Characterization included optimizing enzymatic conditions (pH, temperature, NaCl concentration), identifying metalloprotease types, and analyzing enzyme kinetics and thermodynamics. The study successfully produced protease using a halophilic medium with casein and 5% NaCl. After 96 hours of incubation, the protease exhibited a specific activity of 654.737 U/mg. Optimal activity was observed at pH 7, 50°C, and 10% NaCl, with stability between 2.5% and 15% NaCl concentration. Enzyme kinetics revealed a high affinity for casein, with a KM value of 0.164 mg/mL and Vmax of 13.182 µmol/mL·min. Thermodynamic analysis indicated high stability, as shown by a positive ΔGi value (+105.84 kJ/mol), a low inactivation constant (ki = 0.0031 min-1), and a long half-life (t½ = 223.548 minutes). EDTA chelation tests confirmed that the protease is a metalloprotease. The halostable protease from Bacillus clausii J1G-0%B shows significant potential for industrial applications and bioremediation in high-salinity environments.

Change of ectropion surgery on astigmatic vector and ocular biometry

Aim: To evaluate corneal astigmatism change and intraocular lens (IOL) power values following ectropion surgery. Materials and Methods: This comparative prospective research included patients with involutional ectropion who were divided using the snap-back test. The severity of ectropion increased progressively to reach the highest levels in Group 4. Patients underwent lateral tarsal strip procedures, and preoperative and 3-month postoperative biometry measurements were performed. Results: While the mean flattest keratometry (K1), steepest keratometry (K2), and mean keratometry (Km) values exhibited nonsignificant increases at 3 months after surgery in Groups 1 and 2 (p>0.05 for all), the mean values of K1, K2, and Km were increased with statistical significance in Groups 3 and 4 at 3 months postoperatively (p<0.05 for all). Conclusion: According to the snap-back test, ectropion in advanced stages will affect topographic values and IOL power calculations. If surgeons are going to perform cataract or refractive surgery after ectropion repair, they may consider changing the lens power selection accordingly.

Effect of Ionizing Radiation on the Activity of the Enzyme Invertase Studied by Polarimetry

The effect of ionizing radiation on biological systems, and particularly the enzymes which are responsible for most cellular functions, leads to chemical alterations in their molecular structure, and as a subsequent result often causes inhibition of their activities. Of particular importance for understanding the molecular and kinetic mechanisms of action of ionizing radiation on these systems is to find suitable experimental protocols and kinetic models applicable to specific enzymes and their substrates. In the present study we offer a convenient experimental approach by applying polarimetric measurements to study the influence of ionizing radiation on the catalytic action of the enzyme invertase. Following the Michaelis- Menten kinetic model we determined the values of the kinetic constants Km и Vmax for the reactions catalyzed either by the native or irradiated invertase and made some conclusions about possible changes either in the active centre or in the molecular structure of the enzyme under the action of ionizing radiation.

Identification and characterization of archaeal-type FAD synthase as a novel tractable drug target from the parasitic protozoa Entamoeba histolytica.

Flavin adenine dinucleotide (FAD) is an essential cofactor for numerous flavoenzymes present in all living organisms. The biosynthesis of FAD from riboflavin involves two sequential reactions catalyzed by riboflavin kinase and flavin adenine dinucleotide synthase (FADS). Entamoeba histolytica, the protozoan parasite responsible for amebiasis, apparently lacks a gene encoding FADS that share similarity with bacterial and eukaryotic canonical FADS, yet it can synthesize FAD. In this study, we have identified the gene responsible for FADS and thoroughly characterized physiological and biochemical properties of FADS from E. histolytica. Phylogenetic analysis revealed that the gene was likely laterally transferred from archaea. The kinetic properties of recombinant EhFADS were consistent with the notion that EhFADS is of archaeal origin, exhibiting KM and kcat values similar to those of the arachaeal enzyme while significantly differing from the human counterpart. Repression of gene expression of EhFADS by epigenetic gene silencing caused substantial reduction in FAD levels and parasite growth, underscoring the importance of EhFADS for the parasite. Furthermore, we demonstrated that EhFADS gene silencing reduced thioredoxin reductase activity, which requires FAD as a cofactor and makes the ameba more susceptible to metronidazole. In summary, this study unveils unique evolutionary and biochemical features of EhFADS and underscores its significance as a promising drug target in combating human amebiasis.IMPORTANCEFAD is important for all forms of life, yet its role and metabolism are still poorly studied in E. histolytica, the protozoan parasite causing human amebiasis. Our study uncovers the evolutionary unique key enzyme, archaeal-type FADS for FAD biosynthesis from E. histolytica for the first time. Additionally, we showed the essentiality of this enzyme for parasite survival, highlighting its potential as target for drug development against E. histolytica infections.

Gene cloning, IPTG-independent auto-induction and characterization of a novel hyperstable S9 prolyl oligopeptidase having lipolytic activity from Thermotoga naphthophila RKU-10T with applications

A hyperstable lipase from Thermotoga naphthophila (TnLip) was cloned and overexpressed as a soluble and active monomeric protein in an effectual mesophilic host system. Sequence study revealed that TnLip is a peptidase S9 prolyl oligopeptidase domain (acetyl esterase/lipase-like protein), belongs to alpha/beta (α/β)-hydrolase superfamily containing a well-conserved α/β-hydrolase fold and penta-peptide (GLSAG) motif. Various cultivation and induction strategies were applied to improve the heterologous expression and bacterial biomass, but TnLip intracellular activity was enhanced by 14.25- fold with IPTG-independent auto-induction approach after 16 h (26 °C, 150 rev min−1) incubation. Purified TnLip (35 kDa) showed peak activity at 85 °C in McIlvaine buffer (pH 7.0–8.0), and has great stability over a broad range of pH (5.0–10.0), and temperature (40–85 °C) for 8 h. TnLip exhibited prodigious resistance toward various commercial detergents, chemical additives, and salt. TnLip activity was improved by 170.51 %, 130.67 %, 127.42 %, 126.54 %, 126.61 %, 120.32 %, and 116.31 % with 50 % (v/v) of methanol, ethanol, n-butanol, isopropanol, acetone, glycerol, and acetic acid, respectively. Moreover, with 3.0 M of NaCl, and 10 mM of Ca2+, Mn2+, and Mg2+ TnLip activity was augmented by 210 %, 185.64 %, 152.03 %, and 116.26 %, respectively. TnLip has an affinity with various substrates (p-nitrophenyl ester and natural oils) but maximal hydrolytic activity was perceived with p-nitrophenyl palmitate (pNPP, 3600 U mg−1) and olive oil (1182.05 U mg−1). The values of Km (0.576 mM), Vmax (4216 μmol mg−1 min−1), VmaxKm−1 (7319.44 min−1), kcat (1106.74 s−1), and kcatKm−1 (1921.42 mM−1 s−1) were calculated using pNPP substrate. Additionally, TnLip degraded animals' fats and removed oil stains within 3 h and 5 min, respectively. All these features make halo-alkali-thermophilic TnLip as an auspicious contender for laundry detergents (cleaning bio-additive), fat degradation, wastewater treatment and endorse eco-friendly stewardship along with various other biotechnological applications.

Enhancement of β-galactosidase catalytic activity and stability through covalent immobilization onto alginate/tea waste beads and evaluating its impact on the quality of some dairy products

The current study aimed to evaluate the hydrolysis of whole fat milk (WFM) and sweet whey (SW) using β-galactosidase (β-gal) after covalent immobilization onto activated alginate/tea waste (Alg/TW) beads as a novel carrier. The optimum temperature for free and Alg/TW/β-gal was 40 °C and the ideal pH was 7.0. However, Alg/TW/β-gal displayed better stabilities at high temperatures and a wide pH range. Additionally, the value of Km and Vmax for Alg/TW/β-gal was higher than the free enzyme. The Alg/TW/β-gal showed better residual activity (78.6 %) after 90 storage days at 4 °C. The reusability of Alg/TW/β-gal was very good as it conserved its full activity after 15 consecutive cycles and conserved 93 % of its initial activity after 10 cycles with ONPG (O-nitrophenyl-β-D-galactopyranoside) and lactose as a substrate, respectively. The impact of Alg/TW/β-gal on WFM and SW using HPLC analysis revealed a remarkable decrease in lactose concentration and increase of glucose and galactose concentrations. The SW exhibited higher degree of lactose hydrolysis (97.3 %) compared to WFM (62.4 %). Besides, SW had a prominent increase in total phenolic content (96.8 mg/L) compared to WFM (54.3 mg/L). The antioxidant activity had increased after enzyme treatment in both WFM and SW. The GC–MS analysis for volatile compounds identified twenty-five flavour constituents. Finally, Alg/TW/β-gal has a potential application for obtaining healthy, acceptable, and commercial dairy products of low lactose.

Assessing the complexation of dissolved organic matter with heavy metals (Cu2+, Pb2+) in leachate from an old Japanese landfill site using fluorescence quenching.

Dissolved organic matter (DOM) in landfill leachate impacts the toxicity, bioavailability, and migration of heavy metals. The present study investigated the complexation of heavy metals (Cu2+ and Pb2+) with DOM from two landfill leachate samples, representing an old landfill site containing incineration residues and incombustible waste. The logarithms of the stability constant (log KM) and percentage of complexed fluorophores were calculated using both the Ryan-Weber non-linear model and the modified Stern-Volmer model, yielding good agreement. The log KM values (at pH = 6.0 ± 0.1) calculated using both methods for the two sampling points were 5.02-5.13 and 4.85-5.11 for Cu2+-DOM complexation, and 5.01-5.13 and 4.46-4.87 for Pb2+-DOM complexation, respectively. Log KM was slightly higher for binding of DOM with Cu2+ than Pb2+, and the quenching degree was stronger for complexation with Cu2+ (28.5-30.6% and 38.0-45.9%) than Pb2+ (6.5-7.1% and 10.0-15.4%) in both leachate samples. While log KM values were similar, differences in the contributions of functional groups and molecular composition led to varying degrees of quenching. This study reveals the potential for heavy metal binding by DOM in landfill leachate with a unique solid waste composition and emphasizes variations in fluorescence quenching between Cu2+ and Pb2+ despite similar log KM levels. These findings may be useful for assessing heavy metal behavior in landfill leachate and its impacts on the surrounding environment.

Clotrimazole Identified as a Selective UGT2B4 Inhibitor Using Canagliflozin-2'-O-Glucuronide Formation as a Selective UGT2B4 Probe Reaction.

UGT2B4 is a highly expressed drug-metabolizing enzyme in the liver contributing to the glucuronidation of several drugs. To enable quantitatively assessing UGT2B4 contribution toward metabolic clearance, a potent and selective UGT2B4 inhibitor that can be used for reaction phenotyping was sought. Initially, a canagliflozin-2'-O-glucuronyl transferase activity assay was developed in recombinant UGT2B4 and human liver microsomes (HLM) [±2% bovine serum albumin (BSA)]. Canagliflozin-2'-O-glucuronidation (C2OG) substrate concentration at half-maximal velocity value in recombinant UGT2B4 and HLM were similar. C2OG formation intrinsic clearance was five- to seven-fold higher in incubations containing 2% BSA, suggesting UGT2B4 susceptibility to the inhibitory unsaturated long-chain fatty acids released during the incubation. Monitoring for C2OG formation, 180 compounds were evaluated for UGT2B4 inhibition potency in the presence and absence of 2% BSA. Compounds that exhibited an apparent UGT2B4 IC50 of < 1 μM in HLM with 2% BSA were evaluated for inhibition of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, UGT2B10, UGT2B15, and UGT2B17 catalytic activities to establish selectivity suitable for supporting UGT reaction phenotyping. In this study, clotrimazole was identified as a potent UGT2B4 inhibitor (HLM apparent IC50 of 11 to 35 nM ± 2% BSA). Moreover, clotrimazole exhibited selectivity for UGT2B4 inhibition (>24-fold) over the other UGT enzymes evaluated. Additionally, during this study it was discovered that the previously described UGT2B7 inhibitors 16α- and 16β-phenyllongifolol also inhibit UGT2B4. Clotrimazole, a potent and selective UGT2B4 inhibitor, will prove essential during UGT reaction phenotyping. SIGNIFICANCE STATEMENT: To mechanistically evaluate drug interactions, it is essential to understand the contribution of individual enzymes to the metabolic clearance of a drug. The present study describes the development of a UGT2B4 activity assay that enabled the discovery of the highly selective and potent UGT2B4 inhibitor clotrimazole. Clotrimazole can be used in UGT reaction phenotyping studies to estimate fractional contribution of UGT2B4.

Kinetic and thermodynamic parameters of urease in soils with different fertilization regimes

In recent years the growing demand for food led to intensification of agricultural practices, especially by the excessive use of fertilizers, which increased the environmental pollution. Therefore, pollution control by improving the efficiency of fertilizers and reducing their application is of great interest. Urease is one of the most active hydrolases in soil, having an important role in soil N cycle and being used as an indicator of soil quality. The objective of this study was to assess the urease activity in soils with different fertilization treatment, as well as its kinetic and thermodynamic parameters to better understand its driving factors. The results show a better enzymatic activity in soils treated with combined manure and mineral fertilizers. Soil urease has two optimal pH values, in the neutral and basic domains. Enzymatic activity has a steep increase with the temperature in the interval 45–65°C. The KM values increase with temperature from 11 to 23.5 mM, indicating a lower substrate affinity. The activation enthalpies for enzyme-substrate formation as well as for the rate limiting step are 7.59 and 14.18 kcal/mol respectively. The relationship between urease activity and microbial biomass will be further investigated.

Mandelonitrile lyase MDL2-mediated regulation of seed amygdalin and oil accumulation of Prunus Sibirica

BackgroundThe Prunus sibirica seeds with rich oils has great utilization, but contain amygdalin that can be hydrolyzed to release toxic HCN. Thus, how to effectively reduce seed amygdalin content of P. sibirica is an interesting question. Mandelonitrile is known as one key intermediate of amygdalin metabolism, but which mandelonitrile lyase (MDL) family member essential for its dissociation destined to low amygdalin accumulation in P. sibirica seeds still remains enigmatic. An integration of our recent 454 RNA-seq data, amygdalin and mandelonitrile content detection, qRT-PCR analysis and function determination is described as a critical attempt to determine key MDL and to highlight its function in governing mandelonitrile catabolism with low amygdalin accumulation in Prunus sibirica seeds for better developing edible oil and biodiesel in China.ResultsTo identify key MDL and to unravel its function in governing seed mandelonitrile catabolism with low amygdalin accumulation in P. sibirica. Global identification of mandelonitrile catabolism-associated MDLs, integrated with the across-accessions/developing stages association of accumulative amount of amygdalin and mandelonitrile with transcriptional level of MDLs was performed on P. sibirica seeds of 5 accessions to determine crucial MDL2 for seed mandelonitrile catabolism of P. sibirica. MDL2 gene was cloned from the seeds of P. sibirica, and yeast eukaryotic expression revealed an ability of MDL2 to specifically catalyze the dissociation of mandelonitrile with the ideal values of Km (0.22 mM) and Vmax (178.57 U/mg). A combination of overexpression and mutation was conducted in Arabidopsis. Overexpression of PsMDL2 decreased seed mandelonitrile content with an increase of oil accumulation, upregulated transcript of mandelonitrile metabolic enzymes and oil synthesis enzymes (involving FA biosynthesis and TAG assembly), but exhibited an opposite situation in mdl2 mutant, revealing a role of PsMDL2-mediated regulation in seed amygdalin and oil biosynthesis. The PsMDL2 gene has shown as key molecular target for bioengineering high seed oil production with low amygdalin in oilseed plants.ConclusionsThis work presents the first integrated assay of genome-wide identification of mandelonitrile catabolism-related MDLs and the comparative association of transcriptional level of MDLs with accumulative amount of amygdalin and mandelonitrile in the seeds across different germplasms and developmental periods of P. sibirica to determine MDL2 for mandelonitrile dissociation, and an effective combination of PsMDL2 expression and mutation, oil and mandelonitrile content detection and qRT-PCR assay was performed to unravel a mechanism of PsMDL2 for controlling amygdalin and oil production in P. sibirica seeds. These findings could offer new bioengineering strategy for high oil production with low amygdalin in oil plants.